WO2013132272A1 - Erythropoiesis - Google Patents

Abstract

The invention provides means for treating conditions characterised by inappropriate erythropoiesis. The invention also extends to the treatment of conditions involving inappropriate myeloid leukopoiesis. The invention also extends to pharmaceutical compositions for use in treating such conditions, and to methods of treatment.

Description

Erythropoiesis

The present invention relates to erythropoiesis, and particularly, although not exclusively, to the treatment of conditions characterised by inappropriate

erythropoiesis. The invention also extends to the treatment of conditions involving inappropriate myeloid leukopoiesis. The invention also extends to pharmaceutical compositions for use in treating such conditions, and methods of treatment.

The Hedgehog (Hh) family of secreted inter-cellular signalling proteins is essential for the development of many tissues during embryogenesis, and they are also involved in the homeostasis of adult tissues, including skin, gut, bone and thymus. Their role in the regulation of hematopoiesis, however, has proved controversial, with different experimental models supporting opposing interpretations. There are three mammalian Hh proteins, with distinct patterns of expression and functions, namely Sonic Hh (Shh), Indian Hh (Ihh) and Desert Hh (Dhh). Shh and Ihh are each essential for mouse development, whereas Dhh mutant mice are healthy and appear normal, although males are infertile. Shh is the most pleiotrophic of the family members and is essential for development of many tissues and organs, including brain, heart, lungs, limbs and thymus. Ihh has some overlapping functions with Shh, and is essential for bone differentiation and the regulation of thymocyte

differentiation. In contrast, the functions of Dhh seem to be more limited to testis development and Schwann-cell function, hence the viability and lack of obvious phenotype of Dhh-deficient animals. The Hh proteins all share a common signalling pathway, which is initiated when the Hh ligand binds to its cell-surface receptor Patched (Ptch). This releases Ptch's inhibition of the signalling molecule,

Smoothened (Smo), allowing transduction of the Hh signal into the cell, and activation of the Hh-responsive transcription factors, Glii, Gli2 and GI13.

The Hh signalling pathway is regulated by multiple positive and negative feed-backs, and both Ptch and Glii are themselves Hh target genes. Glii is not essential for mouse development or Hh signalling, and as it is itself an Hh target gene, measurement of its transcription can be used as a read-out of Hh signalling in a given population of cells. Studies on the role of the Hh signalling pathway in hematopoiesis have lead to conflicting results. In zebrafish, mutants of the Hh pathway have defects in hematopoietic stem cell (HSC) formation and definitive hematopoiesis. In vitro studies also support the idea that Hh signalling is important for HSC proliferation and hematopoiesis. Likewise, analysis of Ptch+/- mice, in which Hh signalling is increased, showed that Hh pathway activation caused cycling and expansion of bone marrow (BM) hematopoietic cells, leading to HSC exhaustion. In one report, conditional deletion of Smo, the non-redundant Hh pathway signal transduction molecule, suggested that HSC require Hh signals for their homeostasis. This was in contrast to two studies which showed that conditional deletion of Smo had no impact on HSC function or hematopoiesis.

Analysis of mutants in Shh, Ihh, Gli2, GI13 and Smo have shown that Hh signalling seems to be involved in T-cell development. Analysis of Glii-null mice has indicated that Glii also plays a role in HSC homeostasis, myeloid differentiation, stress-induced hematopoiesis and thymocyte development. Ihh has been shown to be involved in the induction of haemaotpoietic cell fate and terminal erythroid differentiation in the embryo. In addition, in a recent report, Ptch mutation or conditional deletion of Smo have shown that Hh signalling is involved for BMP4-dependent stress-induced erythropoiesis in the mouse spleen. Thus, the role of Hh signalling in the regulation of hematopoiesis and erythropoiesis is controversial and requires further

investigation.

Erythrocytes, like other blood lineages, develop from HSC, through a sequence of well-defined intermediates. In mouse, HSCs have been defined by their absence of lineage-specific markers, and expression of Sca-i and cKit, and are therefore referred to as Lin-Sca-i⁺ckit⁺ (LSK) stem cells. LSK give rise to a number of progenitor cells, including common myeloid progenitor cells (CMP), which in turn give rise to granulocyte/macrophage progenitor cells (GMP) and megakaryocyte/erythroid progenitor cells (MEP). The MEP then give rise to the erythroid lineage, first by differentiating into burst forming unit cells (BFU-E), the first erythroid committed cells, and then colony forming units (CFU-E). BFU-E and CFU-E cannot be identified by cell surface markers, and so are quantified by their ability to produce colonies in functional assays in vitro. These cells then develop through a series of erythroblast stages, which are defined by surface expression of CD71 and Terii j. Teni9^medCD7i^M (population I, proerythroblast) precursors differentiate to Τεπΐ9^Μϋθ7ΐ^Μ (population II, basophilic erythroblast), which then down-regulate CD71 to become

Terii9^hiCD7i^med (population III, polychromatophilic erythroblast) and then

Terii9^hiCD7i- (population IV, orthochromatic erythroblast) cells. At the later erythroblast stages, the nucleus progressively shrinks and is shed before the cells become mature erythrocytes. The developmental program from HSC to mature erythrocyte is regulated by complex transcriptional networks and by environmental signals. In the adult, most erythropoiesis occurs in the BM, but under conditions of erythropoietic stress (anaemia, hypoxia), the number of erythrocytes is increased, and this process of stress-induced erythropoiesis occurs predominantly in the spleen.

