WO2012051572A1 - Modèle mammifère non humain humanisé de la malaria et utilisations de ce dernier - Google Patents

Modèle mammifère non humain humanisé de la malaria et utilisations de ce dernier Download PDF

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Publication number
WO2012051572A1
WO2012051572A1 PCT/US2011/056425 US2011056425W WO2012051572A1 WO 2012051572 A1 WO2012051572 A1 WO 2012051572A1 US 2011056425 W US2011056425 W US 2011056425W WO 2012051572 A1 WO2012051572 A1 WO 2012051572A1
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human
human mammal
mammal
malaria
cytokines
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PCT/US2011/056425
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English (en)
Inventor
Anburaj Amaladoss
Qingfeng Chen
Ming Dao
Peter Preiser
Jianzhu Chen
Subra Suresh
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Massachusetts Institute Of Technology
Nanyang Technological University
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Publication of WO2012051572A1 publication Critical patent/WO2012051572A1/fr

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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
    • A01K67/0271—Chimeric vertebrates, e.g. comprising 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
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • A01K2267/0337—Animal models for infectious diseases
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Malaria is one of the world's most devastating human infections with 300-500 million clinical cases each year. It is caused by the parasites of the Plasmodium species, which are transmitted by the bite of infected mosquitoes. Plasmodium species are highly host specific and therefore difficult to model in laboratories. Most experimental in vivo studies on malaria have been carried out with various Plasmodium species specific to mice and rodents.
  • Immunodeficient mouse-human chimeras provide a novel and practical means to study the interaction between microbial pathogens and human cells, tissues, and organs. Plasmodium falciparum has been studied in severe combined immunodeficient (scid) mice grafted with human red blood cells (RBCs) with limited success (Badell, 2000; Morena et al., 2001; Morena et al., 2006). This 'grafting' involves repeated injection of large volumes of human RBCs into recipient mice, which are often treated with chemical and immunomodulators to suppress clearance of human RBCs from the circulation.
  • the present disclosure is based on the successful development of a mouse model for infection of human malarial parasite (e.g., Plasmodium falciparum), i.e., immunodeficient mice carrying human erythrocytes differentiated from human hematopoietic stem cells (HSCs), which are introduced into the immunodeficient mice, in the presence of one or more o human cytokines.
  • human malarial parasite e.g., Plasmodium falciparum
  • HSCs human hematopoietic stem cells
  • one aspect of the present disclosure relates to a method of producing a non-human mammal (e.g., a mouse) that is a model for human malaria infection.
  • the method comprises: introducing into an immunodeficient non-human mammal (i) human HSCs, (ii)5 one or more human cytokines that promote differentiation of the human HSCs into human erythrocytes in the non-human mammal, and (iii) a malarial parasite that infects human erythrocytes; and maintaining the non-human mammal under conditions in which the human HSCs differentiate into functional human erythrocytes in the non-human mammal; wherein the malarial parasite infects the human erythrocytes, thereby producing a non-human mammal
  • HSCs can be introduced into the non-human mammal intracardially or through a facial vein.
  • the one or more human cytokines e.g., human erythropoietin, human IL-3, human granulocyte-macrophage colony stimulating factor and/or human stem cell factor
  • the one or more human cytokines are introduced into the non-human mammal after the HSCs have been introduced 5 into the same non-human mammal.
  • the one or more human cytokines are introduced into the non-human mammal by delivering (via, e.g., hydrodynamic injection) one or more nucleic acids (e.g., naked DNAs (i.e., DNA delivered free from agents which promote transfection, such as DNA inserted into a plasmid alone) or vectors such as expression vectors) encoding the one or more human cytokines and maintaining the non- 0 human mammal under conditions allowing expression of the one or more nucleic acids (i.e., producing the encoded cytokines in the non-human mammal).
  • nucleic acids e.g., naked DNAs (i.e., DNA delivered free from agents which promote transfection, such as DNA inserted into a plasmid alone) or vectors such as expression vectors
  • the one or more nucleic acids encoding the one or more human cytokines are one or more naked DNAs or one or more vectors.
  • Vectors encoding the human cytokines can be plasmids, viral vectors (e.g., lentiviral vectors or adenoviral vectors), or combinations thereof.
  • the non-human mammal that may be used in the methods 5 described herein is a non-human primate, a rodent, a canine, a feline or a ruminant.
  • the non-human mammal is a mouse. Examples include, but are not limited to, a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation (NOD/scid mouse); a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation (NOD/scid mouse); a non-obese diabetic mouse that carries a severe combined
  • the malarial parasite that may be used in the methods described herein preferably is a malarial parasite species capable of infecting human erythrocytes. Examples include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, P. ovalae, P. malariae, P.
  • a malarial parasite is introduced
  • the method described above can further comprise introducing into the non-human mammal an agent that depletes macrophages (e.g., clodronate-liposome).
  • an agent that depletes macrophages e.g., clodronate-liposome.
  • Such a macrophage-depleting agent can be introduced into the non-human mammal prior to infection 2 o with the malarial parasite.
  • the human HSCs are introduced into the non-human mammal intracardially or by a facial vein.
  • Another aspect of the present disclosure relates to a non-human mammal (e.g., a mouse such as those described herein) that is a model for human malarial infection.
  • a non-human mammal e.g., a mouse such as those described herein
  • non-human mammal preferably immunodeficient, comprises human erythrocytes infected with a malarial parasite (e.g., those described herein).
