WO2000035471A1

WO2000035471A1 - Soluble haemopoietin receptor nr6 (clf-1) and uses thereof

Info

Publication number: WO2000035471A1
Application number: PCT/AU1999/001119
Authority: WO
Inventors: Warren Scott Alexander; Donald Metcalf
Original assignee: The Walter And Eliza Hall Institute Of Medical Research
Priority date: 1998-12-17
Filing date: 1999-12-17
Publication date: 2000-06-22
Also published as: AUPP776298A0

Abstract

The present invention relates generally to a method for the treatment or prophylaxis of disease conditions associated with reduced levels of a soluble haemopoietic receptor. The disease conditions include conditions associated with dysfunctional haemopoietic regulation as well as an inability or reduced ability for infant mammals to suckle. More particularly, the present invention provides a method for facilitating haemopoietic progenitor cell production by the administration of a soluble or solubilized form of a haemopoietin receptor or receptor-like molecule or an agonist thereof or by inducing, up-regulating or otherwise facilitating expression of a gene encoding said soluble haemopoietin receptor. The present invention further comprises compositions useful in facilitating haemopoietic progenitor cell production comprising a soluble or solubilized form of a haemopoietin receptor or receptor-like molecule and/or agonists thereof or inducing up-regulating or otherwise facilitating expression of a gene encoding said soluble haemopoietin receptor. The composition of the present invention is also useful for inducing or otherwise facilitating postnatal survival in mammals and in particular humans such as by stimulating the ability for an infant to suckle.

Description

SOLUBLE HAEMOPOIETIN RECEPTOR NR6 (CLF-1) AND USES THEREOF

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.

The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research into the medical and allied health fields. Cytokine research is of particular importance, especially as these molecules regulate the proliferation, differentiation and function of a wide variety of cells. Administration of recombinant cytokines or regulating cytokine function and/or synthesis is becoming increasingly the focus of medical research into the treatment of a range of disease conditions. However, despite the identification of a range of cytokines and other secreted regulators of cell function, comparatively few cytokines are directly used or targeted in therapeutic regimens. One reason for this is the pleiotropic nature of many cytokines.

Cytokines with four alpha-helical bundles represent a major class of cytokines. This class encompasses the major haemopoietic regulators including interleukins (IL) -2, -3, -4, -5,-7, -9, -10, -11, -12, -13 and -15, granulocyte, granulocyte-macrophage and macrophage colony- stimulating factors (G-CSF, GM-CSF and M-CSF, respectively), erythroprotein (EPO) and thrombopoietin (TPO), as well as ciliary neurotrophic factor (CNTF) and more generally acting factors such as growth hormone (GH) and prolactin, interferon-γ (IFN-γ), leukaemia inhibitory factor (LIF), IL-6, oncostatin-M (OSM) and cardiotrophin (CT-1) [1]. Although these proteins share a common predicted tertiary structure, they show little primary amino acid similarity.

Cytokines exert their effects by binding to specific receptors expressed on the surface of target cells. Members of the haemopoietin receptor family are class I transmembrane proteins that include the receptors for the majority of four alpha-helical bundle cytokines and are defined by the presence of a conserved 200 amino acid extracellular domain known as the haemopoietin receptor domain (2). This domain contains two 100 amino acid subdomains which are structurally related to fibronectin type III repeats, being composed of seven beta strands which form a sandwich (3) . The defining features of the haemopoietin domain are four conserved cysteine residues found in the N-terminal subdomain and the five amino acid motif Trp-Ser-Xaa-Trp-Ser (WSXWS) found in the C-terminal subdomain; the remainder of the domain shows little strict sequence conservation.

The relatively low level of sequence similarity exhibited by members of the haemopoietin receptor family reflects not only their ability to bind cytokines which are themselves only distantly related but also the variety of roles that this receptor family plays in cytokine binding and signal transduction. Some members of the haemopoietin receptor family including the receptors for G-CSF, EPO, TPO and GH function as homodimers which bind a single cytokine molecule. Others act as heteromers with ligand-specific α-subunits binding a cytokine at low affinity and the cytokine/ -chain complex substantially interacting with additional β chains to form a high affinity complex which can initiate signal transduction. Receptors of this class include the α-chains of the GM-CSF, IL-3 and IL-5 receptors which use the shared βc signalling chain, the receptors for IL-6, IL-11, LIF, CNTF, OSM and CT- 1, each of which combines ligand-binding α-chains with the LIF receptor and/or gpl30 to form an active signalling complex, and the receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, which share the common gamma chain (γc)[l].

Although the vast majority of haemopoietin receptors contain transmembrane domains, exceptions exist, such as the CNTF receptor, which is anchored to the membrane via a glycosyl phosphatidylinosital (GPI) linkage (4). Indeed, for the IL-6 and IL-11 receptor - chains, attachment to the membrane is not absolutely required for function since a soluble complex of cytokine and receptor -chain is capable of productively interacting with cell- surface gpl30 to initiate signal transduction (5; 6). This theme is carried a step further in the case of IL-12. IL-12 is an unusual cytokine in that it is secreted as a disulphide-linked heterodimer comprising a p35 subunit, which shares homology to four-alpha helical bundle cytokines, and a p40 subunit which is a truncated member of the haemopoietin receptor family (7). IL-12 acts by binding to a cell surface receptor composed of two highly related members of the haemopoietin receptor family, IL-12 receptor βl and β2 (8). Interestingly, the p40 subunit of IL-12 is also secreted as homodimer which acts as an IL-12 antagonist (9). A second apparently soluble member of the haemopoietin receptor family, EBI3, was recently isolated in a screen for genes transcribed in response to Epstein Barr Virus (EBV) infection. Although the EBI3 protein shares little primary amino acid sequence similarity with IL-12 p40, it has been shown to interact with IL-12 p35 when overexpressed (10; 11).

A receptor termed "NR6" has been identified and its genetic sequences cloned (see

International Patent Application No. PCT/GB97/02479; filed 11 September, 1997). NR6 contains a signal sequence, an immunoglobulin-like domain and a classic haemopoietin receptor domain, but no transmembrane domain, indicating that NR6 is a soluble member of the haemopoietin receptor family. In work leading up to the present invention, the inventors identified a new biological role of NR6 by an analysis of tissue and developmental expression patterns and the generation of mice in which the gene for NR6 has been functionally deleted. It is apparent that NR6 is required for haemopoietic regulation and, hence, has a role in haemopoietic progenitor cell production. NR6 knock-out mice also have an impairment in the ability to suckle. Accordingly, NR6 has an indirect role in facilitating postnatal survival by inducing or facilitating young animals to suckle.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.

One aspect of the present invention contemplates a method of modulating production of haemopoietic progenitor cells in a mammal, said method comprising administering to said mammal, a modulating effective amount of NR6 or an isoform, derivative, splice variant, agonist, antagonist, homologue, chemical analogue or mimetic thereof or administering an expression-modulating effective amount of an agent capable of modulating expression of a gene encoding NR6 or its derivatives or homologues.

Another aspect of the present invention provides a method of facilitating production of haemopoetic progenitor cells in a mammal, said method comprising administering to said mammal a production-facilitating effective amount of NR6 or an isoform, derivative, splice variant, agonist, homologue, chemical analogue or mimetic thereof or administering to said mammal an NR6- encoding nucleotide sequence-expression facilitating effective amount of an agent which induces or otherwise facilitates expression of a genetic sequence encoding NR6 for a time and under conditions sufficient to induce, increase or otherwise facilitate production of haemopoetic progenitor cells.

Yet another aspect of the present invention is directed to a method of enhancing, promoting or otherwise facilitating postnatal survival of a mammalian offspring said method comprising administering to said postnatal offspring an effective amount of NR6 or an isoform, derivative, splice variant, agonist, homologue, chemical analogue or mimetic thereof or administering an agent capable of inducing or enhancing expression of NR6 genetic sequences to thereby increase levels of NR6 in vivo.

Still another aspect of the present invention contemplates a method of facilitating production of haemopoietic progenitor cells in a mammal said method comprising contacting stem cells in said mammal or cells intermediate of stem and committed lineage progenitor cells with an effective amount of NR6 or an isoform, derivative, splice variant, agonist, homologue, chemical analogue or mimetic thereof or facilitating an effective amount of expression of NR6 genetic sequences in cells of said mammal said effective amounts providing sufficient NR6 to facilitate production of myeloid cells from stem cells.

Yet still another aspect of the present invention provides a composition comprising NR6 or an isoform, derivative, splice variant, agonist, homologue, chemical analogue or mimetic thereof and one or more pharmaceutically acceptable carriers and/or diluents.

Even yet another aspect of the present invention contemplates a method for detecting NR6 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for NR6 or its immunointeractive derivative or homologue for a time and under conditions sufficient for an antibody-NR6 complex to form, and then detecting said complex.

Another aspect of the present invention provides a genetically modified embryonic stem cell which carries a defective gene encoding for NR6.

Yet another aspect of the present invention provides a genetically modified mammal comprising an NR6-/- genotype and/or the phenotype of an NR6-/- genotype wherein said phenotype comprises reduced levels of haemopoietic progenitor cells compared to a corresponding NR6+/+ mammal.

SUMMARY OF SEQ ID NOs.

DESCRIPTION SEQ ID NO.

