Transgenic immunodeficient mouse expressing human SIRP-alpha

Abstract

The present invention provides a transgenic mouse which comprises a deficiency for murine T lymphocytes, B lymphocytes and NK cells, a deficiency for murine MHC class I and MHC class II molecules, and a functional xenogenic SIRP transgene. This mouse is useful for in vivo screening of various compounds, including immuno-therapeutic agents and vaccines. The said mouse is also useful for testing the in vivo metabolism of xenobiotic compounds.

Claims

1. A transgenic mouse, said mouse having a genome comprising: a) a genetic background that is FVB/N, C57BI/6, 129, or C3H, or a mixture of at least two genetic backgrounds selected from the group consisting of FVB/N, C57BI/6, 129, and C3H; and b) a transgene encoding human SIRP, wherein the human SIRP is functionally expressed in the mouse; c) an inactivated 2-microglobulin (.sub.2-m) gene, wherein the mouse does not express functional major histocompatibility complex (MHC) I proteins; d) an inactivated I-A.sup.b gene, wherein the mouse does not express functional MHC II proteins; e) a homozygous disruption of a Rag2 gene (Rag2.sup./) and a homozygous disruption of a common receptor y (y.sub.c.sup./) gene, wherein the mouse lacks functional mouse T lymphocytes, B lymphocytes, and natural killer NK cells; f) a transgene encoding a human MHC I protein and a transgene encoding a human MHC II protein, wherein the mouse functionally expresses the human MHC I and II proteins; and wherein said transgenic mouse supports development of human myeloid and lymphoid lineages comprising T cells, B cells, NK cells, macrophages and dendritic cells following engraftment of human cord blood hematopoietic stem cells (HSCs).

2. A transgenic mouse of claim 1, where the inactivated 2-microglobulin gene is a homozygous disruption of the 2-microglobulin gene (.sub.2m.sup./).

3. The transgenic mouse of claim 1, wherein the inactivated I-A.sup.b gene is a homozygous disruption of an H-2.sup.b-Agene (H-2.sup.b-A.sup./).

4. The transgenic mouse of claim 1, wherein the MHC I protein is human leukocyte antigen (HLA) A2 (HLA-A2) and the MHC II protein is HLA-DR1.

5. The transgenic mouse of claim 1, wherein said mouse has a genotype of Rag2/, .sub.2-m.sup./, y.sub.c.sup./, and I-A.sup.b/, and optionally HLA-A2.sup.+/+and/or HLA-DR1.sup.+/+.

6. The transgenic mouse of claim 1, wherein said mouse does not express a functional C5 protein.

7. The method of producing a transgenic mouse comprising human myeloid and lymphoid lineages comprising T cells, B cells, and NK cells, macrophages and dendritic cells, said method comprising transplanting human cord blood HSCs into the transgenic mouse of claim 1.

8. The transgenic mouse comprising human myeloid and lymphoid lineages comprising T cells, B cells, NK cells, macrophages and dendritic cells made by the method of claim 7.

Description

FIGURE LEGENDS

(1) FIG. 1: Analysis of SIRP expression on mC11b+ cells in three different transgenic founders.

(2) FIG. 2: Analysis of human T and NK cells in the thymus of the CH1-2hSa host 5 months after engraftment.

(3) FIG. 3: Analysis of human lymphocytes in the bone marrow of the CH1-2hSa host 5 months after engraftment.

(4) FIG. 4: Analysis of human myeloid cells (macrophages and dendritic cells, DC) and hematopoietic stem and progenitor cell (HSPC) in the bone marrow of the CH1-2hSa host 5 months after engraftment.

(5) FIG. 5: Analysis of human lymphocytes subsets in the spleen of the CH1-2hSa host 5 months after engraftment.

(6) FIG. 6: Amino acid sequence of the human SIRP protein.

(7) FIG. 7: Nucleotide sequence of the human SIRP gene.

(8) FIG. 8: Analysis of the presence of human T, B and NK cells in the bone marrow, spleen and gut of the CH1-2hSa host 6 months after engraftment (gated on human CD45.sup.+ cells).

(9) FIG. 9: Analysis of reconstitution efficiency in CH12hSa and NSG hosts (gated on human CD45.sup.+ cells).