  • the human erythrocytes are differentiated from human HSCs introduced into the non-human mammal in the presence of one or more human cytokines that promote differentiation of the human HSCs into human erythrocytes (e.g., human erythropoietin, human IL-3, human granulocyte-
  • the non-human mammal that is a model for human malarial infection can be prepared by any of the methods described herein for producing such animal models.
  • the present disclosure provides a method of using the non-human mammal described herein for identifying anti-malaria agents.
  • This method comprises: (a) 5 introducing into any of the non-human mammals that is a model for human malarial infection as described herein a candidate agent (e.g., a vaccine candidate), (b) determining a level of malarial parasite infection of the human erythrocytes in the non-human mammal; and (c) assessing whether the candidate agent is an anti-malaria agent.
  • a candidate agent e.g., a vaccine candidate
  • a reduced level of malarial parasite infection of the human erythrocytes in the non-human mammal as relative to a o control non-human mammal indicates that the agent is an anti-malaria agent. If malarial parasite infection of the human erythrocytes is not detected in the non-human mammal, this indicates that the candidate agent is an anti-malaria agent that can treat or prevent malarial infection in a human.
  • the method further comprises the steps of any of the 5 methods provided for producing the non-human mammal such that it is a model for human malaria infection.
  • the non-human mammal malaria model is treated with a macrophage-depleting agent prior to infection with a malarial parasite.
  • an anti-malaria agent comprising: (a) introducing into an immunodeficient non-human mammal (e.g., a o mouse such as one of those immunodeficient mice described herein) (i) human hematopoietic stem cells (HSCs), (ii) one or more human cytokines (e.g., human erythropoietin, human IL- 3, human granulocyte-macrophage colony stimulating factor and/or human stem cell factor) that promote differentiation of the human HSCs into human erythrocytes in the non-human mammal, (iii) a malarial parasite that infects human erythrocytes (e.g., Plasmodium
  • a candidate agent e.g., a vaccine candidate
  • a reduced level of malarial parasite infection of the human erythrocytes in the non-human mammal as relative to a control non-human mammal indicates that the candidate agent is an anti-malaria agent. If malarial parasite infection of the human erythrocytes is not detected in the just-noted non-human mammal, this indicates that the candidate agent is an anti-malaria agent that can treat or prevent malarial infection in a
  • the non-human mammal that is a model for human malaria infection is a non-human mammal produced by any of the methods for producing a non-human mammal provided herein.
  • the one or more human cytokines o are introduced into the non-human mammal after the HSCs has been introduced into the same non-human mammal.
  • the one or more human cytokines are introduced into the non-human mammal by delivering (via, e.g., hydrodynamic injection) one or more nucleic acids encoding the one or more human cytokines into the non-human mammal and maintaining the non-human mammal under conditions in which the one or more5 nucleic acids are expressed.
  • the one or more nucleic acids encoding the one or more human cytokines are one or more naked DNAs or vectors, such as expression vectors.
  • Vectors encoding the human cytokines may be plasmids, viral vectors (e.g., lenti viral vectors or adenoviral vectors), or combinations thereof.
  • the non-human mammal is a non-human primate, a rodent, a o canine, a feline or a ruminant. In some embodiments, the non-human mammal is a mouse.
  • Examples include, but are not limited to, a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation (NOD/scid mouse); a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation and lacks a gene for the cytokine- receptor ⁇ chain (NOD/scid IL2R ⁇ _/ ⁇ mouse), or a Balb/c rag _/ ⁇ c ' ⁇ mouse.
  • NOD/scid mouse a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation
  • NOD/scid IL2R ⁇ _/ ⁇ mouse a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation and lacks a gene for the cytokine- receptor ⁇ chain
  • Balb/c rag _/ ⁇ c ' ⁇ mouse a non-obese diabetic mouse that carries a severe combined immunodeficiency
  • the malarial parasite that may be used in the methods described herein preferably is a malarial parasite species capable of infecting human erythrocytes. Examples include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, P. ovalae, P. malariae, P.
  • a malarial parasite is introduced into the non-human mammal noted above after human erythrocytes have been generated in 0 the mammal.
  • the method described above can further comprise introducing into the non-human mammal an agent that depletes macrophages (e.g., clodronate-liposome).
  • macrophage-depleting agent can be introduced into the non-human mammal prior to infection with the malarial parasite.
  • the human HSCs are introduced into the non-human mammal intracardially or by a facial vein.
  • an anti-malaria agent identified from the methods described herein is provided.
  • the anti-malaria agent can be used for treating or preventing malaria in humans.
  • compositions comprising one or more of the anti-malaria agents provided are also provided.
  • such compositions may further comprise one or more pharmaceutically acceptable carriers.
  • Such compositions may be for use in treating or preventing malaria in humans and the uses of such agents for the manufacture of medicaments for human malaria treatment or prevention.
  • methods of treating or preventing malaria with such anti-malaria agents and compositions comprise administering one or more of the anti-malaria agents or compositions provided to a subject that has, is suspected of having or is at risk of having a malarial infection. In other embodiments, the methods further comprise administering another agent to treat or prevent malarial infection in the subject.
  • Figure 1 is a diagram showing the presence of human erythrocytes (indicated as CD235 + ) in NSG mice introduced with human HSCs. Left panel: in the absence of human cytokines; Right panel: in the presence of human cytokines. The data demonstrate improved reconstitution of human erythrocytes. Humice were hydrodynamically injected with empty pcDNA vector (Ctrl) or pcDNA vectors expressing human EPO and IL-3. Four weeks after injection, blood was stained for human CD235ab. Shown are CD235ab versus DAPI staining profiles of all blood cells. Representative data from one of three mice are shown. The numbers indicate percentages of cells in the gated region.