Nucleotide sequence of antisense NR6 oligonucleotide 1 Amino acid sequence of C-terminal FLAG eptitope tag 2

Amino acid sequence of N-terminal region of NR6 peptide 3

Amino acid sequence of WSXWS motif 4

Amino acid sequence of N-terminus of NR6 produced by 5 KUSA cells Nucleotide sequence of NR6.1¹ 6

Amino acid sequence of NR6.1 7

Nucleotide sequence of NR6.2² 8

Amino acid sequence of NR6.2 9

Nucleotide sequence of NR6.3³ 10 Amino acid sequence of NR6.3 11

The polyadenylation signal AATAAATAAA is at nucleotide position 1451 to 1460; NR6.1 (SEQ ID NO:6) and NR6.2 (SEQ ID NO:8) are identical to nucleotide 1223 encoding Q407, the represents the end of an exon. NR6.1 splices out an exon present only in NR6.2 and uses a different reading frame for the final exon which is shared with NR6.2; this corresponds to amino acids VLPAKL at amino acid residue positions 408-413. The region of 3 '-untranslated DNA shared by NR6.1, NR6.2 and NR6.3 is from nucleotide 1240 to 1475. The WSXWS motif is at amino acid residues 330 to 334.

The polyadenylation signal AATAAA is at nucleotide positions 1494 to 1503. The WSXWS motif is at amino acid residues 330 to 334. NR6.1 and NR6.2 are identical to nucleotide 1223 encoding Q407 which represents the end of an exon. NR6.2 splices in an exon beginning at amino acid residue D408, nucleotide 1224 and ends at residue G422, nucleotide 1264. The region of 3' untranslated DNA shared by NR6.1, NR6.2 and NR6.3 is from nucleotide position 1283 to 1517.

The nucleotide and amino acid numbering corresponds to SEQ ID NO: 6 and 8. The WSXWS motif is at amino acid residues 330 to 334. The polyadenylation signal AATAAATAAA is from nucleotide 1781 to 1780. NR6.1, NR6.2 and NR6.3 are identical to nucleotide 1223 encoding Q407, this represents the end of an exon. NR6.3 fails to splice from this position and, therefore, translation continues through the intron, giving rise to the C-terminal protein region from amino acid residues 408 to 461. The region of 3' untranslated DNA shared by NR6.1, NR6.2 and NR6.3 is from nucleotide 1469 to 1804. The following single and three letter abbreviations are used for amino acid residues:

Amino Acid Three-letter One-letter Abbreviation Symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N Aspartic acid Asp D

Cysteine Cys C

Glutamine Gin Q

Glutamic acid Glu E

Glycine Gly G Histidine His H

Isoleucine He I

Leucine Leu L

Lysine Lys K

Methionine Met M Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Tip w Tyrosine Tyr Y

Valine Val V

Any residue Xaa X BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photographic representation showing expression of NR6 in adult issues and embryos. Northern blots of RNA extracted from tissues of adult mice, whole embryos at various gestational ages and KUSA cells. The 1.8kb NR6 species arrowed was detected with an NR6 cDNA hybridisation probe that included the entire coding region. The blots were stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase cDNA fragment (GAPDH) as a control for RNA integrity. Sal. gland, salivary gland; MLN, mesenteric lymph node; int, intestine; BM, bone marrow.

Figure 2 is a photographic representation showing expression of NR6 in cell lines. Northern blot analysis of cells line RNA is shown. The NR6 panels were generated using an NR6 cDNA hybridisation probe that included the entire coding region. The blots were stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase cDNA fragment (GADPH) as a control for RNA integrity. Murine cell lines: Ml, WEHI-3BD, 32D, FDC-P1, 416B, RAW264; J774: myeloid/macrophage; Ba/F3:pro-B; 7OZ, WEHI231, ABLS-8.1: pre-B; WEHI279, 129B: B; MPC11; plasmacytoma; CTLL-2, YAC, BW2, 52DA20, BW5147, EL4, WEHI703: T-cell; F4N, DP16, TS5: erthroleukemia; P815: mastocytoma; WEHI11.13; fibrosarcoma; KUSA, 729, Sr7, scI19, BAdAl l, 798, Ac621b: bone marrow/fetal liver stromal; 3T2-L1: fibroblast. Human cell lines: U937: monocytic; HL60: promyelocytic; HepG2: hepatoma; Allen- 1: Ewing sarcoma. COS cells are monkey fibroblasts. LPS B and ConA T are primary B and T blast cells generated from spleen by LPS and concanavalin A stimulation, respectively.

Figure 3 is a photographic representation showing expression of NR6 in the early embryo. Embryos at days 7.5-11.5 post-coitus were incubated with digoxigenin-labelled antisense NR6 riboprobes. A. 9.5 dpc embryo showing expression in the mesonephric duct (md) and low level expression in the forelimb bud (lb) and the first branchical arch (bal) B, C. An 11.5 dpc embryo showing intense expression of NR6 in the nasal processes (np) and first branchial arch, in an asymmetric pattern in the limb buds, and in dermatomyotome (dm). D. Transverse section of the caudal region of a 9.5 dpc embryo, showing intense NR6 expression in the mesonephric ducts. E. Oblique section of an 11.5 dpc embryo, at the level of the forelimb, showing NR6 expression in the dermatomyotome. da, dorsal aorta; g, gut; nc, notocord; nt, neural tube. Scale bars: D, 20 m; E, 10 m. Control experiments using NR6 sense probes were negative.

Figure 4 is a photographic representation showing expression of NR6 in 14.5 and 18.5 dpc embryos. Sagittal sections of embryos were hybridised with 33P-labelled antisense NR6 riboprobes. A. 14.5 dpc embryo showing expression of NR6 in lung (1), kidney (k), genital tubercle (gt), percartilaginous condensations of the digital metacarpals, (d), intervetebral discs (id), tongue (t) and infacial mesenchyme. A control experiment, using NR6 sense probes did not show specific hybridisation at these sites B, C. Serial sagittal sections of the head an 18.5 dpc embryo, hybridised with sense (B) and antisense (C) probes. NR6 expression is seen int he cortex (c) and hippocampus (hi), as well as in facial mesenchyme, developing teeth (th) and salivary gland (sg). E. Coronal section through the head of an 18.5 dpc embryo showing expression of NR6 in the cortex and hippocampus. Expression is also prominent in the cartilage of the external auditory meatus (earn), p, pons. Scales bars, 1 mm.

Figure 5 is a photographic representation showing NR6 expression in embryonic organs. Lightfield (A, C, E) and darkfield (B, D, F) images of NR6 expression in embryonic tissues, detected by in situ hybridisation of 33P-labelled anti-sense NR6 riboprobes to histological sections. A, B. NR6 expression in the lung bronchi at 14.5 dpc, but not in surrounding parechyma. C, D. NRt6 expression in the kidney at 14.5 dpc, restricted to the tips of collecting ducts. E, F. Expression in precartilaginous mesenchymal condensations in the developing metacarpals and the proximal phalanges of the forelimb at 14.5 dpc. Scale bars, 10 m.

Figure 6 is a photographical representation showing that NR6 is a secreted homodimer.

Western blot analysis of proteins in the medium conditioned by KUSA cells transfected with a FLAG epitope tagged version of the murine NR6.2 cDNA. Proteins eluted from an anti- FLAG affinity matrix were resolved in polyacrylamide gels under nonreducing (-DTT) or reducing (+DTT) conditions, transferred onto membrane and blotted with a monoclonal anti- FLAG antibody 9M2) or a rabbit anti-NR6 polyclonal antiserum raised against the N-terminal region of NR6 (NR6).

Figure 7 is a diagrammatic representation showing: A. Genomic structure and gene targeting of the NR6 locus. A. Structure of the human NR6 gene deduced from the sequence of cosmid R30292 with exons boxed and coding regions filled. B. Structure and map of murine NR6 gene with exons boxed and coding regions filled. The map was constructed from restriction analysis of three overlapping genomic clones shown above; the entire gene was sequenced to determine the positions of the exons. The targeting vector used to generate NR6-null mice is also shown, as is the predicted structure of the targeted allele following homologous recombination. The Spel fragments used to distinguish the normal and targeted alleles Southern blot analysis are indicated by broken arrows.

Figure 8 is a representation showing the initiation of NR6 transcription. A. Primer extension products from KUSA RNA and control tRNA were electrophoresed in polyacrylamide gels and visualised by autoradiography. The major product of 108 nucleotides (nt), corresponding to a site of transcription initiation 129 nt upstream of the translation initiation codon, is indicated (see panel B). DNA sequencing products (G, A, T, C) were used as size standards. B. Sequence of the murine NR6 5 '-flanking region. The transcription initiation site identified by the primer extension experiment is indicated + 1 , with transcribed sequences in upper case and translated sequence bolded. Consensus binding motifs for the TFIID, ISL-1, PTF-β, cEBP/β and Spl transcription factors were underscored. The oligonucleotide (complementary to the sequence between positions +22 and +41 relative to the translation initiation site) used in primer extension experiments is indicated by broken underscoring.

Figure 9 is a photographic representation showing Southern blot of Spel digested genomic

DNA extracted from the tails of mice derived from a cross between heterozygous (NR6 +/-) mice. The blot was initially hybridised with probe NR6 A, which is situated in the NR6 locus outside just 3' of the targeting vector, to allow a distinction between endogenous (9.9kb) and mutant NR6 (7.1kb) alleles. This signal was stripped away and the filter was then hybridised with a neo probe and finally with NR6 B, which is part of the NR6 gene deleted by homologous recombination, to demonstrate the loss of genetic material in NR6 -/- mice.

