WO2016144642A1 - Genetically engineered host cells for malaria research - Google Patents

Abstract

In some embodiments of the present invention, an erythroleukemic cell includes exogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B-cell lymphoma/leukemia 11A (BCL11A). In some embodiments of the present invention, a cell culture includes an erythroleukemic cell, including exogenous or endogenous expression of at least one selected from the group consisting of Ourfy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B-cell lymphoma/leukemia 11A (BCL11A); and hemin. In some embodiments, the cell culture also includes JQ1 and TGFβ1. In some embodiments of the present invention, a method of inducing hemoglobinization and differentiation in an erythroleukemic cell.

Description

GENETICALLY ENGINEERED HOST CELLS FOR MALARIA RESEARCH

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under Grant No. W81XWH-12- C-0100 awarded by the Department of Defense. The government has certain rights in this invention.

FIELD

[0002] The present invention relates to methods and compositions for producing genetically modified cell lines capable of supporting the invasion and growth of the malarial Plasmodium species.

BACKGROUND

[0003] Plasmodium falciparum and Plasmodium vivax, transmitted by Anopheles mosquitoes are the two most prevalent malarial parasites that afflict humans. Outside the African continent, P. vivax is the most common in malaria-endemic countries of Asia and Latin America, causing approximately between 70 and 80 million civilian cases of malaria per year.

[0004] Most of the efforts to study malarial Plasmodium have been directed at the cultivation of the erythrocytic stages in the Plasmodium life cycle that are associated with the pathogenesis of malaria and thus, a major target for vaccine development. Studies have shown that the broad invasiveness of P. falciparum in humans is driven by its interaction with glycophorin A (GlyA) and basigin, two surface proteins expressed by erythroblasts as well as mature red blood cells (RBCs). Specifically, the P.falciparum 175 kilodalton erythrocyte binding protein (EBA-175) and the reticulocyte-binding protein homologue (RH)5 bind respectively to the GlyA and basigin receptor proteins of the host cell. However, while in vitro culture of P. falciparum is more readily achieved using mature red blood cells as cell hosts, resistance monitoring and development of new therapeutics have been hindered for culturing of P. vivax because this parasite preferentially invades young erythrocytes

(reticulocytes), which are a small minority of RBCs (0.5-1.5%) and are difficult to obtain consistently in high numbers.

[0005] The lack of a practical and continuous culture system for P. vivax in the laboratory has limited the understanding of this widespread malaria species. One restriction is that P. vivax infection is restricted to Duffy positive (Fy+) individuals. As such, despite P. vivax being widespread throughout the tropical and subtropical world, its low incidence in West Africa is attributed to the fact that the majority of the population (more than 95%) is

Duffy negative. This observed parasite resistance in Duffy negative West Africans led to the extensive characterization and genetic sequencing of both the parasite ligand- P. vivax Duffy binding protein (PvDBP) - and the cognate DBP receptor on host reticulocytes - the Duffy antigen receptor chemokine (DARC). In addition to its low incidence in Duffy negative populations, the reticulocyte preference of P. vivax results in a lower in vitro infectious rate of human peripheral blood for this species compared to P. falciparum, thereby further limiting study of this parasite species.

[0006] Currently used methodologies for cultivating Plasmodium spp. depend on a program of volunteer donors in order to obtain either mature erythrocytes (e.g. P. falciparum) or stem cells (e.g. P. vivax). In the latter, stem cell harvesting is followed by a complex process of cell selection and isolation, and further culturing with relatively high levels of cytokines, rendering a prohibitively laborious and expensive process. Moreover, the yield of reticulocytes is variable, unpredictable, and dependent on the individual donors used.

Accordingly, there is a need for an engineered host cell capable of maintaining a culture of malarial P. vivax and P. falciparum, as well as being genetically modified in order to provide the specific erythrocyte protein expression that regulates the host and Plasmodium spp. interactions.

SUMMARY

[0007] In some embodiments of the present invention, an erythroleukemic cell includes exogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B- cell lymphoma/leukemia 11 A (BCL11A).

[0008] In some embodiments of the present invention, a cell culture includes an erythroleukemic cell, including exogenous or endogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B-cell lymphoma/leukemia 11A (BCL11A); and hemin. In some embodiments, the cell culture also includes JQ1 and ΤΰΡβ

[0009] In some embodiments of the present invention, a method of inducing

hemoglobinization and differentiation in an erythroleukemic cell having exogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B-cell lymphoma/leukemia 11A (BCL1 1A) includes culturing the erythroleukemic cell in the presence of hemin and JQ1 to form a hemoglobinized and differentiated erythroleukemic cell. In some embodiments, the method also includes culturing in the presence of ΤΰΡβ In some embodiments, the differentiated erythroleukemic cell is immortalized. [0010] In some embodiments of the present invention, a differentiated erythroleukemic cell capable of being invaded by Plasmodium species (spp.), the differentiated

erythroleukemic cell having exogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta- globin, alpha-globin, and B-cell lymphoma/leukemia 1 1A (BCL11A), and differentiated by culturing in the presence of hemin and JQ1. In some embodiments, the differentiated erythroleukemic cell is capable of being invaded by P. falciparum and/or P. vivax.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0012] These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings.

[0013] FIG. 1A is a schematic depicting a lentiviral vector (LV) encoding DARC and green fluorescent protein (GFP), the LV-DARC-GFP vector being transduced into an erythroleukemic cell, whereby transduction is monitored by intracellular GFP (green) and expression of DARC on the cell surface is monitored by an anti-CD34-allophycocyanin (APC) antibody (red), according to embodiments of the present invention.

[0014] FIG. 1 B is a schematic depicting lentiviral vectors encoding DARC (LV-DARC), beta-hemoglobin (LV-Hbb), and BCL1 1A (LV-BCL11A) each vector being transduced and expressed in an erythroleukemic cell, followed by induction of hemoglobinization and differentiation of the erythroleukemic cell, thereby rendering the differentiated

erythroleukemic cell capable of infection by Plasmodium vivax by the binding of the P. vivax Duffy binding protein (PvDBP) to the DARC receptor, according to embodiments of the present invention.

[0015] FIG. 1 C is a schematic depicting lentiviral vectors encoding GlyA (LV-GlyA), Hbb (LV-Hbb), and BCL11A (LV-BCL11A), each vector being transduced and expressed in an erythroleukemic cell, followed by induction of hemoglobinization (Hb) and differentiation of the erythroleukemic cell, thereby rendering the differentiated erythroleukemic cell capable of infection by Plasmodium falciparum by the binding of the P. falciparum EBA-175 protein to the GlyA protein, according to embodiments of the present invention.

[0016] FIG. 2A shows the amount of fluorescence by FACS analysis of GFP (green) and anti-CD234-APC (red) in untransduced K562 cells (mock), according to embodiments of the present invention. [0017] FIG. 2B shows the amount of fluorescence by FACS analysis of GFP (green) and mouse lgG2a-APC (isotype control antibody) (red) in K562 cells transduced with LV-DARC- GFP vector, according to embodiments of the present invention.

[0018] FIG. 2C shows the amount of fluorescence by FACS analysis of GFP (green) and anti-CD234-APC (red) in K562 cells transduced with a LV-DARC-GFP vector, according to embodiments of the present invention.

[0019] FIG. 2D shows the amount of fluorescence by FACS analysis of GFP (green) and anti-CD234-APC (red) in K562 cells transduced with a LV-DARC-GFP vector and a LV-Hbb- OFP, according to embodiments of the present invention.

[0020] FIG. 2E shows the amount of fluorescence by FACS analysis of GFP (green) and OFP (orange) in K562 cells transduced with a LV-DARC-GFP vector and a LV-Hbb-OFP, according to embodiments of the present invention.

[0021] FIG. 3A shows the amount of fluorescence by FACS analysis of GFP (green) and anti-CD234-APC (red) in untransduced KU812 cells (mock), according to embodiments of the present invention.

[0022] FIG. 3B shows the amount of fluorescence by FACS analysis of GFP (green) and mouse lgG2a-APC (isotype control antibody) (red) in KU812 cells transduced with a LV- DARC-GFP vector, according to embodiments of the present invention.

[0023] FIG. 3C shows the amount of fluorescence by FACS analysis of GFP (green) and anti-CD234-APC (red) in KU812 cells transduced with a LV-DARC-GFP vector, according to embodiments of the present invention.

[0024] FIG. 4A shows the amount of fluorescence by FACS analysis of GFP (green) and anti-CD234-APC (red) in untransduced KMOE-2 cells (mock), according to embodiments of the present invention.

[0025] FIG. 4B shows the amount of fluorescence by FACS analysis of GFP (green) and mouse lgG2a-APC (isotype control antibody) (red) in KMOE-2 cells transduced with a LV- DARC-GFP vector, according to embodiments of the present invention.