  • Figure 2 is a photo showing Plasmodium falciparum invasion of human RBCs generated in humanized mice (humice). 1 million schizonts were used to infect 500 million RBCs from humice or NSG control mice. A: Before percol gradient. B: purified schizonts.
  • Figure 3 is a photo showing ex vivo reinvasion of parasites (1 st cycle) into human RBCs generated in humice. Thin smear was taken 16 hours later ( ⁇ 6 hours post invasion) and stained with Geimsa. About 0.2% ring stage parasites were observed.
  • Figure 4 is a photo showing maturation of ring stage parasites into trophozoites and schizonts at approximately 30 hours post invasion.
  • A human RBCs.
  • B RBCs from humice.
  • Figure 5 is a photo showing that schizonts from RBCs of humanized mice produced viable merozoites that entered into 2 nd cycle of reinvasion.
  • A human RBCs.
  • B RBCs from humice.
  • Figure 6 is a photo showing maturation of ring stage parasites into trophozoites and schizonts during 2nd cycle.
  • A Human RBCs.
  • B RBCs from humice.
  • Figure 7 is a photo showing ring stage parasites in 3 rd cycle reinvasion.
  • A human RBCs.
  • B RBCs from humice.
  • Figure 8 is a photo showing detection of parasites by nested PCR in blood samples from humanized mice infected with various parasite strains at different time points, -ve: negative control.
  • Figure 9 is a diagram showing characterization of reticulocytes in humice.
  • Percentage of human RBCs (numbers in gated regions) shown from 8 different humanized mice.
  • Human RBCs were stained with a FITC-conjugated antibody specific to CD235, a major surface protein marker on the human red blood cells.
  • B Co- staining of human reticulocytes with the anti-CD235 antibody and Thiozol Orange (TO).
  • Figure 10 is a photo showing detection of parasites by nested PCR in blood samples from humanized mice infected with viable (M1-M3) or freeze-thawed parasites (M4-M6) at different time points.
  • Figure 11 is a photo showing detection of parasites by nested PCR in blood samples from humanized mice infected with 3 different parasite strains selected based on the preliminary screening, -ve: negative control.
  • Figure 12 is a diagram showing infection of humanized mice with mouse-adapted parasite strains.
  • A a photo showing detection of parasites by nested PCR in blood samples from humanized mice infected with mouse-adapted parasite strains at different time points.
  • Ve negative control.
  • B a photo showing Geimsa smear at the 3 rd cycle.
  • Figure 13 is a photo showing blood smear from ex vivo cultured parasites stained with anti-CD235 antibody from mice and detected with anti-mice secondary antibody conjugated to FITC.
  • Figure 14 demonstrates the detection by nested PCR from blood in vivo infected humice.
  • Pf + humice 100 ul whole blood from humanized mice were collected; 5 million schizonts were added to the culture. The parasitemia ranged from 0.5 to 1.6%. The culture was injected into the same mice and animals were sacrificed at timed intervals for bleeding.
  • Pf+ NSG control mice 600 ml blood were collected from humanized mice; culture was set up with 30 million schizonts. The parasitemia was about 0.2%. Culture corresponding to 100 ul blood was injected into 6 different NSG mice. Animals were sacrificed at timed intervals for bleeding.
  • Figure 15 is a photo showing that P. falciparum cultured in vivo in selected small RBCs are able to sustain 2 rounds of invasion in humanized mice.
  • Malaria is one of the major health problems today. Efforts to combat this disease over the last 30 year have mainly focused on Plasmodium falciparum, the parasite responsible for the most severe form of malaria in humans. P. falciparum is estimated to cause around 300-
  • mice are useful for studying human cells and biological processes in vivo.
  • the engraftment is achieved and maintained by daily intra peritoneal injections throughout the experiment (Jimenez Diaz, 2009).
  • NOD-scid I12rg _/ ⁇ mice developed recently bears a targeted mutation at the interleukin-2 receptor (I1-2R) o gamma chain locus (Shultz et al, 2005). These mice have been reported to support greatly increased engraftment of human tissue, HSCs and PBMCs (Shultz et al, 2007).
  • I1-2R interleukin-2 receptor
  • the present disclosure provides non-human mammal models of human malaria and uses thereof for, e.g., identifying anti-malaria agents and developing new diagnostic approaches. These malaria models are suitable for studying infections of both P. vivax and P. 5 falciparum, as well as other malarial parasite species. Other advantages of the non-human mammal models of malaria and methods described herein include, but are not limited to, the following.
  • the non-human mammal models of malaria described herein carry human erythrocytes that can be infected with a human malarial parasite and can be used to assess the efficacy of new compounds for preventing and/or treating infection by the
  • Vaccine means a composition of matter that improves the immune response to a particular pathogen,5 in this case a malaria parasite.
  • a vaccine may contain factors that stimulate a subject's
  • Anti-malaria agents are any agents that prevent, treat, ameliorate or alleviate malaria infection, malaria infectivity or the symptoms thereof. o Non-human Mammal Models for Human Malaria
  • a non-human mammal model for human malaria is a living, non-human mammal that may be used for the research and investigation of human malaria infection.
  • the non-human mammal chosen meets a determined taxonomic equivalency to humans, so as to react to malaria infection and/or its treatment in a way that resembles human physiology as 5 needed.
  • non-human mammal models for human malaria infection described herein are non-human mammals (e.g., mice), preferably immunodeficient, that carry human
  • erythrocytes are differentiated from human hematopoietic stem cells (HSCs) introduced into the non-human mammals, preferably in the presence of one or more human cytokines capable of promoting erythrocyte differentiation from HSCs.