Figure 10 is a photographic representation of lack of NR6 expression in NR6 -/- mice. A. Northern blot analysis of RNA extracted from the lungs, kidneys, heads and limbs of neonatal NR6-/- and control wild-type (+/+) and heterozygous (+/-) mice. After hybridisation with a full length NR6 cDNA probe, the filter was stripped and hybridised with a glyceraldehyde- 3 -phosphate dehydrogenase cDNA fragment (GAPDH) as a control for RNA integrity. B. NR6-/- (left) and wild-type (right) embryos at days 9.5 post-coitus were incubated with digoxigenin-labelled antisense NR6 riboprobes. Expression in the limb buds and the first branchial arch of the normal embryo is clearly absent in NR6-deficient embryos.

Figure 11 is a photographic representation showing failure to suckle in NR6-/- mice. A litter of living newborn mice from a cross between NR6+/- parents, showing the empty stomachs of three NR67- mice (above). Wild-type or NR6+/- mice, shown below, suckle normally.

Figure 12 is a photographic representation of a Western blot under (A) non-reducing and (B) reducing conditions. Clone 4/99- 1H4 producing a monoclonal antibody to NR6 was tested against purified full length NR6-Flag tag. Fractions (Fr) were collected from a Sephadex column. The Sephadex column was loaded with a sample for a Flag column. M2, positive control for flag; Ig2b, isotopye control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the elucidation of the biological properties associated with NR6. Reference herein to "NR6" includes its isoforms and splice variants such as NR6.1, NR6.2 and NR6.3 as well as derivatives, homologues, chemical analogues, mimetics and functional equivalents of NR6 or its isoforms, splice variants or hybrids at either the nucleotide or protein levels. A "hybrid" includes a heterodimer as well as a complex formed with the NR6 receptor ligand, CLC. The nucleotide sequence encoding CLC is represented in genbank AR002595, AC005849 and AF172854 (see also U.S. Patent No. 5,741,772 and International Publication No. WO99/00415). The nucleotide sequences encoding splice variants NR6.1, NR6.2 and NR6.3 are represented in SEQ ID NOs: 6, 8 and 10, respectively. The corresponding amino acid sequences are respectively shown in SEQ ID NOs: 7, 9 and 11.

A "homologue" of NR6 is taken to include any haemopoietin receptor having an extracellular domain comprising the amino acid sequence Tip Ser Xaa Trp Ser [SEQ ID NO:4] where Xaa is any amino acid residue or a functional, agonising or antagonising derivative thereof but which lacks transmembrane and cytoplasmic domains.

Reference herein to a "derivative" includes single or multiple nucleotide or amino acid substitutions, deletions and/or additions as well as parts, fragments and portions thereof. The present invention further contemplates nucleic acid or amino acid fusion and hybrid molecules.

Conveniently, a derivative of NR6 includes a molecule encoded by a nucleotide sequence capable of hybridising to the nucleotide sequence of NR6.1 (SEQ ID NO:6), NR6.2 (SEQ ID NO:8) and/or NR6.3 (SEQ ID NO: 10) under low stringency conditions at 42°C.

Reference herein to a low stringency at 42 °C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about IM to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31 % v/v to at least about 50% v/v formamide and from at least about 0.0 IM to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions. In general, washing is carried out T_m = 69.3 + 0.41 (G+C)% (25). However, the T _mof a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (26).

Alternatively, or in addition to, a derivative or hybrid may be defined at the level of nucleotide or amino acid similarity to the nucleotide or amino acid sequence corresponding to NR6.1, NR6.2 and/or NR6.3. Various mutants, derivatives and splice variants of NR6 are described in International Patent Application No. PCT/GB97/02479 and corresponding US applications USSN 08/928,720 and USSN 09/037,657 filed 11 September, 1997 and 10 March, 1998, respectively and wherein each of which is incorporated herein by reference. Generally, an NR6 molecule or its derivative or homologue comprises an amino acid or nucleotide sequence similarity to one or more of SEQ ID NOs: 6-11 of from about at least 30% to about 100%, such as at least above 40%, 50%, 60%, 70%, 80% or 90% .

To identify critical biological roles of NR6, mice lacking a functional NR6 gene were generated. These mice are referred to herein as "NR6-/-" mice. NR6-/- mice were born without obvious anatomical abnormalities, but failed to suckle and died within 12-24 hours of birth. Furthermore, NR6-/- mice contained fewer haemopoietic progenitor cells then normal littermates indicating that NR6 has important functions in haemopoietic regulation.

Accordingly, one aspect of the present invention contemplates a method of modulating production of haemopoetic progenitor cells in a mammal, said method comprising administering to said mammal, a modulating effective amount of NR6 or an isoform, derivative, splice variants, agonist, antagonist, homologue, chemical analogue or mimetic thereof or administering an expression-modulating effective amount of an agent capable of modulating expression of a gene encoding NR6 or its derivatives or homologues.

Reference hereinafter to "NR6" is taken to include its isoforms, splice variants (eg. NR6.1, NR6.2 and NR6.3), derivatives, agonists, homologues, chemical analogues and mimetics.

The term "modulating" includes inducing, enhancing, increasing or otherwise facilitating production of haemopoietic progenitor cells as well as inhibiting, reducing, retarding or otherwise decreasing production of haemopoietic progenitor cells. Although facilitating progenitor cell production is a preferred embodiment in accordance with the present invention, decreasing production may also be important in the treatment of certain cancers or to induce apoptosis of particular cell types.

The term "expression" is used in its broadest context and may be measured at the level of transcription and/or translation. Accordingly the expression of a nucleotide sequence may be determined by a measurable phenotypic change involving transcription and translation into a proteinaceous product which in turn has a phenotypic effect or at least contributes to a phenotypic effect or its absence results in a phenotypic effect. Increased expression includes both increased efficiency in transcription and/or increased stability of transcript.

Accordingly, in a preferred embodiment, the present invention provides a method of facilitating production of haemopoetic progenitor cells in a mammal, said method comprising administering to said mammal a production-facilitating effective amount of NR6 as hereinbefore defined or administering to said mammal an NR6- encoding nucleotide sequence- expression facilitating effective amount of an agent which induces or otherwise facilitate expression of a genetic sequence encoding NR6 for a time and under conditions sufficient to induce, increase or otherwise facilitate production of haemopoetic progenitor cells.

The term "mammal" as used herein includes but is not limited to a human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), laboratory test animal (e.g. mouse, rat, rabbit, guinea pig, hamster), companion animal (e.g. dog, cat) or captive wild animal (e.g. game animal, fox, deer, bear). Preferably, however, the mammal is a human, primate or murine species. Most preferably, the mammal is a human.

Mice deficient from the NR6 gene also exhibited a reduced capacity or total inability to suckle. Ultimately, this leads to death unless intervention occurs.

Accordingly, another aspect of the present invention is directed to a method of enhancing, promoting or otherwise facilitating postnatal survival of a mammalian offspring said method comprising administering to said postnatal offspring an effective amount of NR6 as hereinbefore defined or administering an agent capable of inducing or enhancing expression of NR6 genetic sequences to thereby increase levels of NR6 in vivo.

It is proposed herein that NR6 regulates haemopoiesis in a mammal and, hence, modulates haemopoietic progenitor cell production. More specifically mice deficient for NR6 (i.e. NR6- /- mice) carry fewer clonogenic cells capable of responding to stem cell factor (SCF), M-CSF or a combination of SCF, IL-3 and EPO compared to NR6+/+ mice. Although not intending to limit the present invention to any one theory or mode of action, it is proposed herein that NR6 does not directly affect lineage committed cells but rather stem cells or cell types intermediate of stem cells and committed progenitor cells such as but not limited to colony forming units I (CFUI) and II (CFUII). All such non-committed progenitor cells are encompassed by the term "stem cell" or "stem cells". The target of NR6 protein or genetic therapy includes stem cells and lineage-committed and prior-committed progenitor cells. It is also proposed that the NR6-/- genotype leads to an inability or reduced capacity for young mammals to suckle. The administration of NR6 may be an appropriate means to induce suckling in young animals.

Another aspect of the present invention contemplates a method of facilitating production of haemopoietic progenitor cells in a mammal said method comprising contacting stem cells in said mammal or cells intermediate of stem and committed lineage progenitor cells with an effective amount of NR6 as hereinbefore described or facilitating an effective amount of expression of NR6 genetic sequences in cells of said mammal said effective amounts providing sufficient NR6 to facilitate production of myeloid cells from stem cells.

The identification of NR6 as hereinbefore defined as a regulator of haemopoiesis in mammals enables the production of compositions useful in modulating and more particular facilitating production of haemopoietic progenitor cells in a mammal such composition may also be useful in facilitating postnatal survival in mammals by inducing or promoting suckling.

Accordingly, another aspect of the present invention provides a composition comprising NR6 as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents.

The preferred composition of the present invention is in the form of a pharmaceutical composition.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with NR6 and optionally other active ingredients as required, followed by filtered sterilization or other appropriate means of sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying and the freeze-drying technique which yield a powder of NR6 plus any additionally desired ingredient.

When NR6 is suitably protected, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet or administered via breast milk. For oral therapeutic administration, NR6 may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1 % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound. Alternative dosage amounts include from about 1 μg to about 1000 mg and from about 10 μg to about 500 mg. These dosages may be per individual or per kg body weight. Administration may be per hour, day, week, month or year.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and except insofar as any conventional media or agent is incompatible with the active ingredient, their use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

As stated above, dosages may be expressed per body weight of the recipient. For example, from about 10 ng to about 1000 mg/kg body weight, from about 100 ng to about 500 mg/kg body weight and for about 1 μg to above 250 mg/kg body weight may be administered.