(10) FIG. 10: HLA-A2 restricted anti-HBV human CD8 T cells responses upon vaccination of CH12hSa chimera.

(11) FIG. 11: Analysis of the migration of human T, B and NK cells to the liver of the CH1-2hSa host 6 months after engraftment (gated on human CD45.sup.+ cells).

EXAMPLES

(12) Mouse Genetic Background

(13) The CH1-2 mice derived from the crossing of 129 (Rag2.sup./ c.sup./, C5.sup./) X FVB (HLA-DR1.sup.+, C5.sup./) X B6 (2 m.sup./, I-A.sup.b/, HLA-A2) laboratory inbred mouse strains as described in WO 2008/010099 and WO 2008/010100. More precisely, the Rag2.sup./, .sub.c.sup./ mice (129 background) were crossed with I-A.sup.b/ (C57B1/6 background) and the F1 progeny were crossed successively with .sub.2m.sup./ (C57B1/6 background), HLA-DR1 transgenic mice (DR1.sup.+, FVB background) and HLA-A2.1 transgenic mice. I-A.sup.b+ (essential for murine MHC class II.sup./ phenotype) and C5.sup./ progenies (both 129 and FVB strains are constitutively C5.sup./) were selected from each crossing.

(14) FVB, 129 and B6 are well defined backgrounds which are well known to the person of skills in the art (Ridgway et al., Nature Immunol; 8: 669-673, 2007). Among these strains, the 129 strain was used for its ability to be transgenized and give rise to ES cells. FVB strain was used because its fertilized eggs contain large and prominant pronuclei, which facilitate the microinjection of DNA during transgenesis, and because they survive much better than pure B6 eggs. FVB and B6 backgrounds were used for their vigorous reproductive performance with large litters.

(15) Transgenesis

(16) The hCD172 cDNA encoding the membrane receptor SIRP (accession number NM_001040022) was introduced downstream a 7.2 Kb sequence corresponding to the murine c-fms promoter. All the parts were cloned into a pGL2B vector. The resulting 11 kb construct was purified on agarose gel.

(17) Superovulated CH1-2 3-week females were mated with CH1-2 males. Unicellular embryos were then microinjected with the construct (in collaboration avec F. Langa, IP). This procedure avoids the generation of mosaic mice upon transgenesis. Transfected embryos were then implanted into the recipient mice. Twenty-five newborn mice were obtained, of which 11 (4 males and 7 females) tested positive by PCR, indicating that the transgene had integrated into their genome

(18) Expression of a SIRP Transgene by Rag.sup./ c.sup./ 2m.sup./ IA-.sup.b/ HLA-A2.sup.+ HLA-DR1.sup.+ Mice

(19) Some of these animals were then checked by flow cytometry for human SIRP expression at the surface of macrophages in the periphery (double staining for hCD172 and mCD11bexpressed on murine macrophages surface). All transgenic founders were found by flow cytometry to express homogeneously SIRP. FIG. 1 shows 3 mice expressing the hCD172 transgene on most of the mCD11b.sup.+ cells. Expression of the human hSIRP receptor was detected in both macrophages (CD11b.sup.+) and dendritic cells (CD11c.sup.+) and some GR1.sup.+ cells.

(20) The transgenized mice were immediately mated. The resulting new-born mice were irradiated and then grafted intra-hepatically with hematopoietic precursor cells of human blood chord. The level of reconstitution found in human SIRP transgenic hosts engrafted with human hematopoietic progenitors is comparable if not superior to that of NOD SCID c.sup./ (NSG) hosts, showing high levels of T, B, NK and myeloid cells.

(21) Hematopoietic reconstitution in Rag.sup./ c.sup./ 2m.sup./ IA-.sup.b/ HLA-A2.sup.+ HLA-DR1.sup.+ huSIRP.sup.+ mice (CH1-2huSa)

(22) Newborn CH1-2huSa mice were first irradiated, then intra-hepatically grafted with human cord blood CD34.sup.+ hematopoietic progenitors. The reconstitution by human hematopoietic cells (CD45.sup.+ cells population targeted upstream in order to exclude Ly5.2+ murine cells) was monitored by flow cytometry 5 months after engraftment.