[0026] FIG. 4C shows the amount of fluorescence by FACS analysis of GFP (green) and anti-CD234-APC (red) in KMOE-2 cells transduced with a LV-DARC-GFP vector, according to embodiments of the present invention.

[0027] FIG. 5A shows the amount of fluorescence by FACS analysis of unstained UT7- EPO cells, according to embodiments of the present invention.

[0028] FIG. 5B shows the amount of fluorescence by FACS analysis of UT7-EPO cells stained with Pacific blue conjugated anti-glycophorin A/anti-CD235a (anti-GlyA) antibody, according to embodiments of the present invention.

[0029] FIG. 5C shows the amount of fluorescence by FACS analysis of unstained differentiated UT7-EPO cells, according to embodiments of the present invention. [0030] FIG. 5D shows the amount of fluorescence by FACS analysis of differentiated UT7-EPO cells stained with Pacific blue conjugated anti-GlyA antibody, according to embodiments of the present invention.

[0031] FIG. 6A shows the amount of fluorescence by FACS analysis of unstained UT7- EPO cells, according to embodiments of the present invention.

[0032] FIG. 6B shows the amount of fluorescence by FACS analysis of UT7-EPO cells stained with Alexa-fluor 488 conjugated anti-basigin/anti-CD147 antibody, according to embodiments of the present invention.

[0033] FIG. 6C shows the amount of fluorescence by FACS analysis of unstained differentiated UT7-EPO cells, according to embodiments of the present invention.

[0034] FIG. 6D shows the amount of fluorescence by FACS analysis of differentiated UT7-EPO cells stained with Alexa-fluor 488 conjugated anti-basigin/anti-CD147 antibody, according to embodiments of the present invention.

[0035] FIG. 7A shows forward scatter analysis (FSC-A) and side scatter analysis (SSC- A) on untransduced UT7-EPO cells (mock), according to embodiments of the present invention.

[0036] FIG. 7B shows FACS analysis of GFP in untransduced UT7-EPO cells (mock), according to embodiments of the present invention.

[0037] FIG. 7C shows FACS analysis of GFP in UT7-EPO cells transduced with a LV- DARC-GFP vector with a gate on cells having high GFP expression, according to embodiments of the present invention.

[0038] FIG. 8 shows microscopy images of isolated clones of UT7-EPO cells transduced with LV-DARC-GFP isolated from the high GFP expressing gated cells shown in FIG. 7C, with the left image showing brightfield illumination and the right showing GFP fluorescence, according to embodiments of the present invention.

[0039] FIG. 9 is a schematic depicting a lentiviral vector encoding beta hemoglobin-OFP (LV-Hbb), being transduced and expressed in an erythroleukemic cell, whereby transduction is monitored by OFP expression (orange), according to embodiments of the present invention.

[0040] FIG. 10A shows the amount of fluorescence from OFP (orange) in untransduced K562 cells (mock) and K562 cells transduced with LV-Hbb-OFP, according to embodiments of the present invention.

[0041] FIG. 10B shows the amount of fluorescence from OFP (orange) in untransduced KU812 cells (mock) and KU812 cells transduced with LV-Hbb-OFP, according to

embodiments of the present invention.

[0042] FIG. 10C shows the amount of fluorescence from OFP (orange) in untransduced KMOE-2 cells (mock) and KMOE-2 cells transduced with LV-Hbb-OFP, according to embodiments of the present invention. [0043] FIG. 11 A shows the amount of fluorescence by FACS analysis of GFP (green) and anti-CD234-APC (red) in untransduced K562 cells (mock) and K562 cells transduced with a LV-DARC-GFP vector, according to embodiments of the present invention.

[0044] FIG. 11 B shows confocal fluorescence micrographs of K562 cells transduced with LV-DARC-GFP, in which transduction of GFP is indicated (arrows) by intracellular green fluorescence and cell surface expression of DARC is indicated (arrows) by cell surface red fluorescence from an anti-CD235-APC antibody, according to embodiments of the present invention.

[0045] FIG. 12A shows the amount of fluorescence by FACS analysis from OFP

(orange) in untransduced K562 cells (mock) and K562 cells transduced with a LV-Hbb-OFP vector, according to embodiments of the present invention.

[0046] FIG. 12B shows confocal fluorescence micrographs of K562 cells transduced with LV-Hbb-OFP, in which transduction of LV-Hbb-OFP is indicated by intracellular orange fluorescence as indicated (arrows), according to embodiments of the present invention.

[0047] FIG. 13A shows the mean fluorescence intensity (MFI) corresponding to the amount of fetal hemoglobin (HbF) expressed in unstained (no anti-HbF-APC antibody) control K562 cells, stained K562 cells, and stained K562 cells treated with 100 μΜ hemin, in which the MFI corresponding to HbF is quantified using an anti-HbF-APC antibody, according to embodiments of the present invention.

[0048] FIG. 13B shows the mean fluorescence intensity (MFI) corresponding to the amount of fetal hemoglobin (HbF) expressed in unstained (no anti-HbF-APC antibody) control KU812 cells, stained KU812 cells, and stained KU812 cells treated with 100 μΜ hemin, in which the MFI corresponding to HbF is quantified using an anti-HbF-APC antibody, according to embodiments of the present invention.

[0049] FIG. 13C shows the mean fluorescence intensity (MFI) corresponding to the amount of fetal hemoglobin (HbF) expressed in unstained (no anti-HbF-APC antibody) control KMOE-2 cells, stained KMOE-2 cells, and stained KMOE-2 cells treated with 100 μΜ hemin, in which the MFI corresponding to HbF is quantified using an anti-HbF-APC antibody, according to embodiments of the present invention.

[0050] FIG. 14 shows field-of-view images of benzidine-stained UT7-EPO cells at 1 , 2, 3, 4, and 8 days of differentiation treatment, in which from left to right, the first column shows control untreated cells, the second column shows cells treated with JQ1 and hemin, and the third column shows JQ1 , hemin and TGF-βΙ treated cells as indicated, according to embodiments of the present invention.

[0051] FIG. 15A is a panel of 4 graphs, each graph showing the distribution of UT7-EPO cells relative to the intensity of benzidine staining (arbitrary units - AU) in which the graphs from top to bottom represent Day 1 , Day 2, Day 3, and Day 4, respectively, the blue line represents untreated UT7-EPO cells (control), the green line represents UT7-EPO cells treated with hemin and JQ1 , and the red line represents UT7-EP0 cells treated with hemin, JQ1 ,and ΤΘΡβΙ , according to embodiments of the present invention.

[0052] FIG. 15B is a graph showing the percentage of benzidine positive UT7-EPO cells at Day 0, Day 1 , Day 2, Day 3, and Day 4, for the conditions shown in FIG. 15A, in which "benzidine positive" cells are set at a minimum of 140 AU, in which the blue line represents untreated UT7-EPO cells (control), the red line represents UT7-EPO cells treated with hemin and JQ1 , and the green line represents UT7-EPO cells treated with hemin, JQ1 ,and ΤΰΡβΙ , according to embodiments of the present invention.

[0053] FIGs. 16A- 16D show field of view images of Giemsa-stained UT7-EPO cells at Day 5 of the differentiation protocol: FIGs. 16A and 16C show low and high magnification, respectively for untreated UT7-EPO cells, and FIGs. 16B and 16D are low and high magnification, respectively, for treated (e.g. hemin, JQ1 and ΤΰΡβΙ) UT7-EPO cells, according to embodiments of the present invention.

[0054] FIG. 17A shows confocal microscopy images of GFP-positive P. falciparum in human red blood cells (hRBCs), in which from left to right, the images show A: overlay of 4'- 6-diamidino-2-phenylindole (DAPI) (nuclei) staining (blue) and GFP (green), A1 : DAPI staining only, A2: GFP only, according to embodiments of the present invention.

[0055] FIG. 17B shows confocal microscopy images of UT7-EPO cells after co-culturing under invasion conditions (e.g., hemin, JQ1 , and ΤΰΡβΙ in 50% differentiation medium and 50% complete medium for P. falciparum) with P. falciparum for 7 days, in which from left to right, the images show B: overlay of DAPI (nuclei) staining (blue) and GFP (green), B1 : DAPI staining only, B2: GFP only, according to embodiments of the present invention.

[0056] FIG. 17C shows confocal microscopy images of UT7-EPO cells after co-culturing under invasion conditions (e.g., hemin, JQ1 , and ΤΰΡβΙ in 50% differentiation medium and 50% complete medium for P. falciparum) with P. falciparum for 7 days, in which from left to right, the images show C: overlay of DAPI (nuclei) staining (blue) and GFP (green), C1 : DAPI staining only, C2: GFP only, according to embodiments of the present invention.