The compositions according to the aspects of the present invention may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating NR6 expression or NR6 activity. The vector may, for example, be a viral vector.

The identification of the role of NR6 in regulation of haemopoiesis permits the identification of potential disease conditions which could lead to postnatal death or impairment. For example, expectant mothers could undergo foetal testing for NR6 or expression of NR6 genetic sequences. Alternatively, immediate postnatal babies may be monitored from NR6 production or expression of NR6 genetic sequences. Any indication of low NR6 levels is then responded to by the administration of NR6 as hereinbefore defined.

Accordingly, another aspect of the present invention contemplates a method for detecting NR6 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for NR6 or its immunointeractive derivative or homologue for a time and under conditions sufficient for an antibody-NR6 complex to form, and then detecting said complex.

Accordingly, another aspect of the present invention provides an antibody or a fragment, antigenic binding portion, synthetic form or functional equivalent thereof, wherein said antibody is capable of interacting with NR6 on an antigenic or epitope-containing portion thereof. Preferably the antibody specifically and/or exclusively binds to NR6 or an antigenic or epitope-containing portion thereon. One such antibody is designated herein "4/99-1H4". Antibodies may readily be obtained by any number of means. For example, in relation to 4/99- 1H4, a purified N-terminal FLAG-tagged, full length NR6 molecule from a CHO cell line was used to immunize mice (e.g. C57/B16, Balb/C or C3H/J) and sera tested using ELISA. Spleen cells were then fused with SP₂O myeloma cells using PEG. The fusions were plated out on 96 well flat bottom plates and again screened using ELISA. Supernatants were then tested by Western blot. Any number of variations may be made to the antibody producing protocol. Furthermore, the present invention extends to both polyclonal and monoclonal antibodies although monoclonal antibodies are preferred.

The presence of NR6 may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody to NR6 is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-NR6 complex, a second antibody specific to NR6, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of another complex of antibody-NR6-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of NR6. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is one which might contain NR6 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.

In the typical forward sandwich assay, a first antibody having specificity for the NR6 or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2- 40 minutes or overnight if more convenient) and under suitable conditions (e.g. from about room temperature to about 37 °C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hap ten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

In another alternative method, the NR6 ligand is immobilised to a solid support and a biological sample containing NR6 brought into contact with its immobilised ligand. Binding between NR6 and its ligand can then be determined using an antibody to NR6 which itself may be labelled with a reporter molecule or a further anti- immunoglobulin antibody labelled with a reporter molecule could be used to detect antibody bound to NR6.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fiuorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-NR6 complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-NR6-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. The fluorescent labelled antibody is allowed to bind to the first antibody-NR6 complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and enzyme immunoassay techniques are both very well established in the art. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The present invention also contemplates genetic assays such as involving PCR analysis to detect the NR6 gene or its derivatives. Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformational polymorphisms analysis (SSCP) as specific oligonucleotide hybridisation, as methods such as direct protein truncation tests.

A further aspect of the present invention contemplates the use of NR6 as hereinbefore defined in the manufacture of a medicament for the treatment of haemopoietic regulatory dysfunction.

The present invention further contemplates genetically modified mammals such as laboratory test animals (e.g. mice) which are deficient for one (NR6+/-)or both (NR6-/-)alleles for NR6.

Preferably, the genetically modified mammals (e.g. mice) are deficient for both alleles. Such mammals (e.g. mice) are particularly useful as live models to screen for agents (e.g. NR6) which can facilitate regulation of production of haemopoietic progenitor cells.

Accordingly, another aspect of the present invention provides a genetically modified embryonic stem cell which carries a defective gene encoding for NR6.

Preferably, the embryonic stem cell is of murine origin.

Preferably, the embryonic stem cell is used in the production of NR6-/- mammals such as mice.

Yet another aspect of the present invention is directed to a genetically modified mammal comprising an NR6-/- genotype and/or the phenotype of an NR6-/- genotype wherein said phenotype comprises reduced levels of haemopoietic progenitor cells compared to a corresponding NR6+/+ mammal.

Preferably, the mammal is a murine species such as a mouse.

In one embodiment, NR6 is in the form of a homodimer. However, the present invention extends to heteromultimers of NR6 such as but not limited to heterodimers. A heteromultimer contemplated by this aspect of the present invention include a first component comprising a single NR6 polypeptide and a second or further component comprising another polypeptide such as but not limited to the p40 or p35 components of IL-12 or functional or structural homologues of these molecules. Reference herein to "NR6" includes reference to both forms of the molecule.

Monomeric forms of the NR6 molecule may also be useful in selecting for binding partners such as homomeric or heteromeric components. For example, a monomeric NR6 molecule may be immobilised onto a solid support and this used as a screening device for binding partners. Such binding partners are encompassed by the present invention. The present invention is further described by the following non-limited Examples.

EXAMPLE 1 cDNA CLONING

Mouse testis, brain (Stratagene) and KUSA cell (12) cDNA libraries were screened with an oligonucleotide complementary to the sequence encoding the WSXWS motif as previously described (13). Four cDNA clones (68.1, 68.2, MBC-1 and K166) were obtained which appeared to encode a novel member of the haemopoietin receptor family, NR6. The cDNA libraries were then screened with the 1.0 kb 68.1 or the 1.6 kb K166 cDNA inserts or with NR6-specific oligonucleotides derived from their sequence. Fourteen additional murine NR6 cDNA clones were isolated 68-5, 68-35,68-41, 68-51, 68-77, 73-23, K166.2, K166.7, K166.9 and K166.10. Murine cDNA inserts were used as hybridisation probes under low stringency conditons to isolate human NR6 cDNAs from foetal liver, fetal kidney and placental libraries (Stratagene).

EXAMPLE 2

GENOMIC CLONING AND PRIMER EXTENSION

Two NR6 clones (NR6-57.3, NR6-2.2) were insolated from a murine 129/Sv genomic library (Stratagene) using the NR6 68-1 cDNA insert as the hybridisation probe. A clone extending further 5' (NR6-57.3.1) was subsequently isolated with an exon 1 probe. A radiation map was generated from these clones and the entire gene was sequenced to allow positioning of intro/exon boundaries. The 5' end of the gene was determined by primer extension of first- strand NR6 cDNA was previously described (24). Briefly, a specific antisense NR6 oligonucleotide (5'-GTGACTGCAACGCCGGTATG-3' [SEQ ID NO:l]) situated at +22 to +41 in the cDNA sequence was end-labelled with ³ P, annealed with 5μg poly-A+ RNA extracted from KUSA cells and extended with reverse transcriptase. Reaction products were electrophoresed in 8% w/v poly aery lamide/7M urea gels and visualised by autoradiography. DNA sequencing products primed from genomic NR6 clones by the above oligonucleotide were used to determine the precise size of the primer extension product. The nucleotide and corresponding amino acid sequence for the NR6 isoforms, NR6.1, NR6.2 and NR6.3 are represented in SEQ ID NOs: 6 and 7, 8 and 9 and 10 and 11, respectively.

EXAMPLE 3 DATABASE SEARCHES

The NCBI genetic sequence database (Genbank), which encompasses the major database of expressed sequence tags (ESTs) and the TIGR database of human expressed sequence tags were searched for sequences with similarity to human and mouse NR6 using the TFASTA algorithm (14). Using the software package SRS (15), ESTs that exhibited similarity to NR6 (and their partners derived from sequencing the other end of cDNAs) were retrieved and assembled into contigs using Autoassembler (Applied Biosystems, Foster City, CA). Consensus nucleotide sequences derived from overlapping ESTs were then used to search the various databases using BLASTN (16). Again, positive ESTs were retrieved and added to the contig. This process is repeated until no additional ESTs could be recovered. Final consensus nucleotide sequences were then translated using Sequence Navigator (Applied Biosystems, Foster City, CA).

EXAMPLE 4 RNA EXPRESSION ANALYSES

Northern blot analysis of poly-A+RNA were performed as described (17). Probes used were: the 1.6kb EcoRI insert from murine NR6 cDNA clone 73-12 and a 1.2 kb Pstl chicken glyceraldehyde-3-phosphate dehyrogenase (GAPDH) fragment. For in situ hybridisation analyses, tissues were fixed in 4% v/v paraformaldehyde, dehydrated, embedded in paraffin and sections required embryos and sections being treated with 20 μg/ml of proteinase K (Boehringer, Mannheim). The full length NR6 cDNA was used as probe. Whole mount in situ hybridisation was performed at 70°C and in situ hybridisation to tissue sections was performed at 50°C. EXAMPLE 5 GENERATION OF NR6-DEFICIENT MICE