(23) As shown in FIG. 2, human thymocytes developed normally in the murine CH1-2huSa thymic environment. Both CD8.sup.CD4.sup. (DN) and CD8.sup.+CD4.sup.+ (DP) immature thymocytes and CD4.sup.+CD8.sup. (SPCD4) and CD8.sup.+CD4.sup. (SPCD8) mature thymocytes were present. As expected, only the SP4 and SP8 populations showed a high level of TCRaB expression, thus confirming their maturity. Likewise, CD56.sup.+, CD56.sup.+ CD16.sup.+ and CD16.sup.+ NK cells were detected.

(24) Human B cells (CD19.sup.+) were present in the chimera bone marrow with an IgM IgD expression profile similar to the one of cells engaged in development (FIG. 3). Human CD3.sup.+ T cells were also detected in the chimera bone marrow as well as CD56.sup.+, CD56.sup.+CD.sup.16+ and CD16.sup.+ NK cells.

(25) Macrophages and dendritic cells were detected in the bone marrow by co-staining for CD11c and CD14 (FIG. 4). Interestingly, a population of CD34+ cells was identified in the chimera bone marrow. These cells could correspond to the maintenance in chimera marrow of a population of human hematopoietic precursor cells (CD34.sup.+CD117.sup.+).

(26) As shown in FIG. 5, CD3.sup.+ CD4.sup.+ and CD3.sup.+ CD8.sup.+ human T cells, and CD19.sup.+ human B cells with an IgM IgD profile similar to mature human B cells, and human NK cells (mostly CD56.sup.+ CD16.sup.+) were detected.

(27) Presence of Human T, B and NK Cells to the Bone Marrow, Spleen and Gut of CH1-2hSa Chimeras

(28) CH1-2hSa irradiated newborns were engrafted intra-hepatically with 50 000 CD34.sup.+ human cord blood hematopoietic progenitors. Five month later, the percentages of human huCD45.sup.+ mCD29.sup. hematopoietic cells were evaluated by flow cytometry (gated on CD45.sup.+ cells) in the bone marrow, the spleen and the gut.

(29) As shown on FIG. 8, human cells were found in the 3 sites, indicating that they were able to move through the circulation to other organs, including lymphoid organs.

(30) Differential Ability of CH12 Versus RAG.sup./ c.sup./ Murine Thymic Anlage at Inducing the Development of Mature Human T Cells.

(31) Fetal thymuses from either CHb12 or RAG.sup./ c.sup./ HLA-mice were seeded with human cord blood CD34.sup.+ hematopoietic progenitors in the presence of human factors. At the end of the Fetal Thymic Organ Culture (FTOC), human CD45.sup.+ CD4.sup.+ and CD8.sup.+ cells were analyzed for the expression CD3 as marker of maturity. As shown in the table below, HLA expression dramatically increases the % of mature CD4 and CD8 human T cells in the thymus of chimera.

(32) Thymus were removed from day 14 embryos and each thymic lobe was incubated in Terasaki wells for 2 days in 25 l of complete medium: RPMI 1640 supplemented with 10% heat-inactivated human serum, 5% fetal calf serum, 100 IU/ml penicillin, 100 g/ml streptomycin, 2 mml-glutamine. 10-30 000 CD34.sup.+ cells purified from cord blood were added to each well. The plates were immediately inverted to allow the formation of hanging drops and incubated undisturbed in a humidified incubator (5% CO.sub.2 in air, 37 C.). After 48 h, thymic lobes were transferred onto floating nucleopore filters (Isopore membrane, 25 mm in diameter, pore size 8 m, Millipore SA, France) in six-well plates in 2.5 ml of complete medium and cultured for 28-35 days at 37 C. in air supplemented with 5% CO.sub.2 with a weekly medium change. Cytokines [rhu-IL-2 (5 ng/ml), 20 ng/ml rhu-IL-7 and 50 ng/ml rhu-SCF] were included only during the first 48 h, mainly to prevent apoptosis of the human progenitors and of early T-cell progenitors. Cells were then extracted from the different lobes by mechanical disruption of the lobes, and were pooled to be analyzed by flow cytometry.