[0057] FIG. 18 is a schematic depicting a UT7-EPO cell of the present invention, genetically engineered using the CRISPR/cas9 technology to integrate a GFP reporter transgene (tg-GFP) under the native ubiquitous β8θίίη promoter resulting in Engineered Cell for Malaria (ECeM), which is then hemoglobinized and differentiated as described herein (Diff Protocol), resulting in a platform cell (ECeM-Tg+) enabling facile analysis of malarial target genes (e.g., Gene of interest) of the host cell, according to embodiments of the present invention.

[0058] FIG. 19A is a schematic depicting a representative HIV transfer vectors encoding the nucleic acid sequences for DARC, GlyA, beta-globin and BCL1 1A genes operably linked to the constitutive elongation factor 1-alpha (EF1-alpha), according to embodiments of the present invention. [0059] FIG. 19B shows a restriction enzyme map of the initial lentiviral backbone, according to embodiments of the present invention.

[0060] FIG. 19C shows a restriction enzyme map of the pCDH-EF1-DARCisoA-IRES- EGFP (Lenti-DARC or LV-DARC or LV-DARC-GFP), according to embodiments of the present invention.

[0061] FIG. 19D shows a restriction enzyme map of the pCDH-EF1-bA-globin-IRES- OFP (Lenti-Hbb or LV-Hbb-OFP), according to embodiments of the present invention.

[0062] FIG. 20 shows schematics of cloning strategies using the CRISPR/Cas9 technology for transforming UT7-EPO cells to ECeM cells using the CRISPR/Cas technology, in which A shows integration of enhanced GFP (EGFP) (in a "repair cassette") at the native β8θίίη locus of UT7-EPO cells targeted by Cas9 and guide RNA (gRNA) for β8θίίη to form "ECeM" cells; and B shows integration of transgene (Tg) of interest having left and right homologous arms (LHA) and (RHA) for replacement of the EGFP reporter gene in the ECeM cells, according to embodiments of the present invention.

DETAILED DESCRIPTION

[0063] Aspects of embodiments of the present invention relate to the unmet need for differentiated self-maintaining erythroleukemic cells (e.g., erythroid cells-erythroblasts and/or reticulocytes) that have been genetically modified to allow for the ex vivo invasion and growth of Plasmodium spp. parasites. The creation of a low cost continuous supply of host erythroid cells in culture is needed in order to (1) establish long term cultures of P. vivax, (2) obviate the need for fresh donor erythrocytes from blood for propagation of Plasmodium spp., (3) provide genetically identical cell populations for consistent results, (4) offer a discovery platform for further genetic modification, and (5) ultimately, identify critical host targets for development of anti-malarial therapeutics.

[0064] In aspects of embodiments of the present invention, maintenance of Plasmodium spp. in differentiated erythroleukemic host cells in culture is achieved through sufficient expression of cell surface receptors (e.g., DARC, GlyA, and/or basigin) for parasite attachment and entry, as well as by methods of producing sufficient levels of hemoglobin for intracellular parasitic growth and reproduction. The present invention addresses this need by providing such methods, as well as transfer vectors and hemoglobinization and differentiation factors to obtain the requisite differentiated erythroleukemic host cell for Plasmodium invasion.

[0065] As used herein, erythroleukemic cell, and like terms, refer to a cell derived from a human erythroleukemic subject. An erythroleukemic cell of the present invention is a self- renewing cell having the capacity to differentiate into the erythrocytic pathway.

[0066] As used herein, exogenous expression, exogenously expressed, and like terms, refer to the expression of a cloned gene product (e.g., a protein) that has been introduced into a host cell. For example, exogenous expression of the Duffy antigen receptor chemokine (DARC) in an erythroleukemic host cell includes introduction of a DARC transgene into the erythroleukemic host cell.

[0067] As used herein, hemoglobinization, and like terms, refer to the accumulation and functional availability of mature hemoglobin. Hemoglobin is the protein molecule in erythrocytes that carries oxygen. Hemoglobin is made up of four protein molecules (globulin chains) that are connected together and each associated with a heme group. Fetal hemoglobin (HbF) is composed of two chains of alpha-globin and two chains of gamma- globin (α2γ2). Adult Hb (HbA) is composed of two chains of alpha-globin and two chains of beta-globin (α2β2).

[0068] As used herein, differentiation, and like terms, refer to the process by which a cell becomes specialized in order to perform a specific function. Natural differentiation is guided by regulated and sequenced gene expression. Differentiation of embodiments of the present invention is induced with directed culturing of cells in the presence of factors known to induce gene expression. More specifically, differentiation of an erythroleukemic cell is the process by which an erythroleukemic cell expresses genes of the erythroid pathway thereby further specializing the erythroleukemic cell to a proerythroblast, a basophilic erythroblast, polychromatic erythroblast, and finally orthochromatic erythroblast, as described in Zeuner et al. 2012, the entire contents of which are herein incorporated by reference. In addition to erythroblasts, a differentiated erythroleukemic cell of the present invention includes reticulocytes which are characterized by the extrusion of the nucleus from an orthochromatic erythroblast.

[0069] As used herein, immortalization, and like terms, refer to the process by which a population of cells from a multicellular organism which would normally not proliferate indefinitely are mutated (e.g., genetically engineered) to evade normal cellular senescence and are capable of undergoing division indefinitely. Accordingly, an immortalized cell obtained by the processes disclosed herein is capable of being cultured for prolonged periods in vitro.

[0070] Embodiments of the present invention address several issues in order to successfully culture Plasmodium spp. (e.g., P. vivax and P. falciparum) in a host cell. (1) A first issue is the use of established erythroleukemic cell lines. These established

erythroleukemic cells lines are capable of producing high numbers of cells from which adequate amounts of erythroblasts and/or reticulocytes may be derived. Non-limiting examples of erythroleukemic cell lines include K562, KU812, KMOE-2, and UT7-EPO, as described respectively in Andersson et al., 1979 Int. J. of Cancer 23: 143-147; Okano H, et al. 1988, J. Biochem. 104: 162-164; Okano et al., 1981 , Cancer Res Clin Oncol,. 102;.49~55; and Komatsu et a!., 1993, Blood, 82:456-464, the entire contents of ail of which are herein incorporated by reference. (2) A second issue is adequate expression of DARC and/or GlyA and basigin on the surface of a differentiated erythroleukemic host cell for interaction with the P. vivax PvDBP (with DARC) or the P. falciparum EBA-175 and RH5 ligands (with GlyA and basigin, respectively) for parasite invasion. Erythroleukemic cell lines having insufficient endogenous DARC expression may be remedied by introduction of exogenous DARC.

Similarly, adequate expression of GlyA and basigin on the surface of the host cell is also necessary for invasion of P. falciparum. Erythroleukemic cells having insufficient

endogenous GlyA and/or basigin expression may be remedied by introduction of exogenous GlyA and/or basigin. (3) A third issue is that once inside the host cell, the Plasmodium parasite requires sufficient levels of hemoglobin for growth and multiplication. The hemoglobinization of erythroleukemic cells is stimulated through differentiation and inducing factors (e.g., hemin, JQ1 , and TGF as disclosed herein). Erythroleukemic cells having insufficient hemoglobinization potential may be remedied by introduction of exogenous hemoglobin. (4) A fourth issue addresses the type of hemoglobin in the cell. It was previously reported that malaria parasitemia requires the adult form of hemoglobin (HbA) within the cell as opposed to the fetal (HbF) form because the latter causes growth retardation of the parasite and explains why reticulocytes from umbilical cord blood do not provide long-term maintenance of P. vivax (Udomsangpetch et al. 2007, Parasitol.ini, 56:65- 69, the entire contents of which are herein incorporated by reference). However, recent data have contradicted this conclusion, as it has been shown that P. falciparum parasites indeed invade and develop normally in fetal cord blood (CB) RBCs, which contain up to 95% HbF (Amaratunga et al., 2011 , PLoS One, 6:e14798, the entire contents of which are herein incorporated by reference. Nonetheless, these parasitized CB RBCs are impaired in their cytoadherence interactions, a property correlated with the development of high parasite density and malaria symptoms. As such, many established human erythroleukemic cell lines produce the fetal form of hemoglobin (HbF) caused by methylation and gene silencing of the beta-globin promoter. Accordingly, while Plasmodium spp. appears to be able to invade erythroleukemic cells producing HbF, increased expression of HbA (either beta-globin, or alpha-globin) in erythroleukemic cells may increase parasite growth and density.