To construct the NR6 targeting vector, 4.1kb of murine genomic NR6 DNA containing exons 2 through to 6 was deleted and replaced with a G418-resistance cassette, leaving 5' and 3' arms of NR6 homology of 2,9 and 4.5 kb, respectively. A 4.5 kb Xhol fragment of the murine genomic NR6 clone 2.2 containing exons 7, 8 and 3' flanking sequence was subcloned into the Xhol site of pBluescript generating pBSNR6Xho4.5. A 2.9 kb Notl-Stul Fragment within NR6 intron 1 from the same genomic clone was inserted into Notl-Stul fragment within NR6 intron 1 from the same genomic clone was inserted into Notl and EcoRV digested pBSNr6Xho4.5 creating pNR6-Ex2-6. This plasmid was digested with Clal, the recognition site for which was situated between the two NR6 fragments, and following blunt ending, was ligated with a blunted 6 kb Hindlll fragment from placZneo, which contains the lacZ gene and a PGKneo cassette, to generate the final targeting vector, pNRόlacZneo (Figure 7). pNRόlacZneo was linearised with Notl and electroporated into W9.5 embryonic stem cells. Colonies of cells resistant to 175 μg/ml G418 were picked and expanded after 8 days in selection medium. Clones in which the targeting vector had recombined with the endogenous NR6 gene were identified by hybridising Spel-digested genomic DNA with a 0.6kb Xhol-Stul genomic NR6 fragment (Figure 7), which distinguished between the endogenous (9.9 kb) and targeted (7.1 kb) NR6 loci. Homologous recombination at the NR6 locus was observed in 19 of 158 clones analysed (12%). Two targeted ES cell lines, W9.5NR6-2-44 and W9.5- NR6-4-2, were injected into C57B1/6 blastocysts to generated chimeric mice. Male chimeras were mated with C57B1/6 females to yield NR6 heterozygotes which were subsequently interbred to produce wild-type (NR6 + /+), heterozygous (NR6+/-) and mutant NR6 (-/-) offspring. The genotypes of offspring were determined by Southern blot analysis of genomic DNA extracted from tail biopsies. DNA extraction, digestion with restriction endonucleases and processing of Southern blots were performed as described (17).

EXAMPLE 6 HISTOLOGICAL AND HAEMATOLOGICAL ANALYSIS Neonatal NR6-/- mice were compared histologically with normal littermates using whole animal serial saggital sections following fixation in Bouin's fixative and staining with haematoxylin and eosin (H&E). Morphological sites of the brain were conducted on H&E- stained serial coronal sections of tissues fixed in 4% v/v paraformaldehyde.

Peripheral blood was collected by retro-orbital bleeding and diluted into 3 % v/v acetic acid containing methylene blue (white cells) or 1 % w/v ammonium oxalate (platelets) for manual cell counts using hemocytometer chambers and standard microscopy. Manual 100-400 cell leukocyte differential counts of peripheral blood, bone marrow, liver and spleen were performed on smears or cytocentrifuge preparations stained with May-Gruenwald-Giemsa. Megakaryocytes were enumerated by microscopic examination of hematoxylin and eosin- stained histological sections of liver. The clonal culture of hematopoietic progenitor cells were performed in 1 ml cultures of 104 (fetal liver) or 2 x 104 (neonatal bone marrow, spleen or liver) cells in 0.3% w/v agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% w/v fetal calf serum (FCS), 10 ng/ml murine IL-3, 100 ng/ml murine (stem cell factor) SCF and 4 U/ml human EPO as previously described (18). Parallel cultures were stimulated using 100 ng/ml of SCF or 10 ng/ml of M-CSF. Agar cultures were fixed and sequentially stained fro acetylcholinesterase, Luxol fast Blue and hematoxylin, and the composition of each colony was determined at 100- to 400-fold magnifications.

EXAMPLE 7 PROTEIN EXPRESSION AND PURIFICATION

The polymerase chain reaction was used to incorporate a derivative of the murine NR6 cDNA (NR6.2, that included a C-terminal FLAG epitope tag (DYKDDDDK [SEQ ID NO:2]), into the mammalian expression vector pEF-BOS (19). The construct was fully sequenced and then co-transfected into KUSA cells with a plasmid expressing the neo resistance gene from a phosphoglycerate kinase promoter as previously described (13). The transfected KUSA cells were cultured in Iscove's Modified Dulbecco's medium containing 10% v/v foetal bovine serum and 800 μg/ml G418 (Life Technologies). The conditioned medium was harvested 3-5 days after the cells were grown to confluence. Aliquots (1 ml) of the conditioned medium were incubed with 20 μl of anti-FLAG antibody M2 affinity gel (Scienfitic Imaging Systems) for 1 h at 4°C. The affinity beads were washed three times with 1 ml PBS containing 0.02% v/v Tween 20 and then the bound proteins were eluted by incubation with 20 μl of 100 μg/ml FLAG peptide for 15 min. at room temperature. The eluted proteins were resolved on a 7.5% w/v polyacrylamide gel under both nonreducing and reducing conditions and electrotransferred onto a prewetted polyvinylidene difluoride membrane (PVDF- Plus, Micron Separation Inc.). After blocking with 1 % w/v bovine serum albumin, the membrane was incubated with 2μg/ml of either anti-FLAG M2 antibody or a rabbit anti-NR6 polyclonal antibody raised against an N-terminal NR6 peptide (VISPQDPTLLIGSSLQATCSIHGDTP [SEQ ID NO:3]), followed by incubation with a sheep anti-mouse or rabbit Ig polyclonal antibody conjugated with horseradish peroxidase (AMRAD Corporation Limited, Melbourne, Australia). The protein was visualized by autoradiography using the ECL system (Amersham Pharmacia Biotech).

For N-terminal sequencing, the KUSA conditioned medium (450 ml) was incubated with 0.6 ml of anti-FLAG antibody M2 affinity resin for 4 h at 4°C. The M2 beads were then recovered by centrifugation and washed extensively with PBS containing 0.02% v/v Tween 20. The bound protein was eluted with 9 x 0.4 ml of 50 μg/ml FLAG peptide. the second, third and fourth fractions eluted were pooled and concentrated to 80 μl using a Ultrafree-MC centrifugal filter unit (Millipore) with a regenerated cellulose membrane (molecular weight cut-off, 10 000). The sample volume was further reduced to 20 μl by centrifugal lyophilization. The concentrated protein was mixed with an equal volume of 2 x SDS sample buffer containing 0.2 M DTT, resolved on a 4-20% w/v polyacrylamide gel (Novex) and electrotransferred onto a PVDF membrane. The membrane was stained for 5 min with 0.1 % w/v Coomassie blue in 50% v/v methanol and destained in 50% v/v methanol and 10% v/v acetic acid. The membrane was then washed extensively with deionized water purified by a tandem Milli-RO and Milli-Q system (Millipore) and air-dried completely. The band corresponding to NR6 protein was removed and subjected to N-terminal sequencing on a Hewlett Packard G 1005 A protein sequencer. For metabolic labelling, the transfected KUSA cells were seeded in a 6- well plate and grown to 70-90% confluence. After removing the culture medium, the cells were washed once with 5 ml of fresh DME medium followed by incubation with 5 ml of DME medium containing 10% v/v foetal bovine serum, 800 μg/ml geneticin and 100 μCi/ml [³⁵S] protein labelling mixture containing both [³⁵S] methionine and [³⁵S] cysteine for 3 days. Aliquots (1 ml) of the labelled conditioned medium were precleared with 30 μl of protein G-Sepharose beads (Amersham Pharmacia Biotech) and then incubated with 6 μg of either anti-FLAG M2 antibody or the rabbit anti-NR6 peptide polyclonal antibody, followed by the addition of 20 μl of protein G beads. The immunocomplexes were eluted from the beads with nonreducing SDS sample buffer, resolved on a 7.5% v/v polyacrylamide gel under both nonreducing and reducing conditions and electrotransferred to a PVDF membrane. The labelled protein was visualized by Phosphor Imager analysis (Molecular Dynamics).

EXAMPLE 8 CLONING OF NR6 cDNA

Using an oligonucleotide encoding the WSXWS [SEQ ID NO:4] motif, a novel cDNA denoted NR6 was isolated from murine testis, brain and KUSA cell line cDNA libraries (see above and International Patent Application No. PCT/GB97/02479). The majority of cDNA clones contained a long open reading frame (NR6.1) of 1275 nucleotides. The 425 amino acid sequence predicted from this open reading frame is consistent with that of a member of the haemopoietin receptor family: a potential signal sequence and immunoglobulin-like domain precede a single haemopoietin domain (HD) containing the expected conserved cysteine pairs and a WSEWS motif, and a sequence with loose homology to part of the fibronectin type III repeat is evident at the C-terminus. Independent clones were also isolated with deduced open reading frames (NR6.2, NR6.3) that contained divergent sequences C-terminal of the HD. Human NR6 cDNAs, all of which were homologues of murine NR6.1, were also isolated using low stringency hybridisation of murine probes to human foetal kidney, fetal liver and placental libraries. No hydrophobic sequences typical of a transmembrane domain were evidence in any of the predicted murine or human proteins, suggesting that NR6 is a soluble member of the haemopoietin receptor family. The primary amino acid sequences of human and mouse NR6.1 show 98% identity. The nucleotide and corresponding amino acid sequences for NR6.1, NR6.2 and NR6.3 are shown in SEQ ID NOs: 6 and 7, 8 and 9 and 10 and 11, respectively.

EXAMPLE 9 EXPRESSION OF NR6 mRNA

Northern blot analysis of poly A + RNA extracted from adult mice tissues revealed a 1.8 kb NR6 transcript, consistent in size with the isolated cDNAs, that was limited in expression to the salivary gland and lungs of both male and female mice and was also present in the testis.