  • HSCs human hematopoietic stem cells
  • human blood lineage cells have been successfully reconstituted in immunodeficient mice, which were found to be o suitable for infection with human malaria parasite such as Plasmodium falciparum, leading to schizonts development and reinvasion of human RBCs from such humanized mice ex vivo.
  • Humanized mice are either immunodeficient mice repopulated with human blood cell lineages or mice genetically engineered to express human genes and are useful for studying human cells and biological process in vivo.
  • a non-human mammal is a non-human vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals).
  • mammalian species that can be used to construct the animal models described o herein include, but are not limited to, non-human primates (e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs), canines, felines, and ruminants (e.g., goats, sheep, cows, pigs, horses).
  • the non-human mammal is a mouse.
  • the non- human mammal used in the methods described herein can be adult, newborn (e.g., ⁇ 48 hours old; pups) or in utero.
  • the non-human mammal described herein can be an immunodeficient non-human mammal, i.e., a non-human mammal that has one or more deficiencies in its immune system. That is, the immune system' s capacity to fight infectious diseases is compromised or entirely absent.
  • the level of immune activity in an immunodeficient subject can be 10- 80% of that in a healthy counterpart.
  • Immunodeficient mammals allow reconstitution of 0 human blood cell lineages when human HSCs are introduced.
  • an immunodeficient mammal lacks its own T cells, B cells, NK cells or a combination thereof.
  • the immunodeficient non-human mammal is an immunodeficient mouse, e.g., NSG or NOD scid gamma (NOD. Cg-Prkdcscid Il2rgtml Wjl/SzJ) mouse.
  • an immunodeficient mouse e.g., NSG or NOD scid gamma (NOD. Cg-Prkdcscid Il2rgtml Wjl/SzJ) mouse.
  • mice Other types of immonodeficient mice include, but are not limited to, a non-obese diabetic mouse 5 that carries a severe combined immunodeficiency mutation (NOD/scid mouse); a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation and lacks a gene for the cytokine-receptor ⁇ chain (NOD/scid IL2R ⁇ -/- mouse); and a Balb/c rag-/- c-l- mouse, severe combined immunodeficiency (scid) mice, non-obese diabetic NOO)-scid mice, lL2rg ⁇ mice (e.g., NOD/LySz-sdd lL2rg ⁇ mice, NOD/Shi- scid lL2rg ⁇ mice (NOG o mice), B ALB/c- Rag ' lL2rg ' mice, Ya A -Rag ' lL2rg
  • HSCs Hematopoietic Stem Cells
  • HSCs are introduced into the non-human mammals noted above for making the human malaria animal model described herein.
  • HSCs e.g., human HSCs
  • HSCs are self-renewing stem cells that, when engrafted into a recipient, can "repopulate” or “reconstitute” the
  • HSCs are multipotent stem cells that give rise to (differentiate into) blood cell types including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) and lymphoid lineages (e.g., T-cells, B-cells, NK- o cells) or any combinations thereof).
  • myeloid e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages e.g., T-cells, B-cells, NK- o cells
  • a human erythrocyte refers to a human erythrocyte at all stages of development, e.g., immature human red blood cells (human reticulocytes); mature human red blood cells
  • HSCs express the cell marker CD34 and are commonly referred to as "CD34+”. As understood by those of skill in the art, HSCs can also express other cell markers, such as 5 CD 133 and/or CD90 ("CD133+", “CD90+”). In some instances, HSCs are characterized by markers that are not expressed, e.g., CD38. Thus, in one embodiment of the invention, the human HSCs used in the methods described herein are CD34+, CD90+, CD133+,
  • CD133+CD90+CD38- CD34+CD133+CD90+CD38-, or any combination thereof.
  • the HSCs are both CD34 ("CD34+”) and CD 133+ ("CD133+”), also referred to herein as "double positive” or "DP" cells or "DPC”.
  • the HSCs are CD34+CD133+, and can further be CD38- and/or CD90+.
  • HSCs are found in bone marrow such as in femur, hip, ribs, sternum, and other bones of a donor (e.g., vertebrate animals such as mammals, including humans, primates, pigs, mice, etc.).
  • a donor e.g., vertebrate animals such as mammals, including humans, primates, pigs, mice, etc.
  • Other sources of HSCs for clinical and scientific use include umbilical cord blood, placenta, fetal liver, mobilized peripheral blood, non-mobilized (or unmobilized) peripheral blood, fetal spleen, embryonic stem cells, and aorta-gonad-mesonephros (AGM), or a combination thereof.
  • AGM aorta-gonad-mesonephros
  • mobilized peripheral blood refers to peripheral blood that is enriched with HSCs (e.g., CD34+ cells).
  • HSCs e.g., CD34+ cells
  • Administration of agents such as chemotherapeutics and/or G-CSF mobilizes stem cells from the bone marrow to the peripheral circulation.
  • G-CSF granulocyte colony- stimulating factor
  • a 30- fold enrichment of circulating CD34+ cells is observed with peak values occurring on day 5 after the start of G-CSF administration.
  • the number of circulating CD34+ cells is very low, estimated between 0.01 to 0.05% of total mononuclear blood cells.
  • the human HSCs for use in the methods can be obtained from a single donor or multiple donors.
  • the HSCs used in the methods described herein can be freshly isolated HSCs, cryopreserved HSCS, or a combination thereof.
  • HSCs can be obtained from these sources using a variety of methods known in the art.