Furthermore, a decrease in the expression of HbF may also increase parasite growth and density. For example expression of the B-cell lymphoma/leukemia 11 A (BCL1 1A) protein suppresses expression of gamma-globin (γ-globin) subunit of fetal hemoglobin. As such, exogenous expression of BCL1 1A in erythroleukemic cells may promote the preferential expression of adult hemoglobin, by suppression of the HbF γ-globin. Exogenous Expression of Requisite Malarial Host Transgenes

[0071] Sufficient expression of the host proteins that enhance and/or are required for Plasmodium parasite invasion and growth may be provided in erythroleukemic cells through the introduction of the desired transgene. As used herein, transgene, and like terms, refers to a gene or genetic material that has been transferred by any of a number of genetic engineering techniques form one organism to another. Examples of transgenes as disclosed herein, include the nucleotide sequence encoding for any functional polypeptide of DARC, GlyA, basigin, beta-globin, alpha-globin, or BCL1 1A. As used herein, a functional polypeptide, includes the full length wild type protein as well as any functional fragment that renders a wild type function.

[0072] In some embodiments, any suitable method of introducing the transgene into the host cell may be used. Non-limiting examples of transgene transfer include viral

transduction and transposon transformation. Viral vector transduction is well known in the art as described, for example, in Kotani et al., 1994, Hum Gene Ther., 5: 19-28; Cavazzana- Calvo et al., 2010, Nature, 467: 318-322.) In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the lentiviral vector is a human immunodeficiency virus (HIV) vector, a simian immunodeficiency virus (SIV) vector, or an equine infectious anaemia virus (EIAV) vector. In some embodiments, a transposon may be used to integrate the transgene into the host cell genome. Transposon-mediated gene transfer is known in the art, as described, for example in Wilson et al., 2007, Molecular Therapy, 15: 139-145, the entire contents of which are incorporated by reference.

[0073] In some embodiments, a transfer vector includes a polynucleotide sequence of the transgene encoding the desired polypeptide that is operably linked to a promoter sequence. For example, a transfer vector may encode the nucleotide sequences of DARC, GlyA, basigin, beta-globin, alpha-globin, or BCL11A for introduction into an erythroleukemic cell. In certain embodiments, the DARC, GlyA, basigin, beta-globin, alpha-globin, or BCL11 A polypeptides are co-expressed with at least one reporter polypeptide. A reporter polypeptide includes any suitable fluorescent peptide tag, as disclosed in Smith, 2007, Nature Methods, 4:755-761 , the entire contents of which are herein incorporated by reference. For example, green fluorescent protein (GFP), orange, fluorescent protein (ORP), mCherry fluorescent protein, red fluorescent protein (RFP), and yellow fluorescent protein (YFP).

[0074] In some embodiments, the polynucleotide of the transfer vector encodes the desired transgene (e.g., DARC, GlyA, basigin, beta-globin, alpha-globin, or BCL1 1A) and a reporter polypeptide both of which are operably linked to the same promoter sequence. In certain embodiments, the polynucleotide sequences encoding the desired transgene and the reporter polypeptide are operably linked to different promoter sequences. In certain embodiments, the promoter or promoters are constitutive promoters.

[0075] In some embodiments, the desired polypeptide and reporter protein may be expressed by an internal ribosome entry site (IRES) encoded in the transfer vector.

Methods of insertion of a transgene using an IRES are known in the art, and described, for example, in Roth et al., 2015, Virus Res. 196: 170-180 and Yu et al., 2003, Mol. Therapy, 7:827-838, the entire contents of both of which are herein incorporated by reference. In certain embodiments, the transfer vector encoding the DARC, GlyA, basigin, beta-globin, alpha-globin, and/or BCL11A polypeptides and said reporter polypeptide are expressed using a translational 2A signal sequence. An example of a 2A co-expression system is described, for example, in Minskaia et al. BMC Biotechnology, 2013, 13:67, the entire contents of which are herein incorporated by reference.

[0076] In some embodiments of the present invention, as depicted schematically in FIG.

IA, a lentiviral vector encoding DARC and GFP (LV-DARC-GFP) is transduced into an erythroleukemic cell, whereby transduction is monitored by GFP expression (green) in the nucleus and expression of DARC protein on the cell surface is monitored by an anti-CD234- allophycocyanin (APC) antibody. This anti-CD234 monoclonal antibody is specific to DARC. As shown and used herein, anti-CD234 antibody is conjugated to allophycocyanin (APC) which fluoresces red. Accordingly, the cell surface expression of DARC may be

fluorescently monitored.

[0077] In some embodiments of the present invention, as depicted schematically in FIG.

I B, lentiviral vectors encoding DARC, (LV-DARC), beta-globin (LV-Hbb), and BCL11A (LV- BCL11A) are transduced into an erythroleukemic cell to thereby provide sufficient expression of DARC on the cell surface for invasion by P. vivax, and binding of the P. vivax PvDBP protein, as well as increase levels of adult hemoglobin (HbA) with the exogenous expression of beta-globin and decreased expression of HbF with exogenous expression of BCL1 1A that suppresses the HbF gamma-globin subunit.

[0078] In some embodiments of the present invention, as depicted schematically in FIG.

I C, lentiviral vectors encoding GlyA, (LV-GlyA), beta-globin (LV-Hbb), and BCL1 1A (LV- BCL11A) are transduced into an erythroleukemic cell to thereby provide sufficient expression of GlyA on the cell surface allowing for invasion by P. falciparum and binding of the P.

falciparum EBA-175 protein, as well as increase levels of adult hemoglobin (HbA) with the exogenous expression of beta-globin and decreased expression of HbF with exogenous expression of BCL11A that suppresses the HbF gamma-globin subunit.

[0079] In some embodiments of the present invention, any erythroleukemic cell may be used for endogenous or exogenous expression of DARC, GlyA, basigin, beta-globin, alpha- globin and/or BCL1 1A. In particular embodiments, an erythroleukemic cell is derived from hematopoietic stem cells and/or progenitors or from immortalized cells with erythroid characteristics. A person having ordinary skill in the art understands the advantages of using an established erythroleukemic cell line, however, any erythroleukemic cell may be used. Non-limiting examples of erythroleukemic cells include K562, KU812, KMOE-2, and UT7-EPO.

[0080] Fluorescence-activated cell sorting (FACS) analysis of erythroleukemic cells expressing exogenous DARC are shown in FIGs. 2A-4C. Specifically, K562 cells transduced with LV-DARC-GFP or controls are shown in FIGs. 2A-2E; KU812 cells transduced with LV-DARC-GFP or controls are shown in FIGs. 3A-3C; and KMOE-2 cells transduced with LV-DARC-GFP or controls are shown in FIGs.4A-4C.

[0081] To the extent a selected erythroleukemic cell has sufficient endogenous expression of DARC for the invasion of P. vivax and/or GlyA or basigin for the invasion of P. falciparum, or high levels of adult hemoglobin, some or all of these desired transgene proteins of the present invention (e.g., DARC, GlyA, basigin, beta-globin, alpha-globin, and BCL11 A) may not need to be exogenously expressed in the host cell. For example, UT7- EPO cells express sufficient amounts of endogenous GlyA and basigin, as shown in FIGs. 5A-6D. Furthermore, the endogenous expression of GlyA and basigin was maintained under differentiation conditions (hemin, JQ1 , and ΤΰΡβΙ), as shown in FIGs. 5D and 6D. These UT7-EPO cells were transduced with LV-DARC-GFP as shown in FIGs. 7A-7C and 8, thereby rendering an erythroleukemic cell expressing sufficient amounts of DARC, GlyA, and basigin.

[0082] In some embodiments of the present invention, as depicted in FIG. 9, beta-globin is exogenously expressed in an erythroleukemic cell. The erythroleukemic cells K562, KU812, and KMOE-2 were transduced with LV-Hbb-OFP, and analyzed by FACS for expression of the orange fluorescent protein, as shown in FIGs. 10A-10C. As shown in

FIGs. 11A-11 B, K562 cells were transduced with LV-DARC-GFP, in which the number of cells having both GFP and APC fluorescence shown in FIG. 11 A, and transduction and expression of the vector confirmed by green fluorescence in the nucleus (FIG. 11 B) and expression and cell surface targeting of DARC confirmed by anti-CD234-APC at the cell surface (FIG. 11 B). Similar analysis of Hbb-OFP expression in K562 cells in shown in FIGs. 12A-12B.

Hemoglobinization and Differentiation

[0083] Induction of hemoglobinization by hemin. Hemin is Protoporphyrin IX containing a ferric iron ion (Heme B) with a chloride ligand. Hemin is an alternative to heme, the small molecule found free or bound to hemoglobin in mammalian blood. In erythroid precursors derived from Diamond-Blackfan Anemia patients, hemin significantly stimulated growth and hemoglobinization (Fibach and Aker, 2012, Anemia, 4 pages, Article ID 940260, the entire contents of which are herein incorporated by reference). High levels of hemoglobinization support growth of Plasmodium species.