In RNA extracted from embryos, this transcript was not detected at 9 days post-coitus (dpc), but became evident at 10 dpc and remained expressed until birth (Figure 1). An extensive survey failed to detect NR6 expression in the majority of haemopoietic cell lines examined: of over thirty lines examined, only FDC-P1, Ba/F3, 70Z, MPC11 and Allenl weakly expressed the 1.8kb NR6 transcript (Figure 2). A smaller transcript of approximately 1.0 kb, the precise nature of which is unclear, was expressed in 70Z, I29B, MPC11, BW5147 and

F4N. In contrast, several adherent and stromal cell lines expressed NR6 prominently, including embryonic stem cells, NIH3T3, COS, STO, 3T3 LI and primary mouse embryonic fibroblasts, the WEHGI-11.13 fibrosarcoma and BAd.All, 729, PA6, OP9, AFT024 and

KUSA bone marrow stromal cell lines (Figure 2).

To better define NR6 expression during embryonic development, embryos at 7.5-11.5 dpc were examined by whole mount in situ hybridisation with digoxigenin-labelled riboprobes. After hybridisation, embryos were serially section and the anatomical location of NR6 transcripts determined. NR6 expression was first detected at 9.5 dpc in the first branchial arch, the developing forelimb bud and mesonephric duct (Figure 3A). At 10.5 and 11.5 dpc, intense expression was seen in nasal processes and the maxillary and mandicular components of the first branchial arch. NR6 expression was also seen in the limbs, in the mesenchyme overlying the otic vesicle and in the dermatomyotome (Figure 3B-E). Expression of NR6 in 12.5, 14.5 and 18.5 dpc embryos was examined by in situ hybridisation of NR6 sense and antisense 33P-labelled riboprobes to tissue sections. At each embryonic stage studied, NR6 was expressed prominently in the cranio-facial mesenchyme and in tissues derived from the first branchial arch. No NR6 expression was evident in the olfactory epithelium. At 14.5 and 18.5 dpc, NR6 transcripts were also observed in dental papillae, in the tongue and throughout mesenchyme beneath the oral and nasal epithelia (Figure 4A, C). Expression was observed in the secretory buds and ducts of the submadibular salivary gland from 14.5 dpc and in the lacrimal glands at 18.5 dpc (Figure 4C). At 12.5 and 14.5 dpc the ecoderm of Rathke's pouch expressed NR6, although transcripts were not detected in the pituitary gland. NR6 expression was seen in the mesonephric (Wolffian) duct at 12.5 dpc and in the growing tips of the collecting ducts throughout embryogenesis (Fig. 5C, D). It is not expressed in uninduced or induced metanephric mesenchyme, nor in mesenchymally derived nephric elements. Mature nephrons did not express NR6, nor did the rest of the collecting tubule. NR6 expression was observed in the genital tubercle at the three timepoints examined but was not detected in other reproductive organs (Figure 4A). NR6 transcripts were detected in the lung buds at E12.5 and marked expressed was observed in the major bronchi, segmental and terminal bronchi, but not in lung parenchyma, at 14.5 dpc (Figure 5 A, B). By 18.5 dpc, NR6 expression in the bronchi was reduced but still detectable. Expression was not seen in the terminal sacs nor in the alveoli. From El 2.5 , NR6 expression was observed in all precartilaginous membranous blastema. At later timepoints, expression was prominent in tissues adjacent to forming cartilage. For example, at 18.5 dpc NR6 expression was seen around the cartilaginous nasal septum, the external auditory meatus, the intermediate digits of hindlimb (Figure 5E, F) and the articular surfaces of synovial joints.

In situ hybridisation studies were also undertaken to examine NR6 expression within the central nervous system. ³ P-labelled NR6 riboprobes were hybridised to coronal, sagittal and transverse sections of murine brain from 11.5 dpc to birth. NR6 transcripts were not detected prior to 17.5 dpc. At this time, expression was observed in the nuclear zone of the neopallial cortex and in the hippocampus (Figure 4C,D). Rare NR6-positive cells were also observed in the midbrain. By birth, expression of NR6 in the brain was no longer detectable. EXAMPLE 10 NR6 IS A SECRETED PROTEIN

To assess the structure of the NR6 protein, the murine NR6.2 coding sequence, into which a C- terminal FLAG epitope tag had been incorporated, was expressed in KUSA cells. As shown in Figure 6 the native NR6 signal sequence efficiently mediated secretion of the recombinant protein into the KUSA cell conditioned medium. Soluble NR6 was detected predominantly as an 112kDa protein under nonreducing Western blot conditions with both an anti-FLAG antibody as well as with a rabbit antiserum raised against an N-terminal NR6 peptide. A small amount of protein with molecular mass of approximately 200 kDa or larger was also consistently observed. All these proteins migrated with a molecular mass of 56 kDa under reducing conditions, suggesting that the 112 kDa and the higher molecular mass species were disulphide- linked dimers, tetramers or higher order aggregates of a 56 kDa NR6 monomer. Because the calculated molecular mass of the unmodified NR6 protein is 48 kDa, the 56 kDa protein secreted from KUSA cells is probably glycosylated. Purification and N-terminal sequencing of the NR6 protein secreted from KUSA cells generated a single amino acid sequence, AHTAVISPQD [SEQ ID NO:5], consistent with the mature amino acid sequence predicted from cDNA sequencing. Experiments in which transfected KUSA cells were metabolically labelled with ⁵S-labelled amino acids also resulted in secretion of NR6 as 112 kDa and 56 kDa species under non-reducing and reducing conditions respectively. This study did not provide an indication that any other proteins were associated with NR6. Similar results were obtained when murine NR6 was expressed in other cell lines including CHO, COS and 293T. When expressed in a variety of cell lines, human NR6 was also secreted as a disulphide-linked homodimer. Taken together these results indicate that NR6 is primarily secreted as a 112 kDa protein that is a disulfide-linked homodimer of 56 kDa subunits.

EXAMPLE 11 GENOMIC STRUCTURE OF THE MURINE NR6 LOCUS

A restriction map of the murine NR6 locus was assembled from three genomic clones, which together encompassed 20 kb of the mouse genome. DNA sequencing was used to determine the intron/exon boundaries and to position each of the exons on the physical map (Figure 7). The NR6 gene was found to be distributed over 9 exons which span 11 kb and are organised in a pattern similar to that observed in the genes for other members of the haemopoietin receptor family (20). Exon 1 contains the 5 untranslated region and sequence encoding the protein signal sequence; exon 2 encodes the Ig-like domain; exons 3-6 encode the haemopoietin domain and exons 7-9 encode FNIII-like sequence. The boundaries of the eight introns conform to the "GT- AG" rule for intron/exon junctions and extended sequence around the splice donor and acceptor sites fits with established consensus motifs (Table 1; 21). This genomic structure provides the molecular basis for the generation of the multiple NR6 transcripts isolated by cDNA cloning. cDNAs encoding NR6.1 , NR6.2 and NR6.3 each contain exons 1 to 7 with differential splicing of exons 8 and 9 producing alternative 3' ends. The structure of the human NR6 gene was deduced from sequence retrieved from databases (Example 3). Although intron sizes diverge, the human gene is similar to that of the mouse, also containing 9 exons spread over 17kb of the human genome (Figure 7).

The site of initiation of murine NR6 RNA transcription was mapped by primer extension using a 32P-labelled oligonucleotide complementary to sequence 22-41 nt upstream of the ATG protein initiation codon. A major product of 108nt was observed which corresponds to a putative transcription initiation site 129nt upstream of the initiation ATG (Figure 8). The nucleotide sequence of the putative NR6 promoter region extending 883nt 5 ' to the proposed transcription initiation site was determined. No TATA or CAAT motifs were evident. However, the sequence around the initiation site fits the Py-Py-A+1-N-T/A-Py-Py transcriptional initiator (Inr) consensus for RNA polymerase II, which has been shown to mediate similar functions to TATA elements (22) with Inr initiators, were also present in the -35 to - 160 region of the putative NR6 promoter. Consensus binding sites for the c-Ets-2, Isl- 1 and Ptf-β, C/EBP and c/EBPβ transcription factors were also present (Figure 8).

EXAMPLE 12 PERINATAL LETHALITY IN MICE LACKING NR6

To examine the biological role of NR6 in vivo, a targeting vector was generated in which sequence encoding the NR6 Ig-like and haemopoietin domains was deleted and replaced with a cassette encoding resistance to G418 (Figure 7). Using homologous recombination in embryonic (ES) cells, this vector was used to generate mice in which the NR6 gene had been functionally deleted. A number of mice in litters born of heterozygous NR6+Λ parents died within 24 hours of birth. Southern blot analysis of DNA extracted from tissue samples revealed that these mice were homozygous for the targeted NR6 allele, while their healthy littermates were heterozygotes or wild-type mice. The use of probes from the region of the NR6 gene deleted by homologous recombination also confirmed that the homozygous mutant mice lacked exons 2 to 6 (Figure 9). Genotype analysis of a larger cohort of neonatal animals revealed that NR6-/- mice were represented in numbers expected from normal Mendelian segregation of alleles (24:40:20 for NR6+/+:NR6+/-:NR6-/-). As no NR6-/- mice survived beyond 24 hours after birth, these data indicate that the loss of NR6 does not compromise embryonic survival but is lethal during the first day of life.

To confirm that the NR6 gene had been functionally deleted, RNA was extracted from tissues of neonatal NR6-/- mice as well as from wild-type and heterozygous littermates. As anticipated, NR6 transcripts were detected in Northern blot analysis of lung, kidney, head and limb RNA from NR6+/- or wild-type mice, but were absent in samples from homozygous mutants (Figure 10). This result was confirmed in while mount in situ hybridisation studies: while evident in the facial mesoderm and developing limbs of a normal embryo, NR6 expression was not detected in an NR6-/- embryo (Figure 10B).