  • HSCs can be obtained directly by removal from the bone marrow, e.g., in the hip, femur, etc., using a needle and syringe, or from blood following pre-treatment of the donor with cytokines, such as granulocyte colony- stimulating factor (G-CSF), that induce cells to be released from the bone marrow compartment.
  • cytokines such as granulocyte colony- stimulating factor (G-CSF)
  • cytokines are introduced into the non-human mammal model of human malaria to promote human HSC differentiation into human erythrocytes, which can be infected by a human malarial parasite.
  • cytokines are proteins that stimulate or inhibit differentiation, proliferation or function of immune cells.
  • human cytokines capable of promoting human HSC differentiation into erythrocytes include, but are not limited to, interleukin 3 (IL-3; e.g., GenBank accession number AAC08706), erythropoietin (EPO; e.g., GenBank accession number CAA26095), garanulocyte- macrophage colony stimulating factor (GM-CSF; e.g., GenBank accession number
  • IL-3 interleukin 3
  • EPO erythropoietin
  • GM-CSF garanulocyte- macrophage colony stimulating factor
  • Methods for obtaining cytokine peptides and/or their coding nucleic acid sequences are routine in the art and include isolating the peptide and/or nucleic acid (e.g., cloning) from a variety of sources (e.g., serum), producing the peptide and/or nucleic acid recombinantly or obtaining the peptide and/or nucleic acid from commercial sources.
  • sources e.g., serum
  • Malarial parasite species capable of infecting humans are known in the art, e.g.,
  • the non-human mammal models for human malaria may be infected with one or a combination of any of these parasites, which can be obtained by methods known in the art, e.g., in vitro culturing.
  • a non-human mammal preferably an immunodeficient mammal such as an immunodeficient mouse
  • human HSCs live into the non-human mammal
  • the human erythrocytes differentiated from the human HSCs constitute at least 2% (e.g., 3%, 5%, 8%, 10%, or higher) of the total erythrocytes in the blood of the non- human mammal.
  • the non-human mammal can be first treated or manipulated prior to, e.g., introduction of the human HSCs, for example, to further enhance reconstitution of the human HSCs and/or enhance engraftment and/or reconstitution of the human HSCs.
  • the non-human mammal is irradiated prior to introduction of the HSCs.
  • one or more chemotherapeutics are administered to the non-human mammal prior to introduction of the HSCs.
  • the HSCs for use in the methods noted above can be introduced into the non-human mammal directly as obtained (e.g., unexpanded) or manipulated (e.g., expanded) prior to introducing the HSCs into the non-human mammal.
  • the HSCs are expanded prior to introducing the HSCs into the non-human mammal.
  • a population of HSCs can be expanded by co-culturing the HSCs with mesenchymal stem cells (MSCs) in the presence of growth factors (e.g., angiopoietin-like 5 (Angplt5) growth factor, IGF-binding protein 2 (IGFBP2), stem cell factor (SCF), fibroblast growth factor (FGF), thrombopoietin (TPO), or a combination thereof) to produce a cell culture.
  • growth factors e.g., angiopoietin-like 5 (Angplt5) growth factor, IGF-binding protein 2 (IGFBP2), stem cell factor (SCF), fibroblast growth factor (FGF), thrombopoietin (TPO), or a combination thereof
  • the cell culture is maintained under conditions in which an expanded population of HSCs is produced (e.g., see Maroun, K., et al., ISSCR, 7th Annual Meeting, Abstract No. 1401 (Jul
  • the HSCs can be introduced into the non-human through a routine administration route as known in the art.
  • the HSCs are injected intracardially into the non-human mammal. Alternatively, they can be injected via facial vein.
  • one or more human cytokines that can promote HSC differentiation into erythrocytes are introduced into the non-human mammal. All or a biologically active portion of cytokines can be introduced into the non-human mammal as a protein, peptide and/or as nucleic acid (e.g., DNA, RNA) that encodes the one or more cytokines.
  • nucleic acid encoding one or more human cytokines is introduced into the non-human mammal, wherein the human cytokines promote differentiation of the human HSCs into human erythrocytes when expressed in the non- human mammal.
  • the non-human is maintained under conditions in which the one or more cytokines are expressed.
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cytokines are introduced into the non-human mammal.
  • only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cytokines are introduced into the non-human mammal.
  • Peptides, proteins and/or nucleic acids encoding each human cytokine can be introduced simultaneously or
  • each nucleic acid encoding each cytokine can be introduced in its own single plasmid or vector, or can be introduced in multiple plasmids or vectors; alternatively, all the nucleic acids encoding the cytokines to be introduced can be introduced in a single plasmid or vector).
  • the one or more cytokines can be delivered into the non-human mammal through a routine route as long as the nucleic acid(s) is/are expressed in the non-human mammal.
  • proteins and peptides can be injected directly.
  • nucleic acids can be naked DNAs (e.g., DNA fragments), which can be integrated into the genome of the non-human mammal at a suitable position such that the encoded cytokine is produced in non-human mammal.
  • the cytokine-encoding sequence is cloned into a plasmid (e.g., pcDNA3.1(+)) or in a viral vector (e.g., adenovirus, adeno-associated virus, lentivirus, retrovirus and the like), which is introduced into the non-human mammal for expression of the encoded cytokine.
  • a viral vector e.g., adenovirus, adeno-associated virus, lentivirus, retrovirus and the like
  • a vector can be a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked.
  • the vector can be capable of autonomous replication or integrate into a host DNA.
  • examples of the vector include a plasmid, cosmid, or viral vector.