[0084] In some embodiments of the present invention, hemoglobinization of an erythroleukemic cell includes culturing an erythroleukemic cell in the presence of hemin. Specifically, hemin is added to the culture medium of an erythroleukemic cell culture. In some embodiments, the concentration of hemin for induction of hemoglobinization of a cell in a cell culture is about 20 μΜ to about 200 μΜ. In some embodiments, the concentration of hemin for induction of hemoglobinization of a cell in a cell culture is about 50 μΜ to about 150 μΜ. In some embodiments, the concentration of hemin for induction of

hemoglobinization of a cell in a cell culture is about 80 μΜ to about 120 μΜ. In some embodiments, the concentration of hemin for induction of hemoglobinization of a cell in a cell culture is about 100 μΜ.

[0085] As shown in FIGs. 13A-13C, K562, KU812, and KMOE-2 cells incubated in the presence of 100 μΜ hemin show an increase in cellular hemoglobin as measured by

HbF:APC.

[0086] Differentiation. Reaching a complete and irreversible stage of erythrocyte differentiation is necessary for invasion by Plasmodium. That is, in addition to sufficient expression (endogenous or exogenous) of the corresponding host receptor proteins (e.g., DARC or GlyA and basigin) as well as sufficient expression of hemoglobin, (e.g., HbA), the host cell must be a differentiated erythroleukemic cell. More specifically, differentiation of an erythroleukemic cell is the process by which an erythroleukemic cell expresses genes of the erythroid pathway thereby further specializing the erythroleukemic cell to a proerythroblast, a basophilic erythroblast, polychromatic erythroblast, and finally orthochromatic erythroblast, as described in Zeuner et al. 2012, the entire contents of which are herein incorporated by reference. In addition to erythroblasts, a differentiated erythroleukemic cell of the present invention includes reticulocytes which are characterized by the extrusion of the nucleus from an orthochromatic erythroblast. Although it has been shown that P. falciparum can invade any orthochromatic cells, later stages are required to support their growth. Moreover, despite being considered as more restrictive for their choice of hosts, late orthochromatic stages are reported to be sufficient to sustain P. vivax invasion and growth in vitro. In some embodiments of the present invention, a differentiated erythroleukemic cell includes an orthochromatic erythroblast and reticulocytes.

[0087] In some embodiments of the present invention, differentiation is induced in an erythroleukemic cell by the culturing of the cell in the presence of JQ1 ((S)-tert-butyl 2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1 ,2,4]triazolo[4,3-a][1 ,4]diazepin-6-yl)acetate; CAS number 1268524-70-4). In some embodiments, the concentration of JQ1 for induction of differentiation of an erythroleukemic cell in a cell culture is about 0.3 μΜ to about 0.7 μΜ. In some embodiments, the concentration of JQ1 for induction of differentiation of an erythroleukemic cell in a cell culture is about 0.4 μΜ to about 0.6 μΜ. In some

embodiments, the concentration of JQ1 for induction of differentiation of an erythroleukemic cell in a cell culture is about 0.5 μΜ.

[0088] In some embodiments of the present invention, differentiation is induced in an erythroleukemic cell by the culturing of the cell in the presence of JQ1 and tumor growth factor beta 1 (ΤΘΡβΙ). In some embodiments, the concentration of ΤΘΡβΙ for induction of differentiation of an erythroleukemic cell in a cell culture is about 1 ng/ml to about 3 ng/ml. In some embodiments, the concentration of ΤΘΡβΙ for induction of differentiation of an erythroleukemic cell in a cell culture is about 2 ng/ml.

[0089] In some embodiments of the present invention, hemoglobinization and

differentiation are induced in an erythroleukemic cell culture either sequentially or in combination. In some embodiments, hemoglobinization and differentiation are induced in an erythroleukemic cell culture by the culturing of the cell in the presence of hemin and JQ1. In some embodiments, hemoglobinization and differentiation are induced in an erythroleukemic cell culture by the culturing of the cell in the presence of hemin, JQ1 , and ΤΘΡβΙ .

[0090] As shown in FIG. 14, UT7-EPO cells cultured in the absence (control, left column) or presence of hemin and JQ1 (middle column) or hemin, JQ1 and TGFB1 (right column) were stained with the hemoglobin-specific benzidine stain to measure the degree of hemoglobinization at Day 1 , Day 2, Day 3, Day 4, and Day 8 of growth as disclosed herein.

[0091] The benzidine staining in the UT7-EPO cells shown in FIG. 14 was quantified using arbitrary units (A.U.). To do so, three representative images were taken on an inverted microscope. Images were first converted from red, green, blue (RGB) color to grayscale and denoised with a Gaussian filter. The images were then normalized using local background. Cells were identified using an adaptive threshold and labeled. Labeled cells that were significantly larger or smaller than the average cell (cell clumps or debris) were removed. The average intensity of benzidine staining for each labeled cell was then quantified, ranging from 0 (white) to 255 (black). Cells with average benzidine intensity greater than an arbitrary threshold intensity (>140) were considered positive. Using 140 A.U. as the threshold for increased hemoglobinization compared to control, the increase of hemoglobinization was aligned (FIG. 15A) and plotted (FIG. 15B) over 4 days.

[0092] Adoption of orthochromatic cell morphology by Giemsa-stained UT7-EPO cells under differentiating and control conditions is shown in FIGs. 16A-16D. Whereas in the control conditions most of the nuclei appear large and round, a vast majority of nuclei in cells exposed to the differentiating conditions appear more condensed and asymmetrically positioned.

[0093] In some embodiments of the present invention, hemoglobinization and

differentiation is induced in an erythroleukemic cell as disclosed herein. For example, an erythroleukemic cell may express at least one of DARC, GlyA, basigin, beta-globin, alpha- globin, and BCL1 1A.

Invasion of P. falciparum and P. vivax

[0094] In some embodiments of the present invention, a differentiated erythroleukemic cell as disclosed herein is capable of being invaded by Plasmodium. In some embodiments, a differentiated erythroleukemic cell as disclosed herein is capable of being invaded by P. falciparum or P. vivax. As shown in FIGs. 17B-17C, differentiated UT7-EPO cells were infected by P. falciparum.

Immortalization

[0095] immortalization may be achieved by any suitable recombinant method known in the art, as described, for example, in Yang G. et al., Carcinogenesis 2007; Price A.M. and Luftig M.A. Adv. Virus Res., 2014, the entire contents of which are herein incorporated by reference. For some primary cell types, it has been shown that a combinational expression of SV40T antigen and hTERT or other genes such as the human papillomavirus type 18 (HPV16) E6 and E7 (E8/E7) viral oncogenes, have been shown to be required for successful immortalization (Matsumura et al. 2004) and proved to be successful in generating a human erythroid cell line with more desirable properties that that at least partially satisfies P. vivax growth (Wong et al. 2010). immortalization is achieved by inactivating tumor suppressor genes of the host cell, !nactivation of tumor suppresser genes may be carried out by viral induction of EBV, HPV- 6 E6/E7 gene, and/or the Simian Virus 40 (SV40) T antigens. In some embodiments, cell immortalization is through the exogenous expression of human telomerase reverse transcriptase protein (hTERT). When hTERT is exogenously expressed, the host cell is able to maintain sufficient telomere lengths to avoid replicative senescence. Immortalization is also achieved by expression of the ras and myc oncogenes. In some embodiments of the present invention, viral induction of EBV, HPV-16 E6/E7 gene, and/or the Simian Virus 40 (SV40) T antigens may be in combination with expression of hTERT, as well as with expression of mutant Ras, Myc, p53, and/or Rb oncogenes. In some

embodiments, short interfering RNAs (siRNAs) targeting tumor suppressor genes may be used to immortalized cells of the present invention. Examples of siRNAs of tumor suppressor genes, include Myc, p53, Rb, and Ras siRNA.

[0096] In some embodiments of the present invention, a differentiated erythroleukemic cell as described herein expressing DARC, GlyA, basigin, beta-globin, alpha-globin, and/or BCL11A, may be immortalized as described above for continued culturing of a differentiated erythroleukemic cell capable of being invaded by malarial Plasmodium.