Closer examination of mice soon after birth revealed that NR6-/- mice failed to suckle effectively and had stomachs devoid of milk (Figure 11). The NR6-null mice had normal body weights and were otherwise morphologically indistinguishable from their normal littermates. They had a normal respiratory rate, were well oxygenated and responded to touch with vocalisation, righting and rooting reflexes. Neonatal NR6-/- mice were capable of opening and closing their mouths and dissections revealed that the palate, mouth and oesophagus were intact. Histological examination of serial sagittal sections of entire mutant neonates failed to reveal any structural abnormalities, including those that might account for failure to suckle. Previous studies of mutant mice which fail to suckle, including fyn -/- and Brn-3a-/- mice (23) have linked loss or abnormality of specific neural populations to this phenotype. As NR6 is expressed in the brain late in gestation (see above), the inventors completed a histological survey of the brains of newborn NR6-/- mice. The brains of two newborn NR6-null mice and two wild type littermates were serially sectioned in the coronal plane and every fifth section was photographed. In this experiment, although not in other anatomical studies, the histology of the brains of the newborn NR6 null mice was slightly inferior to that of the control, possibly due the failure of NR6-/- mice to feed causing progressive development of metabolic abnormalities. With this caveat, no abnormalities were observed in the anatomy of the NR6-/- brains. The cortex and hippocampus, the two sites of NR6 expression detected in in situ studies, did not differ from the wild type and the olfactory bulb was normal. The complete brain stem was not examined in this study.

EXAMPLE 13 HAEMOPOIETIC PROGENITOR CELL DEFICIENCIES IN NR6-/- MICE

As NR6 belongs to the haemopoietin receptor family and is expressed in haemopoietic stromal cell lines, we sought to examine whether haemopoiesis was perturbed in NR6-/- mice. Analysis of newborn NR6-/- mice revealed that the haematocrit, number of circulating platelets and number and morphological distribution of white blood cells were no different from those observed in normal or heterozygous littermates. The frequencies of most classes of morphologically recognisable cells in cytocentrifuge preparations of bone marrow, spleen and liver of these mice also fell within the normal range, although reduced percentages of lymphocytes were evident in the spleen and live (Table 2). The numbers and lineage commitment of haemopoietic progenitor cells in neonatal NR6-/- mice were enumerated in clonogenic cultures. NR6-/- bone marrow and spleen contained 1.5-2.5 fold fewer clonogenic cells capable of responding to stem cell factor (SCF), M-CSF or the combination of SCF, IL-3 and EPO. The deficiencies did not reflect a reduction in cells committed to any particular lineage; fewer colonies of all types enumerated were evident (Table 3). The numbers and lineage commitment of haemopoietic progenitor cells were normal in the livers of neonatal NR6-/- mice (Table 3) and in similar analyses, no disturbances in fetal liver progenitor numbers or lineage commitment were evident when measured at day 13 of gestation. EXAMPLE 14 DETECTION OF HETERODIMER FORMS OF NR6

Experiments are conducted to ascertain whether or not NR6 may also exist as a heteromultimer such as a heterodimer. In one set of experiments, antibodies directed to NR6 (e.g. NR6.1, NR6.2 or NR6.3) or immunointerative fragments thereof are used to screen biological tissues such as in a Western blot procedure to identify changes in the size of NR6 and/or to capture forms of NR6.

In other experiments, NR genetic sequences are co-expressed with genetic sequences encoding cytokine components such as the p35 and p40 components of IL-12.

EXAMPLE 15 OVER EXPRESSION OR OVER ADMINISTRATION OF NR6

To test the effects of NR6 in normal, NR6+/- mice and NR6-/- mice, recombinant NR6 is injected into mice and the mice analysed such as by determination of haemopoietic progenitor cells. In addition, the effects of tissue specific expression of NR6 is also analysed.

EXAMPLE 16

PLURIPOTENCY OF STEM CELLS

The stem cells of NR6+/- and NR6-/- mice are analysed for their pluripotency potential to ascertain whether the NR6 deficient phenotype adversely affects the ability of stem cells to be pluripotential or to ascertain if the stem cells exhibit restricted or altered pluripotentiality.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

EXAMPLE 17 PREPARATION OF A MONOCLONAL ANTIBODY TO NR6

A monoclonal antibody was produced to NR6 using a purified, N-terminal FLAG tagged, full length NR6 from a CHO cell line. The NR6 was prepared as a 1 mg/ml solution in PBS.

The following immunization protocol was employed. Two eight week old mice from each of C57/B16, Balb/C and C34/J strains were injected with:

1. 20 μg FLAG-NR6 in complete Freund's Adjuvant (0.1 ml subcutaneous);

2. 20 μg FLAG-NR6 in incomplete Freund's Adjuvant (0.1 ml intraperitoneal); and 3. 20 μg FLAG-NR6 in incomplete Freund's Adjuvant (0.1 ml intraperitoneal).

Animal were test bled and the sera screened by ELISA.

The following fusion protocol was employed :-

1. C57/B16 #13 was injected with FLAG-NR6 in PBS (0.1 ml i.p.);

2. C57/B16 #13 was killed by cervical dislocation and ibs spleen removed and a sample of blood collected;

3. The spleen cells were fused with 1 x 10⁸ SP₂O myeloma cells using PEG 1500; 4. The fusion was plated out into 6 x 96 well flat bottom plates.

The cell culture medium comprised :-

Gisco Hybridoma serum free medium; 10% v/v FBS Batch 149; 10% v/v IL-6 condition medium derived from P388D1 cell line; Hypoxanthne, Amnopterin, Thymidne (HAT).

The fusion was re-fed with 100 μl of cell culture. The fusion was screened using ELISA. 7 x 96 well ELISA plates were coated with 8 μg/ml FLAG-NR6 in carbonate buffer (pH 9.6). This antigen used for coating was from the same batch of antigen used for immunization.

6 x 96 well ELISA plates were also coated with 2 μg/ml FLAG-NR4 in carbonate buffer (pH 9.6).

Seven clones were identified as being positive and were moved to 24 well plates.

The clones were expanded and frozen while weaning cells onto Medium with FBS alone.

10 ml of supernatant was then collected from each clone for further screening. The supernatants were tested by Western blot. One clone designated 4/99- 1H4 was found to be positive by Western blot.

Clone 4/99- 1H4 was then isotyped using Boehringer isotype kit and also dilution cloned. The isotype was K, IgG 2b.

100 ml of supernatant was then collected from 4/99- 1H4 and purified over Protein G.

4/99- 1H4 was re-screened using purified full length NR6 FLAG-tag from the NNTI cell line (Chugi). Purification was done over a FLAG column with fractions then run over a Superdex column. Fractions from the column were run reduced and non-reduced and probed with M2 as a positive control for Flag, purified 4/99- 1H4 and Ig2b isotype control.

The results are shown in Figures 12(a) and (b). TABLE 1 Intron-Exon Junctions of the Mouse NR6 Gene

Exon Exon size Donor Intron size Acceptor

(bp) (bp*)

1 253 GC CAA G gtgaggtgga 5195 ctgttctcag AT GTC T

2 282 C CGA GG gtaggttctg 214 gtcccaacag A GAG AA

3 130 GT GTG G gtaagggcat 107 ttgacctcag GC CTG C

4 170 GGA CCA gtgaggacct 1372 ttctccccag GCT CCA

5 158 AT GCA G gtaggtcagt 68 ttgccgacag TG ACA A

6 169 A GAC AG gtaaggctgg 2020 tgattcttag G GAC CC

7 188 AG GCT G gtaagaacct 104 tcccctgcag TG CTC C

8 43 TGG AAG gtactgtcag 181 ctcctctcag GTG CTG

9 252

consensus AG gt^a/g^a9t tc rich-cag G

Intron size correct to 0.1 kb

TABLE 2 Haematological profile of NR6 mutant mice

Genotype

NR6^+/+ NR6^+/ NR6 '

Peripheral Blood

Platelets (xlO Vml) 469+70 398 + 116 529+82

Haematocrit (%) 40+2 40+3 45+3 *

White Cell Count (x ⁶/ml) 3.9 + 1.1 4.6+0.8 4.4 + 1.2

Neutrophils (%) 74+2 75 +4 81 + 10

Lymphocytes (%) 11 + 1 10±4 9±6

Monocytes (%) 14+2 15+2 11 +4

Eosinophils (%) 1 + 1 0 0

Bone Marrow

Blasts (%) 4+2 3 + 1 4±2

Promyelocytes/Myelocytes (%) 4+3 3 + 1 2 + 1

Metamyelocytes/Neutrophils (%) 59+8 59+6 61 +9

Lymphocytes (%) 12+7 9+3 11 +4

Monocytes (%) 9+ 1 8+5 11 +3

Eosinophils (%) 0.3 +0.6 0.3±0.6 1 +0.5

Nucleated erythroid cells (%) 11 +2 17 + 13 11 +3

Spleen

Blasts 4+3 2+2 3 + 1

Promyelocytes/Myelocytes (%) 1 +2 2 + 1 l ± l

Metamyelocytes/Neutrophils (%) 20+4 32 + 18 31 +9

Lymphocytes (%) 20+6 17 + 10 9 + 1 f

Monocytes (%) 4+3 7+2 7+2

Eosinophils (%) 1 + 1 2+2 0

Nucleated erythroid cells (%) 49+8 35 + 10 50 + 12 Genotype

NR6^+/+ NR6^+/" NR6 '

Liver

Blasts (%) 4+2 5+3 4+3

Promyelocytes/Myelocytes (%) 6+3 6+2 4+2

Metamyelocytes/Neutrophils (%) 9+4 8+6 13 +6

Lymphocytes (%) 28+9 15+7 6+3*

Money tes (%) 4+2 4 + 1 4+2

Eosinophils (%) 0.3 +0.6 0.7+0.6 1 + 1

Nucleated erythroid cells (%) 49+6 61 + 10 70+9*

Megakaryocytes (per 20 hpf)# 63 + 17 76+9 69 + 12

Mean + standard deviations of data from 4 to 10 mice of each genotype. Statistical comparisons were performed using Student's test. * p < 0.05, f P < 0.08 for comparison of data from NR6^+/+ and NR6^{+ "} mice. # determined from histological sections at 400x magnification, with a minimum of 20 high pwer fields (hpf) counted per mouse.