  • Viral vectors derived from viruses such as a lentivirus (a lentiviral vector) or an adenovirus (an adenoviral vector), are a tool commonly used for delivering genetic materials into cells.
  • the vectors described herein includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell (e.g., a mouse cell), i.e., producing the protein encoded by the nucleic acid in the host cell.
  • a host cell e.g., a mouse cell
  • the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.
  • regulatory sequences include, but are not limited to, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
  • a cytokine-encoding nucleic acid is introduced in a plasmid using hydrodynamic injection (e.g., into tail vein of a non-human mammal). If necessary, expression of the cytokine(s) in the non-human mammal can be examined by methods well known in the art, e.g., RT-PCR, immunoblot, ELISA, or cytokine activity analysis.
  • the HSCs and the one or more cytokines can be introduced simultaneously or
  • the HSCs are introduced into a newborn pup (e.g., about 48 hours old) and the nucleic acids encoding the cytokines5 are introduced about 1-12 months later (e.g., about 2 months, about 3 months, about 4
  • HSCs and the one or more cytokines e.g., as proteins, peptides and/or as nucleic o acids encoding the one or more cytokines
  • cytokines e.g., as proteins, peptides and/or as nucleic o acids encoding the one or more cytokines
  • methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intrafemoral, intraventricular, intracranial, intrathekal, intravenous, intracardial, intrahepatic, intra-bone marrow, subcutaneous, topical, oral and intranasal routes of administration.
  • Other suitable methods of introduction can also include, in utero injection, 5 hydrodynamic gene delivery, gene therapy, rechargeable or biodegradable devices, particle acceleration devises ("gene guns”) and slow release polymeric devices.
  • the non-human mammal is maintained (e.g., housed) under conditions in which the non-human is reconstituted with the HSCs that differentiate into human erythrocytes.
  • Such conditions 0 under which the non-human animals of the invention are maintained include meeting the basic needs (e.g., food, water, light) of the mammal, and/or specific pathogen free conditions (SPF), all of which are well known to those of skill in the art.
  • the methods described herein can further comprise determining whether the human HSCs are present and/or the human HSCs have differentiated into human erythrocytes via methods well known in the art. For example, flow cytometry analysis using antibodies specific for surface cell markers of human HSCs and/or human erythorcytes (e.g.,
  • CD235ab+ can be used to detect the presence of human HSCs in the non-human mammal.
  • cytokines in addition to cytokines, introduction of other agents (e.g., proteins such as human proteins; human secreted proteins), such as growth factors, steroids, and/or small molecules, can be used in the methods to improve reconstitution and/or function of human cells beyond blood lineage cells.
  • agents e.g., proteins such as human proteins; human secreted proteins
  • growth factors such as growth factors, steroids, and/or small molecules
  • an agonist of one or more of the human cytokines can be introduced into the non-human mammal to enhance reconstitution of the HSCs.
  • the human HSCs differentiate into mature (functional) human erythrocytes and human reticulocytes, which are human immature red blood cells.
  • “functional” (or “biologically active” or “mature") human erythrocytes refer to the fact that the human erythrocytes express one or more, and in some instances all, of the cell surface markers of the corresponding normal (wild type) cell found in humans, and as a result, function similarly in the non-human mammal as they function in a human.
  • Assays for determining the function of human erythrocytes in the non- human mammal are known to those of skill in the art and are described herein.
  • a (one or more) malarial parasite that is capable of infecting human erythrocytes is also introduced into the non-human mammal.
  • erythrocytes at any stage of development, e.g., mature human red blood cells; human reticulocytes
  • a variety of malarial parasites that are capable of infecting human erythrocytes are known. Examples of such malarial parasites include Plasmodium (P.) falciparum, P. vivax, P.ovalae, P. malariae, and P. knowlesi.
  • the non-human mammal can be subject to depletion of human macrophages, e.g., reducing the amount of human macrophages in the blood of the non-human mammal by at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more).
  • an agent capable of reducing the amount of macrophages can be administered into the non-human mammal via a routine route.
  • human macrophages are depleted by liposome encapsulated clodronate, following the method described in Van Rooigen and Sanders, 1994.
  • non-human mammals as models of human malaria as described herein can be used in a variety of ways, e.g., screening for anti-malaria agents, which are capable of ameliorating or alleviating malaria in humans.
  • "ameliorating" or “alleviating” malaria includes inhibiting malarial infection of erythrocytes in an individual, preventing further malarial infection of erythrocytes in an individual, complete or partial removal (e.g., an agent that is toxic to (e.g., kills) a malarial parasite) of the malarial parasite from an individual, and/or lessening and/or preventing one or more symptoms of malarial infection in an individual.
  • Such agents in some embodiments, treat or prevent malaria infection.
  • a non-human mammal carrying human erythrocytes can be exposed to a candidate agent, either before or after infection with a human malarial parasite.
  • the level of malarial parasite infection in human erythrocytes is determined by routine technology, e.g., nested PCR, microscopy, or Geimsa staining.
  • Whether the candidate agent is an anti-malaria agent can be determined by comparing the level of malarial parasite infection with the level of malarial infection in human erythrocytes obtained from a control non-human mammal (the same type of non-human mammal not treated with the candidate agent).
  • the candidate agent can be a vaccine candidate, which can be a biological preparation with the potential to improve immunity against malaria parasite infection in humans (e.g., ameliorating, alleviating, preventing, or reducing the risk for malaria parasite infection).
  • the effectiveness of the vaccine candidate can be assessed by measuring anti- 5 malaria antibody production (B-cell) or T-cell responses ex vivo.