Differentiated Erythroleukemic Cells for Malaria Targets

[0097] The transformed and differentiated erythroleukemic cells of the present invention capable of invasion by Plasmodium, are a useful tool for the study of additional host cell targets by the Plasmodium parasite. In some embodiments of the present invention, the engineered cells (e.g. the differentiated erythroleukemic cells that sufficiently express DARC, GlyA, basigin, beta-globin, alpha-globin, and/or BCL11A) are transformed with a reporter gene to allow for a simple and reproducible knock-in cassette platform for assaying additional targets of a malarial parasite. This engineered malarial host cell line is also referred to as Engineered Cells for Malaria parasites (ECeM). Using the UT7-EPO cells as an example, FIG. 18 shows a schematic using the CRISPR (clustered regularly interspaced short palindromic repeat) -Cas9 (CRISPR-associated nuclease 9) technology for introducing a GFP reporter gene to be replaced by any particular gene of interest. Many known molecular cloning methods may be used to insert a reporter gene into the differentiated erythroleukemic cells of the present invention. The CRISPR-Cas9 technology is well established and described, for example, in Harrison et al., 2014, Genes & Dev., 28: 1859- 1872, the entire contents of which are herein incorporated by reference. In some

embodiments of the present invention, a reporter gene may be any suitable gene that upon disruption will either gain a signal or lose a signal. For example, a GFP reporter gene fluoresces green upon integration and results in a loss of fluoresces upon replacement by a transgene (Tg) of interest in a repair cassette, as shown in FIG. 20.

[0098] As disclosed herein, an erythroleukemic cell according to embodiments of the present invention includes exogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta- globin, alpha-globin, and B-cell lymphoma/leukemia 1 1A (BCL11A).

[0099] In some embodiments of the present invention, the erythroleukemic cell includes exogenous expression of DARC. In some embodiments of the present invention, the erythroleukemic cell includes exogenous expression of DARC and beta-globin.

[00100] In some embodiments of the present invention, the erytholeukemic cell includes exogenous expression of DARC and BCL1 1A. In some embodiments of the present invention, the erythroleukemic cell includes exogenous expression of DARC, beta-globin, and BCL1 1A.

[00101] In some embodiments of the present invention, the erythroleukemic cell includes exogenous expression of GlyA. In some embodiments of the present invention, the erythroleukemic cell includes exogenous expression of GlyA and beta-globin. In some embodiments of the present invention, the erythroleukemic cell includes exogenous expression of GlyA and BCL1 1A. In some embodiments of the present invention, the erythroleukemic cell includes exogenous expression of GlyA, beta-globin, and BCL1 1A

[00102] In some embodiments of the present invention, the erythroleukemic cell includes exogenous expression of GlyA and basigin.

[00103] In some embodiments of the present invention, a erythroleukemic cell includes exogenously expressed immortalization factors together with exogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B-cell lymphoma/leukemia 1 1A (BCL1 1A). In some embodiments of the present invention, a erythroleukemic cell includes exogenously expressed immortalization factors selected from the group consisting of Myc, Ras, HPV-16 E6/E7, SV40-T, hTERT gene, and combinations thereof, in combination with exogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B- cell lymphoma/leukemia 1 1A (BCL1 1A).

[00104] In some embodiments of the present invention, a cell culture includes an erythroleukemic cell, including exogenous or endogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B-cell lymphoma/leukemia 1 1A (BCL1 1A) and combinations thereof, and hemin.

[00105] In some embodiments of the present invention, a cell culture includes an erythroleukemic cell, including exogenous or endogenous expression of one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, B-cell lymphoma/leukemia 1 1A (BCL1 1A), and

combinations thereof; hemin; and JQ1 .

[00106] In some embodiments of the present invention, a cell culture includes an erythroleukemic cell including exogenous or endogenous expression of one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, B-cell lymphoma/leukemia 1 1A (BCL1 1A), and

combinations thereof; hemin; JQ1 ; and tumor growth factor beta 1 (TGFfi^.

[00107] In some embodiments of the present invention, a method of inducing

hemoglobinization and differentiation in a erythroleukemic cell having exogenous or endogenous expression of one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, B-cell lymphoma/leukemia 1 1A (BCL1 1A), and combinations thereof, in which the eythroleukemic cell is cultured in the presence of hemin and JQ1 to form a hemoglobinized and

differentiated erythroleukemic cell.

[00108] In some embodiments of the present invention, a method of inducing

hemoglobinization and differentiation in an erythroleukemic cell includes a erythroleukemic cell having exogenous or endogenous expression of one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, B-cell lymphoma/leukemia 1 1A (BCL1 1A), and combinations thereof, in which the eythroleukemic cell is cultured in the presence of hemin and JQ1 to form a

hemoglobinized and differentiated erythroleukemic cell.

[00109] In some embodiments of the present invention, a method of inducing

hemoglobinization and differentiation in am erythroleukemic cell includes a erythroleukemic cell having exogenous or endogenous expression of one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, B-cell lymphoma/leukemia 1 1A (BCL1 1A), and combinations thereof, in which the eythroleukemic cell is cultured in the presence of hemin, JQ1 , and ΤΘΡβΙ to form a hemoglobinized and differentiated erythroleukemic cell. In some embodiments of the present invention, the formed hemoglobinized and differentiated erythroleukemic cell is a heterochromatic erythroblast or a reticulocyte.

[00110] In some embodiments of the present invention, a method of inducing

hemoglobinization and differentiation in an erythroleukemic cell includes an erythroleukemic cell expressing immortalization factors and having exogenous or endogenous expression of one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, B-cell lymphoma/leukemia 1 1A (BCL1 1A), and combinations thereof, in which the eythroleukemic cell is cultured in the presence of hemin and JQ1 to form an immortalized hemoglobinized and differentiated erythroleukemic cell.

[00111] In some embodiments of the present invention, a differentiated erythroleukemic cell having an integrated reporter gene. In some embodiments, the integrated reporter gene is integrated at the native βΑοίίη gene locus for expression by the βΑοίίη promoter.

[00112] In some embodiments of the present invention, the erythroleukemic cell line is UT7-EPO.

EXAMPLES

[00113] Example 1. Lentiviral vector design and production of Lenti-DARC-GFP, Lenti- GlvA-GFP. Lenti-beta-qlobin(Hbb)-OFP. and Lenti-BCL11A-mCherrv. The vector construction strategy is set forth in FIGs. 19A, 19B, 19C, and 19D and consists of the following:

[00114] Lenti-DARC - (1) Synthesis of DARC(isoformA)-IRES-EGFP (2343 bp) and (2) Subcloning in Xbal/Sall of pCDH-EF1-MCS in order to obtain pCDH-EF1-DARCisoA-IRES- EGFP.

[00115] Lenti-Hbb - (1) Synthesis of βΑ-globin-IRES-OFP (1728 bp) and (2) Subcloning in Xbal/Sall of pCDH-EF1-MCS in order to obtain pCHH-EF1^A-globin-IRES-OFP.

[00116] The vector plasma DNA was used in combination with packaging plasmids (pRSV-Rev and pCgpV) and the envelope plasmid (pCMV-VSV-G) (ViraSafe™ Lentiviral

Packaging System, Cell Biolabs, Inc.) for co-transfection of HEK293LTV (Cell Biolabs, Inc.). This cell line stably expresses the SV40 large T antigen and E1 region of adenovirus (E1a and E1 b) and is specifically selected for high level of lentiviral production. At Day 2 in culture the resultant VSV-G pseudotyped lentiviral particles in the supernatant was harvested and the infectious units (IU) determined by standard titration on HEK 293 cells and respective GFP and OFP fluorescence on a FACS Aria flow cytometer (Flow Cytometry Core Facility, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at University of Southern California). [00117] Erythroleukemic cell line (UT7-EPO) constitutively expresses DARC and beta- globin (FIGs. 1A-B, 2A-2E, 3A-3C, 4A-4C, 7A-7C, 10A-10C, 11A-11 B and 12A-12B). Lentiviral supernatants were used to expose erythroleukemic cells by adding 25 μΙ_ each of Lenti-DARC and Lenti-Hbb to K562, KU812, and KMOE-2 in a 96-well plate at 1 x 105 cells per well with 8 μg/mL protamine sulfate. This was followed by a second hit of 25 μΙ_ each the next day. FACS selection of GFP and OFP expressing cells was performed at two weeks in culture and further expanded over a period of 32 days. A fraction of the different cell populations were removed for direct GFP and OFP determination or for presence of DARC according to anti-CD234 antibody staining. The cells were then passed through a BD LSR Flow Cytometer (USC Flow Cytometry Core Facility) for determination of GFP, OFP and APC (anti-CD234) expression. (FIGs. 2A-2E, 3A-3C, 4A-4C, 7A-7C, 10A-10C, 11A-11 B and 12A-12B). FIGs. 2A-2E, 3A-3C, 4A-4C, and 7A-7C show relative expression of GFP and CD234-APC (both transgenes from the lenti-DARC vector) for the various

erythroleukemic cell lines (i.e. K562, KU812, KMOE-2 and UT7-EPO). In all cases the cells previously gated and sorted on GFP expressing cells from initial lenti-DARC transduction were highly enriched (40-95%) with co-expression of the accompanying DARC transgene. For cells isolated on the basis of OFP expression (from lenti-Hbb vector), the populations were also enriched with 35-86% purity (FIGs. 12A, 12B).