TABLE 3 Haemopoietic progenitor cell profile in neonatal NR6-deficient mice

Stimulus Organ NR6 Number of colonies per 2x10' * cells genotype

Total Blast G GM M Eo E Meg E/Meg Mix

SCF BM +/ + 125 + 13 9±2 37+2 34 + 13 21 + 1 2 + 1 5+6 12+6 4±2 1 +2

+ IL-3 +/- 102+30 6±4 36 + 17 23 +3 11 +3 2 + 1 6+6 9+7 8+3 1 +2

+ EPO -/- 78+23* 5+3 23+3 22 + 10 10+3 2+2 5 + 1 8+2 3±2 1 + 1 spleen +/+ 98+6 8+3 27 + 1 22+4 17+2 2+0 10 + 1 7±5 7 + 1 0.7+0.6

+/- 66 + 12* 3+3 18 + 3 17+6 16+5 1 +2 4+3 3 + 1 3+5 0.7+0.6

-/- 42+9* 4+3 12+4 9+3 6+2 0.3 +0.6 4 + 1 3 + 1 4+3 0.5 +0.6 liver +/ + 63+9 6+2 18+5 14+5 16±9 0 3 + 1 3+4 2 + 1 0.7+0.6

+/- 74+ 13 10±2 18 +3 15+7 18+3 0.7+0.6 7±5 2 + 1 3 +2 0.7+0.6

-/- 62 + 13 5 +2 19+8 15+2 10+4 0 6+3 5 +4 2 + 1 0.5+0.6

SCF BM +/ + 58 + 8 6+ 1 39+9 5 +3 6+2 0 0 2+2 0 0.3 +0.6

+/- 47 + 16 5±4 32 + 8 2 + 1 8+6 0 0 0.3 +0.6 0 0

-/- 35 + 16 4+3 23 + 10 3 + 1 4+3 0 0 0.3+0.6 0 0 spleen +/+ 37+5 5+2 19+2 3 +2 10+4 0 0 0 0 0

+/- 29+3t 3 + 1 17 + 1 2+2 6+4 0 0 0 0 0

-/- 16+9* 3 +3 11 +6 0.8 + 1 1 + 1 0 0 0 0 0 liver +/ + 36 + 14 4+2 19 + 10 3 + 1 9±3 0 0 0 0 1 + 1

+/- 40 + 12 5 + 1 24+6 2±0 9+5 0 0 0 0 0

-/- 17±5 2+3 12+4 0.8+0.5 2+ 1 0 0 0 0 0

M-CSF BM +/ + 58±6 0 2 + 1 10+5 43 + 1 0 0 0 0 0

+/- 54+ 11 0 2+2 6+3 46 + 11 0 0 0 0 0

-/- 28 + 14* 0 2+2 5+2 22 + 10 0 0 0 0 0

Stimulus Organ NR6 Number of colonies per 2xl0⁴ cells genotype

Total Blast G GM M Eo E Meg E/Meg Mix spleen +/ + 35 + 15 0 1 + 1 6±4 26+7 0 0 0 0 0 +/- 26+5 0 1 +2 2+3 23 +6 0 0 0 0 0 -/- 14+6f 0 0.8 + 1 1 + 1 12+5 0 0 0 0 0 liver +/+ 40+ 15 0 0.7+0.6 5 +3 33 + 10 0 0 0 0 0 +/- 45 + 10 0 3 + 1 6+3 36+7 0 0 0 0 0 -/- 30+5 0 l ± l 3 + 1 27+4 0 0 0 0 0

Mean + standard deviations of colony numbers from neonatal NR6^+/+ NR6^{+ "} or NR6 ' bone marrow, spleen or liver. Replicate cultures fro each mouse were stimulated with IL-3 +SCF EPO, SCF alone or M-CSF and incubated for seven days in a humidified atmosphere of 5% C in air. Statistical comparisons were performed using Student's test on total colony data from NR6^{+ "} or NR6^+/+ mice verus NR6^+/+. * p <0.0 f p<0.08. n - 3 or 4 mice of each genotype. BM, bone marrow; G, granulocyte; GM, granulocyte-macrophage; M, macrophage; Eo, eosinoph E, erythroid; Meg, megakaryocyte; E/Meg, mixed erythroid/megakaryocyte; Mix, colonies containing cells of three or more linages; 0 - n detected.

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Claims

1. A method of modulating production of haemopoietic progenitor cells in a mammal, said method comprising administering to said mammal, a modulating effective amount of NR6 or an isoform, derivative, splice variant, agonist antagonist, homologue, chemical analogue or mimetic thereof or administering an expression-modulating effective amount of an agent capable of modulating expression of a gene encoding NR6 or its derivatives or homologues.

2. A method of facilitating production of haemopoietic progenitor cells in a mammal, said mammal comprising administering to said mammal a production-facilitating effective amount of NR6 or an isoform, derivative, splice variant, agonist, homologue, chemical analogue or mimetic thereof or administering to said mammal an NR6-encoding nucleotide sequence-expression facilitating effective amount of an agent which induces or otherwise facilitates expression of a genetic sequence encoding NR6 for a time and under conditions sufficient to induce, increase or otherwise facilitate production of haemopietic progenitor cells.

3. A method of enhancing, promoting or otherwise facilitating post-natal survival of a mammalian offspring, said method comprising administering to said post-natal offspring an effective amount of NR6 or an isoform, derivative, splice variant, agonist, antagonist, homologue, chemical analogue or mimetic thereof or administering an agent capable of inducing or enhancing expression of NR6 genetic sequences to thereby increase levels of NR6 in vivo.

4. A method of facilitating production of haemopoietic stem cells in a mammal, said method comprising contacting stem cells in said mammal or cells intermediate of stem and committed lineage progenitor cells with an effective amount of NR6 or an isoform, derivative, splice variant, agonist, antagonist, homologue, chemical analogue or mimetic thereof or facilitating an effective amount of expression of NR6 genetic sequences in cells of said mammal, said effective amounts providing sufficient NR6 to facilitate production of myeloid cells from stem cells.

5. A method according to Claim 1 or 2 or 3 or 4 wherein the NR6 comprises an amino acid sequence selected from SEQ ID NO: 7, 9 and 11 or an amino acid sequence having at least about 60% similarity to one or more of SEQ ID NO: 7 or 9 or 11.

6. A method according to Claim 1 or 2 or 3 or 4 wherein the NR6 is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:6 or 8 or 10 or a nucleotide sequence having at least about 60% similarity to one or more of SEQ ID NO: 6 or 8 or 10 or a nucleotide sequence capable of hybridizing to one or more of SEQ ID NO: 6 or 8 or 10 under low stringency conditions at 42 °C.

7. A composition comprising NR6 or an isoform, derivative, splice variant, agonist, antagonist, homologue, chemical analogue or mimetic thereof and one or more pharmaceutically acceptable carriers and/or divalents.

8. A composition according to Claim 7 for use in one or more of modulating production of haemopoietic progenitor cells, facilitating production of haemopoietic progenitor cells and enhancing, promoting or otherwise facilitating post-natal survival.

9. A composition according to Claim 7 or 8 wherein the NR6 comprises an amino acid sequence selected from SEQ ID NO:7, 9 and 11 or an amino acid sequence having at least about 60% similarity to one or more of SEQ ID NO: 7 or 9 or 11.

10. A composition according to Claim 7 or 8 wherein the NR6 is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 6 or 8 or 10 or a nucleotide sequence having at least about 60% similarity to one or more of SEQ ID NO: 6 or 8 or 10 or a nucleotide sequence capable of hybridizing to one or more of SEQ ID NO: 6 or 8 or 10 under low stringency conditions at 42 °C. - SO -

11. An agent for use in one or more of modulating production of haemopoietic progenitor cells, facilitating production of haemopoietic progenitor cells and enhancing, promoting or otherwise facilitating post-natal survival comprising NR6 or an isoform, derivative, splice variant, agonist, homologue, chemical analogue or mimetic thereof.

12. Use of NR6 or an isoform, derivative, splice variant, agonist, antagonist, homologue, chemical analogue or mimetic thereof in the manufacture of a medicament for use in one or more of modulating production of haemopoietic progenitor cells facilitating production of haemopoietic progenitor cells, enhancing, promoting or otherwise facilitating postnatal survival.

13. A genetically modified embyonic stem cell which carries a defective gene encoding NR6.

14. A genetically modified mammal comprising an NR6-/- genotype and/or the phenotype of an NR6-/- genotype wherein said phenotype comprises reduced levels of haemopoietic progenitor cells compared to a corresponding NR6+/+ mammal.