  • B-cell anti- 5 malaria antibody production
  • examples of such vaccines include attenuated or inactivated parasite or related organism, subunit vaccines produced recombinantly or synthetically or DNA-based vaccines.
  • the screening method is performed by introducing into an immunodeficient non-human mammal (i) human hematopoietic stem cells (HSCs); (ii) one or o more human cytokines capable of promoting differentiation of the human HSCs into human erythrocytes in the non-human mammal; (iii) a malarial parasite that infects human erythrocytes; and (iv) a candidate agent.
  • HSCs human hematopoietic stem cells
  • a malarial parasite that infects human erythrocytes a malarial parasite that infects human erythrocytes
  • a candidate agent a candidate agent that infects human erythrocytes.
  • the non-human mammal is then maintained under conditions allowing the human HSCs to differentiate into human erythrocytes in the non- human mammal.
  • the level of malarial parasite infection of the human erythrocytes is then5 determined. Based on the infection level
  • a non-human mammal carrying human erythrocytes differentiated from human HSCs introduced into the same animal in the presence of one or more cytokines and infected with a human malarial parasite is treated with a candidate agent. o Following the same procedures described above, whether this candidate agent is an anti- malaria agent can be determined.
  • the screening method described above aims at identifying an agent (e.g., a vaccine candidate) that can be used to prevent or treat malaria in a human, following the procedures described above. If, in the presence of the candidate agent, no
  • this candidate agent can be used to prevent or treat malaria in a human.
  • a suitable control includes an immunodeficient non-human mammal (a counterpart non-human mammal) introduced with hematopoietic stem cells (HSCs), one or more human cytokines capable of promoting differentiation of the human HSCs into functional human erythrocytes when presented in the non-human mammal, and a malarial parasite that infects human erythrocytes, but not the candidate agent.
  • HSCs hematopoietic stem cells
  • candidate agents can be assessed in the methods described herein.
  • Agents to be assessed in the methods include nucleic acids (e.g., DNA, RNA), peptides, proteins, small organic molecules, vaccines (e.g., inactivated organisms (malarial organisms), attenuated organisms and/or subunits thereof), mutated malaria and the like.
  • Known antimalarial drugs include chloroquine, pyrimethamine, atovaquone, and artemisinin.
  • the non- human mammals described herein can be used for targeted validation studies.
  • a candidate agent can be a mutated malaria (mutant malaria parasite), in which one or more of the malarial proteins or peptides are mutated (disabled; knocked out; knocked down).
  • a mutant malaria parasite can be introduced into a non-human mammal model described herein and if the mutant malaria does not infect the human erythrocytes then the mutated protein or peptide of the mutant malaria has been identified as a target (e.g., a therapeutic target) for intervention.
  • An anti-malaria agent identified from any of the screening methods described herein is also provided and may be used for treating or preventing malaria.
  • Treating malaria can be the application or administration of a composition including one or more anti-malaria agents to a subject, who is infected with malaria, has a symptom of malaria infection, is suspected of having malaria infection, or is at risk for malaria infection, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the infection, the symptoms of the infection, or reduce the risk of infection.
  • Preventing malaria can be the application or administration of a composition including one or more anti-malaria agents to a subject at risk for malaria infection or further malaria infection which results in a reduction, inhibition or elimination of malaria infection or malarial infectivity.
  • one or more anti-malaria agents can be formulated as pharmaceutical compositions via routine methods well known in the pharmaceutical industry.
  • Such compositions may include a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” means a pharmacologically inactive material used together with the anti-malaria agents to formulate the inventive compositions.
  • Pharmaceutically acceptable carriers comprise a variety of materials known in the art,
  • saccharides such as glucose, lactose, and the like
  • preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers.
  • saline such as phosphate buffered saline
  • compositions can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • Subject means animals, including warm blooded mammals such as humans and primates; avians; domestic household or farm animals such as cats, dogs, sheep, goats, cattle,5 horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like.
  • Example 1 Development of a Humanized Mouse Model for Malarial Infection in Humans
  • erythrocytes Reconstitution of human erythrocytes was improved as follows.
  • Humice were hydrodynamically injected with empty pcDNA vector (as a vehicle control) or pcDNA vectors expressing human EPO and IL-3.
  • blood collected from the humice was stained to examine CD235ab positive cells (a marker for human RBCs) .
  • 3D7 strain obtained from Malaria Research and Reference Reagent Resource (MR4) was maintained in leukocytes free human erythrocytes as described by Trager and Jensen, 1976.
  • the culture was treated with trypsin and chymotrypsin for an hour at 37°C under shaking conditions and followed by trypsin inhibitor in order to prevent the reinvasion of uninfected red blood cells (RBCs) carried along with late stage schizonts.
  • Late stage schizonts were purified by percol gradient according to Fernandez, 2004.
  • the percentage of human RBCs in blood obtained from the reconstituted mice was quantified by staining with anti-CD235 antibodies conjugated to FITC. Mice with at least 3% human RBC reconstitution were used for the experiment. 100 ul whole blood was collected from facial vein of each reconstituted mouse and washed 2 times with RPMI media. With this blood, a number of 1 ml cultures were set up in 12 well plates as described below:
  • the parasitaemia was counted 16 hours later and only ring stage parasites were considered for re-invasion.