[00118] Example 2. Induction of hemoglobinization by hemin. High levels of

hemoglobinization need to be achieved to support growth of Plasmodium species. K562, KU812, and KMOE-2 cells were incubated (1 x 105 cells in 24-well plate) hemin (100 μΜ). At 10 days in culture, the cells were harvested, counted for viable and dead cells, fixed in 4% paraformaldehyde, permeabilized with "Perm & Wash" solution (BD Biosciences) and stained with 5 μΙ_ APA-C conjugated anti-HbF (mouse anti-HbF, Invitrogen) and 5 μΙ_ PerCP conjugated anti-HbA (mouse anti-HbA, Santa Cruz Biotechnology Inc) for estimating respective levels of fetal and adult hemoglobin production via flow cytometry. No toxicity due to hemin was observed.

[00119] Hemoglobin analysis of the data was based on the fluorescence intensity of the anti-HbF-APC antibody. FIGs. 13A-13C exemplify the variation in HbF expression following addition of the hemin and among K562, KU812, and KMOE-2 cell lines. Addition of hemin in general was not toxic and provided a strong induction of hemoglobin, as measured by HbF expression. The highest level after hemin treatment was seen in KMOE-2 cells with about a 24-fold increase in anti-HbF fluorescence.

[00120] Example 3. Differentiation protocol. The impact of various conditions was tested on the status of (1) hemoglobinization (benzidine staining) and (2) cell morphology (Giemsa staining).

[00121] (1) Presence of hemoglobin in the host cells is necessary for the growth of Plasmodium, as the parasite has been shown to use erythrocyte hemoglobin as a major nutrient source. In order to determine the impact of the differentiation protocol on the hemoglobinization of the UT7-EPO cells, we performed benzidine staining.

[00122] (2) As UT7-EPO cells differentiate into the erythroid pathway, they recapitulate the differentiation process of erythroblasts: from proerythroblasts to basophilic, to

polychromatic/heterochromatic and finally orthochromatic (Zeuner et al., 2012, supra). One key feature of this process is the condensation and asymmetric positioning of the nucleus. This phenomenon occurs prior the extrusion of the nucleus, a trademark of reticulocytes. Although it has been shown that P. falciparum can invade orthochromatic cells, late orthochromatic stages are required to support their growth. Similarly, despite considered to be more restrictive for their choice of hosts, similar stages may be sufficient to sustain P. vivax invasion and growth in vitro (Panichakul et al. 2007, International Journal for

Parasitology 37: 1551-1557, the entire contents of which are herein incorporated by reference.) Hence monitoring the evolution of the shape and size of the UT7-EPO nuclei during differentiation is crucial to determine the possibility of invasion in vitro.

[00123] In order to efficiently differentiate erythroleukemic (UT7-EPO) cell lines, the UT7- EPO cells were cultured in IMDM medium containing 10% FBS, 1 % Glutamine and

Penicillin/Streptavidin and 2 units/ml of EPO, in addition to the hemoglobinization and differentiation factors 100 μΜ hemin, 0.5 μΜ JQ1 , and 2 ng/ml ΤΘΡβΙ . The differentiation medium is replenished with JQ1 and ΤΘΡβΙ at Day 3. By Day 5, the cells have adopted a morphology reminiscent of a late erythroblast stages as determined by Giemsa staining

(FIGs. 16A-16D), and have accumulated high levels of hemoglobin as determined by benzidine staining (FIGs. 14, 15A, and 15B).

[00124] Example 4. Hemoglobinization of UT7-EPO cells under differentiating conditions. In order to determine the impact of the differentiation protocol on the hemoglobinization of the UT7-EPO cells, benzidine staining was performed for 8 days. Briefly, 50 μΙ_ of cell suspension was extracted from the culture and washed with PBS. A stock solution of o- diansidine (8mM) was activated by freshly adding 3% H202 (10: 1). The cell suspensions were then incubated with active o-diansidine (10: 1). Once the reaction has fully developed, the staining was observed under an inverted microscope, and representative pictures were taken (FIG. 14). For quantification purposes, three images of each well were taken. As illustrated in FIG. 14, cells cultured in control conditions only marginally express hemoglobin (FIG. 14, left column). The majority of the cells express a level of benzidine staining around 100-120 AU (FIG. 15A). Based on our arbitrary threshold of 140 AU, which reflects manual quantifications done for two independent experiments, the percentage of benzidine-positive cells in control conditions remains around 5% throughout the experiments (FIG 15B). On the contrary, when UT7-EPO cells are cultured in presence of hemin (100 μΜ) and JQ1 (0.5μΜ), a significant percentage of benzidine positive cells appear as early as Day 1 (on average 27%, FIG. 15B). By Day 3, the percentage of benzidine positive cells has reached a plateau, which is maintained up to Day 8 (around 85%, FIG. 15A). In all experiments, the addition of ΤΘΡβΙ did not appear to significantly affect hemoglobinization of UT7-EPO cells, however, a slight acceleration in the hemoglobinization, with a significantly higher percentage of benzidine positive cells was achieved on Day 2 (FIG. 15B). Nevertheless, by Day 3, no significant difference is observed between the two differentiating conditions (FIG. 15). From three independent experiments, it was concluded that the treatment of UT7-EPO cells with hemin, JQ1 and ΤΘΡβΙ allow for significant hemoglobinization of the UT7-EPO cells.

[00125] Example 5. Adoption of orthochromatic cell morphology by UT7-EPO cells under differentiating conditions. As UT7-EPO cells differentiate into the erythroid pathway, it is expected that the cells recapitulate the differentiation process of erythroblasts: from proerythroblasts to basophilic, to polychromatic and finally orthochromatic, which is represented by condensation and asymmetric positioning of the nucleus. This phenomenon occurs prior the extrusion of the nucleus, a trademark of reticulocytes. Monitoring of the evolution of the shape and size of the UT7-EPO nuclei during differentiation is crucial to determine the most appropriate timing of invasion in vitro. In order to assess if and when the UT7-EPO cells reached the proper erythroid stage under the differentiating conditions, Giemsa staining was performed at Day 5 of the protocol. After fixation with 4%

paraformaldehyde and staining with Giemsa, the shape and size of treated and untreated UT7-EPO cells and nuclei were compared (FIG. 16A-16D). Whereas in the control conditions most of the nuclei appear large and round, a vast majority of nuclei in cells exposed to differentiating conditions appear more condensed and asymmetrically positioned. Indeed, only a small percentage (0.3%) of cells appeared as late orthochromatic in the untreated conditions while 52.5% have reached this staged under the differentiating conditions at Day 5. Similar results were obtained repetitively.

[00126] Example 6. Invasion by P. falciparum. UT7-EPO cells were cultured in I M DM medium containing 10% FBS, 1 % Glutamine and Penicillin/Streptavidin and 2 units/ml of EPO, as well as the differentiating factors 100 μΜ hemin, 0.5 μΜ JQ1 , and 2 ng/ml ΤΰΡβ The differentiation medium was replenished with an additional dose of JQ1 and ΤΰΡβΙ at Day 3. The UT7-EPO culture was started at the cellular density of 2x10⁵ cells/ml in 5 ml (10⁶ cells total) of differentiation medium. UT7-EPO cells were maintained under differentiating conditions for day 5. The day of invasion, human red blood cells (hRBCs) previously infected with P. falciparum were enriched using the magnetic property of the Pf-infected cells

(Spadafora et al., 2011 , Malaria J, 10: 96-99, the entire contents of which are herein incorporated by reference). In order to perform the invasion assay, half of the culture medium for the differentiated UT7-EPO cells was changed to a fresh parasite medium (RPMI Medium 1640, 0.05 mg/ml Gentamicine, 0.014 mg/ml Hypoxantine, 38.4 nM HEPES, 0.20% Sodium Bicarbonate, 0.20% Glucose, 3.5 mM NaOH, 4.2% human serum, 0.20% glutamax), to which the three inducers were added (100 μΜ hemin, 0.5 μΜ JQ1 , and 2 ng/ml ΤΘΡβΙ). In order to increase the probability of invasion, each 5 ml culture of cells was co-incubated with Pf-enriched hRBC suspension at a ratio of 10 to 1. In parallel, a control culture with re- invasion of fresh human RBCs was started. The cultures were placed in a 37 °C incubator, in a sealed flask flushed with a 3% C0₂ mixture. After 3 or 7 days of co-cultures, the samples were collected, washed with PBS and fixed with 2% PFA.