(33) Results

(34) TABLE-US-00001 Origin of Human CD4.sup.+ Human CD8.sup.+ fetal thymi CD3+ cells (%) CD3.sup.+ cells (%) CH12 23 54 RAG.sup.-/- c.sup.-/- 4 13
Ig Production by Reconstituted CH12hSa Chimera: Differential Ability of CH12hSa Versus RAG.sup./ c.sup./ Reconstituted Chimera at Inducing the Production of Human Ig

(35) Serum from either CH12hSa or NOD SCID c.sup./ reconstituted with human cord blood

(36) CD34.sup.+ hematopoietic progenitors were tested for the presence of both human IgM and human IgG. As shown in the table below, IgM titer was strongly increased in CH12hSa chimera compared with NOD SCID c.sup./ chimera. Importantly, IgG were only detected in CH12hSa chimera.

(37) TABLE-US-00002 Chimera strain IgM (ng/ml) IgG (ng/ml) CH12hSa 818 165 NOD SCID c.sup.-/- 116

(38) Comparison of reconstitution efficiency between CH12hSa and NSG hosts.

(39) CH1-2hSa and NOD SCID c.sup./ (NSG) irradiated newborns were engrafted intra-hepatically with 50 000 CD34.sup.+ human cord blood hematopoietic progenitors. Seven weeks later, the percentages of human huCD45.sup.+ Ly5.sup. hematopoietic cells were evaluated by flow cytometry (gated on CD45.sup.+ cells).

(40) As shown on FIG. 9, CH1-2hSa host reconstitution by human cells is higher than the reconstitution of NSG host reconstitution.

(41) Validation of CH12hSa Chimera for the Test of Vaccine Candidates

(42) CH1-2 chimeras transgenic for human SIRPalpha were used for vaccination experiment 5 months after engraftment.

(43) Eight chimeras were injected with cardiotoxin intramuscularly.

(44) Five days later, 4 chimeras were injected intramuscularly with HBV (capsid and env) DNA: the vectors used were pCMV-HBc and pCMV-S2S (Michel et al., Proc. Natl. Acad. Sci. USA, 92: 5307-5311, 1995; Deng et al., Hepatology, 50(5): 1380-1391, 2009) Two weeks later, the injected chimera were reboosted intraperitoneally with a mixture of HBV proteins: HBsAg (env)+(capsid) HBcAg in Alu-S-gel. As control, the 4 left chimera were injected twice with PBS.

(45) Two weeks after the last injection, the chimera were sacrificed and the CD8 anti-HBV HLA-A2 restricted specific T cell response was measured (gated on CD45.sup.+ CD8.sup.+ CD3.sup.+ cells) by staining using a mixture of env and core HLA-A2 pentamers (FIG. 10).

(46) As a control, irrelevant staining using HCV protein HLA-A2 pentamers was performed.

(47) The results were expressed as % of pentamer.sup.+ cells among the total human CD8.sup.+ T cells.

(48) As shown on FIG. 10, vaccinated chimeras were able to mount specific anti-HBV HLA-A2 CD8 T cell response.

(49) No anti-HCV response were detected, neither in vaccinated nor in nave cord blood CD8 T cells, indicating that the response was specific for HBV. In contrast, no response could be detected in the absence of vaccination, further emphasizing the specificity of the response.

(50) CD8 HLA-A2-restricted HBV specific T cells were detected in the spleen of CH1-2hSa chimeras reconstituted with HLA-A2.sup.+ CB CD34.sup.+ human progenitor upon DNA and protein vaccination against both envelop and capsid HBV antigens. CH1-2hSa chimeras are thus capable of mounting CD8-specific T cell response to vaccination in a HLA-restricted manner.

(51) Migration of Human T, B and NK Cells to the Liver of CH1-2hSa Chimeras

(52) CH1-2hSa chimeras were engrafted with human CD34.sup.+ cord blood hematopoietic progenitors. Five months later, the presence of T, B and NK cells were assessed in the liver of the chimera by flow cytometry (gated on CD45.sup.+ cells).

(53) The 3 subsets were present, indicating that they were capable of migrating to the liver independently of vaccination of CH1-2hSa chimeras. The liver T cells were found to exhibit an activated CD45RAlow HLA-DR.sup.+ activated phenotype (FIG. 11).