  • rPLU6 TTA AAA TTG TTG CAG TTA AAA CG (SEQ ID NO: l)
  • rPLU5 CCT GTT GTT GCC TTA AAC TTC (SEQ ID NO:2)
  • rFALl TTA AAC TGG TTT GGG AAA ACC AAA TAT TAT T (SEQ ID NO:3) rFAL2 ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC (SEQ ID NO:4) RESULTS
  • Thin smear was made after 16 hours, stained with Geimsa and observed under microscope for ring stage parasites. 0.2 to 1.6% (average 0.6 + 0.14, n+14) of the total RBCs (up to 40% of human RBCs) were found to be infected by parasites ( Figure 2). However, infection of mice with purified schizonts directly from culture failed to show any parasites in circulation. This may due to the size of the RBCs in mice, which is much smaller (5.5 microns) than that of RBCs in humans (8 microns).
  • the parasites were cultured in whole blood obtained from the humanized mice
  • Late o stage schizonts were prepared from a P falciparum 3D7 culture and incubated with blood samples obtained from 3 different mice to set up ex vivo cultures. Thin smear after 16 hours showed 0.58 to 1.6% (14.5 to 40% normalized to human RBCs) parasitemia.
  • Each culture was injected into the same mice by intravenous injection, and blood samples were collected at various time points. Leucocytes were removed by filtering through plasmodiphur and 5 nested PCR was carried out with genomic DNA made from the blood samples (Snounou et al., 1993).
  • Presence of parasites in the blood samples was detected up to 48 hours (i.e., 2nd cycle), indicating occurrence of reinvasion in vivo.
  • Control experiments were carried out by infecting humanized mice with freeze-thawed (killed) parasites. PCR products were detected only at the 1st time point (0 hours), indicating that the lysed parasites/genomic DNA 0 are rapidly cleared and do not give a PCR product at 48 hours (i.e., 2 nd cycle) ( Figure 8).
  • human RBCs were continuously injected into NSG control mice until they reached around 50% of the total RBCs in the mice (Jimenez-Diaz et al. 2009).
  • Red blood cells are enucleated and the RNA gradually degrades as the cells matures.
  • reticulocytes (young RBCs) carry residual RNA which can be stained with Thiozol Orange (TO). Since leukocytes are removed from the sample, only retciculocytes will be stained. To differentiate human reticulocytes they are co-stained with anti-CD235 antibodies. It has also been observed that human reticulocytes constituted a significant portion of the total human erythrocytes circulating in the humanized mice described herein ( Figure 9). This indicates that these mice are suitable for infection with both P. falciparum and P. vivax.
  • TO Thiozol Orange
  • mice The human RBCs in reconstituted mice were quantified by staining with FITC- conjugated anti- CD235 antibodies. Mice with 2 to 3% human RBCs were used for the experiments. 100 ul whole blood was collected from facial vein of each mouse and washed 2 times with RPMI media. lxlO 6 purified schizonts of P. falciparum 3D7 were mixed with the blood along with 1 ml RPMI media. The newly invaded rings were observed after 16 hours (overnight), and the culture was injected into the humanized mice intravenously. Cultures that were freeze-thawed for several times before injection were used as negative controls. 20 ul blood samples were collected at 0, 48, 96 and 144 hours for smear and gDNA preparation.
  • Parasite clones which are adapted to grow in NSG mice supplemented with human RBCs were obtained from GlaxoSmithKline, Spain and used to infect humanized mice (Angulo-Barturen et al., 2008).
  • Two strains, Pf 3D7 0087/N9 and PF vi/S 0176/N10 were thawed and treated with trypsin/chymo trypsin as they have vast amounts of human red cells before injection into humanized mice. Both strains were able to invade and parasites were detected by nested PCR (top panel) and by Geimsa smear.
  • mice 100 ul blood from humanized mice were cultured ex vivo with 2 million purified schizonts of P. falciparum 3D7 parasites.
  • 3 mice (Ml, M2, M3) received the parasites thus collected and 3 other mice (M4, M5, M6) received the parasites that were freeze-thawed as a negative control.
  • 20 ul blood samples were collected from each treated mouse at 0, 48, 96 and 144 hours and genomic DNA isolated therefrom. Nested PCR was carried out as described above. As shown in Figure 8, PCR products were detected in 48 hours only in the mice that received viable parasites. This result indicates that only viable parasites progressed into the next round of invasion, and the DNAs from lysed parasites are rapidly cleared by the mice.
  • 3D7 knobless clone (3D7KL) does not express surface knobs and is less rigid compared to normal 3D7 parasites.
  • Figure 10 top panel.
  • the 3D7KL (knobless) clone also hosted 2 rounds of re-invasion.
  • Figure 10 bottom panel.
  • macrophages were removed from the blood samples with liposome encapsulated clodronate. More specifically, three humanized mice were injected with 1 dose of clodronate-liposome and then infected with 20 million of Pf Kl clone. Newly invaded ring stage parasites were detected by microscopy for multiple cycles, suggesting that depletion of macrophage improved parasite density in peripheral blood.
  • Jimenez-Diaz MB Mulet T, Viera S, Gomez V, Garuti H, Ibanez J, Alvarez-Doval A,5 Shultz LD, Martinez A, Gargallo- Viola D, Angulo-Barturen I.

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Abstract

L'invention porte sur des modèles mammifères non humains (p.ex. des modèles de souris) de la malaria humaine et sur des utilisations de ces derniers dans, p.ex., l'identification d'agents antipaludiques. L'invention concerne également des compositions et des procédés associés.
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JP7130012B2 (ja) 2014-05-19 2022-09-02 リジェネロン・ファーマシューティカルズ・インコーポレイテッド ヒトｅｐｏを発現する遺伝子改変された非ヒト動物
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