[00127] The invasion of the P. falciparum in the differentiated UT7-EPO cells, was monitored using confocal microscopy. To do so, 100 μΙ of each cell culture sample and 20 μΙ from the hRBC control were smeared on a gelatin-coated microscope slide. Cells were then stained for GFP, using a rabbit anti-GFP (Life Technologies) and a secondary antibody goat anti rabbit Alex488 (Life Technologies). Cells were counterstained with DAPI. The slides were then mounted in ProLong® antifade solution. Single plane images of 2 μηι in thickness were taken using the confocal microscope Zeiss LSM5 (FIGs. 17A, 17B, 17C). The observation of the hRBC control culture clearly demonstrates the presence of GFP-positive parasites. Due to the absence of nucleus in hRBCs, the DAPI signal observed in the hRBC control culture can only emanate from the parasite nuclei (FIG. 17A, A1). Additionally, the low level of background of hRBC allows the observation of the weak GFP signal from the parasites surrounding the nuclei (FIG. 17A, A). On the contrary, the UT7-EPO and differentiated UT7-EPO cells have been shown to present an extremely high level of auto- fluorescence. Despite such a high level of auto-fluorescence, a specific DAPI staining combined to a GFP signal appears strong and specific enough to identify the parasite within the differentiated UT7-EPO cells (FIG. 17B, 17C). At multiple occasions, as indicated in FIGs. 17B, 17C, the presence of small DAPI-positive GFP-positive entities were observed next to the differentiated nuclei and within their cytoplasm, delineated by the GFP

background. The confocal imaging of the cells collected after invasion assays thus indicates the presence of P. falciparum within the differentiated UT7-EPO cells.

[00128] Example 7. Engineered Cells for Malaria (ECeM). As shown in FIG. 18, differentiated erythroleukemic cells as described herein, are transformed with a reporter gene system thereby providing a platform cell line for facile study of genes of interest in the malarial parasitic pathway. As shown in FIGs. 18 and 20, the reporter gene is a GFP (e.g., EGFP) reporter gene integrated into the ubiquitous β8θίίη locus. GFP provides a necessary control of the transgene (Tg) activity throughout erythroid differentiation. It is also used as the universal target for subsequent genetic manipulations to study candidate genes of interest. Specifically, as shown schematically in FIG. 18, ECeM cells with and without a transgene (Tg) of interest is differentiated and tested for Plasmodium (e.g., P. falciparum) invasion and growth. The GFP-Tg is a reproducible and validated target, as well as a screening tool for the Tg integration. [00129] The ECeM cells of the present invention are transformed with a reporter gene using the CRISPR/Cas genome-editing knock-in technology that allows for the abrogation of any locus and replacing it with a functional cassette containing a transgene (Tg) of interest, as described Harrison et al., 2014, Genes & Dev. , 28: 1859-1872, the entire contents of which are herein incorporated by reference. This technology only requires the identification of a specific target sequence of 20 nucleotides, the delivery of a plasmid coding for a guide RNA (gRNA) and the nuclease Cas9. The gRNA recognizes and guides Cas9 endonuclease activity, and the concomitant delivery of a repair cassette introduces the Tg through homologous recombination, as depicted in FIG. 20.

[00130] Using the CRISPR/Cas technology, a GFP reporter gene is integrated under the native ubiquitous β8θίίη promoter (Shmerling et al., 2005, Genesis, 42:229-235, the entire contents of which are herein incorporated by reference) in UT7-EPO cells to create the ECeM cells. The use of ECeM cells provide a double advantage. First the GFP reporter gene allows for validation of the transcriptional activity of the β8θίίη locus, throughout the erythrocyte differentiation pathway, by monitoring the GFP expression in the UT7-EPO cells. Second, the inserted GFP-Tg is then a validated target site for iterative insertion of any Tg of interest (FIGs. 18, 20). Additionally, the successful recombination of the Tg of interest results in the destruction of the initial GFP gene and abolition the GFP expression, which is used as a first screening for the occurrence of the proper Tg insertion.

[00131] As disclosed throughout and evidenced by, for example, FIGs. 5D, 6D, 7C, 8, 14, 15A, 15B, 16D, 17B, and 17C methods and compositions are disclosed for a differentiated erythroleukemic cell line capable of being invaded by Plasmodium spp. Immortalization of the disclosed differentiated erythroleukemic cells allows for culturing malarial Plasmodium spp.

[00132] While the present invention has been illustrated and described with reference to certain exemplary embodiments, those of ordinary skill in the art will understand that various modifications and changes may be made to the described embodiments without departing from the spirit and scope of the present invention, as defined in the following claims.

Claims

WHAT IS CLAIMED IS:

1. An erythroleukemic cell, comprising exogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine (DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B-cell lymphoma/leukemia 11A (BCL11A).

2. The erythroleukemic cell of claim 1 , comprising exogenous expression of

DARC.

3. The erythroleukemic cell of claim 1 , comprising exogenous expression of DARC and beta-globin.

4. The erythroleukemic cell of claim 1 , comprising exogenous expression of DARC and BCL11A.

5. The erythroleukemic cell of claim 1 , comprising exogenous expression of DARC, beta-globin, and BCL11A.

6. The erythroleukemic cell of claim 1 , comprising exogenous expression of

GlyA.

7. The erythroleukemic cell of claim 1 , comprising exogenous expression of GlyA and β -globin.

8. The erythroleukemic cell of claim 1 , comprising exogenous expression of GlyA and BCLIIA.

9. The erythroleukemic cell of claim 1 , comprising exogenous expression of GlyA, β-globin, and BCLIIA.

10. The erythroleukemic cell of claim 1 , comprising exogenous expression of GlyA and basigin.

1 1. The erythroleukemic cell of any one of claims 1-10, further comprising exogenously expressed immortalization factors.

12. The erythroleukemic cell of claim 1 1 , wherein the immortalization factors are selected from the group consisting of Myc, Ras, HPV-16 E6/E7, SV40-T, hTERT gene, and combinations thereof.

13. A cell culture comprising:

an erythroleukemic cell, comprising exogenous or endogenous expression of at least one selected from the group consisting of Duffy antigen receptor chemokine

(DARC), glycophorin A (GlyA), basigin, beta-globin, alpha-globin, and B-cell

lymphoma/leukemia 11A (BCL1 1A); and

hemin.

14. The cell culture of claim 13, wherein the erythroleukemic cell is any of claims

1-10 15. The cell culture of claim 12 or 13, further comprising JQ1.

16. The cell culture of claim 15, further comprising tumor growth factor beta 1 (TGF β1).

17. A method of inducing hemoglobinization and differentiation in the

erythroleukemic cell of any of claims 1-10, comprising:

culturing the erythroleukemic cell in the presence of hemin and JQ1 to form a hemoglobinized and differentiated erythroleukemic cell.

18. The method of claim 17, further comprising:

culturing the erythroleukemic cell in the presence of ΤΘΡβΙ .

19. A hemoglobinized and differentiated erythroleukemic cell made by the method of any one of claims 17-18.

20. The hemoglobinized and differentiated erythroleukemic cell of claim 19, wherein the formed hemoglobinized and differentiated erythroleukemic cell is a

heterochromatic erythroblast or a reticulocyte.

21. The method of any one of claims 17-18, further comprising:

expressing immortalization factors in the hemoglobinized and differentiated erythroleukemic cell to form an immortalized hemoglobinized and differentiated

erythroleukemic cell.

22. An immortalized hemoglobinized and differentiated erythroleukemic cell made by the method of claim 21.

23. The immortalized hemoglobinized and differentiated erythroleukemic cell of claim 22, wherein the formed immortalized hemoglobinized and differentiated

erythroleukemic cell is an immortalized heterochromatic erythroblast or immortalized reticulocyte.

24. A differentiated erythroleukemic cell capable of being invaded by Plasmodium species, the differentiated erythroleukemic cell made by the method of claim 17 or 18.

25. A differentiated erythroleukemic cell capable of being invaded by Plasmodium falciparum and/or Plasmodium vivax, the differentiated erythroleukemic cell made by the method of claim 17 or 18.

26. The differentiated erythroleukemic cell of claim 1 , further comprising an integrated reporter gene.

27. The differentiated erythroleukemic cell of claim 26, wherein the integrated reporter gene is integrated at the native βΑοίίη gene locus for expression by the βΑοίίη promoter.

28. The differentiated erythroleukemic cell of claim 26, wherein the

erythroleukemic cell line is UT7-EPO.

29. The differentiated erythroleukemic cell of claim 24, further comprising an integrated reporter gene.

30. The differentiated erythroleukemic cell of claim 29, wherein the integrated reporter gene is integrated at the native βΑοίίη gene locus for expression by the βΑοίίη promoter.

31. The differentiated erythroleukemic cell of claim 29, wherein the

erythroleukemic cell line is UT7-EPO.