The inventors set out to investigate erythropoiesis in Dhh-null adult mice, and have demonstrated that Dhh surprisingly functions as a negative regulator of normal and stress-induced erythropoiesis, at multiple stages of differentiation, in both the spleen and BM. Given that the three Hh proteins all share a common signalling pathway, the inventors believe that their observations are not limited to Dhh, and can also apply to Shh and Ihh.

Thus, according to a first aspect of the invention, there is provided a modulator of Hedgehog (Hh) signalling, for use in the treatment, amelioration or prevention of a disease characterised by inappropriate erythropoiesis.

In a second aspect, there is provided a method of treating, ameliorating or preventing a disease characterised by inappropriate erythropoiesis in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a modulator of Hedgehog (Hh) signalling.

The function of Hedgehog signalling in hematopoiesis is controversial, with different experimental systems giving opposing results. As described in the Examples, the inventors examined the role of Dhh in the regulation of murine erythropoiesis, and surprisingly show, by analysis of Dhh-deficient mice, that Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the Common Myeloid Progenitor (CMP) population was increased, but differentiation from CMP to Granulocyte/Macrophage progenitor (GMP) was decreased, and the mature granulocyte population was decreased, compared to wild type (WT). In contrast, differentiation from CMP to Megakaryocyte/erythrocyte Progenitor (MEP) was surprisingly increased, and the MEP population was increased. In addition, the inventors have found that erythroblast populations were Dhh-responsive in vitro and ex vivo, and that Dhh negatively regulated erythroblast differentiation. Furthermore, in Dhh-deficient spleen and bone marrow, BFU-Es and later erythroblast populations were increased compared to WT. Finally, the inventors have demonstrated that, during recovery of haematopoiesis following irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared to WT.

These observations are in contrast to what was observed in the prior art, which proposed that Hh renders progenitors BMP4-responsive, leading to the generation of more BFU-Es in the spleen under stress conditions when BMP4 is produced.

Accordingly, the inventors have clearly demonstrated that administration, to a patient, of a modulator of Hedgehog (Hh) signalling, can be used to treat, ameliorate or prevent any disease, which is characterised by inappropriate erythropoiesis.

There are currently three known mammalian Hedgehog (Hh) family proteins, i.e. Sonic Hh (Shh), Indian Hh (Ihh) and Dhh. Accordingly, the modulator maybe capable of modulating Shh, Ihh and/or Dhh signalling. Preferably, however, the modulator is capable of modulating Dhh signalling.

The cDNA sequence (1389 nucleotides) of human Sonic Hh (Shh), having Transcript ID: CCDS5942.1, is provided herein as SEQ ID No:i, as follows.

ATGCTGCTGCTGGCGAGATGTCTGCTGCTAGTCCTCGTCTCCTCGCTGCTGGTATGCTCGGGACTGGCG TGCGGACCGGGCAGGGGGTTCGGGAAGAGGAGGCACCCCAAAAAGCTGACCCCTTTAGCCTACAAGCAG TTTATCCCCAATGTGGCCGAGAAGACCCTAGGCGCCAGCGGAAGGTATGAAGGGAAGATCTCCAGAAAC TCCGAGCGATTTAAGGAACTCACCCCCAATTACAACCCCGACATCATATTTAAGGATGAAGAAAACACC GGAGCGGACAGGCTGATGACTCAGAGGTGTAAGGACAAGTTGAACGCTTTGGCCATCTCGGTGATGAAC CAGTGGCCAGGAGTGAAACTGCGGGTGACCGAGGGCTGGGACGAAGATGGCCACCACTCAGAGGAGTCT CTGCACTACGAGGGCCGCGCAGTGGACATCACCACGTCTGACCGCGACCGCAGCAAGTACGGCATGCTG GCCCGCCTGGCGGTGGAGGCCGGCTTCGACTGGGTGTACTACGAGTCCAAGGCACATATCCACTGCTCG GTGAAAGCAGAGAACTCGGTGGCGGCCAAATCGGGAGGCTGCTTCCCGGGCTCGGCCACGGTGCACCTG GAGCAGGGCGGCACCAAGCTGGTGAAGGACCTGAGCCCCGGGGACCGCGTGCTGGCGGCGGACGACCAG GGCCGGCTGCTCTACAGCGACTTCCTCACTTTCCTGGACCGCGACGACGGCGCCAAGAAGGTCTTCTAC GTGATCGAGACGCGGGAGCCGCGCGAGCGCCTGCTGCTCACCGCCGCGCACCTGCTCTTTGTGGCGCCG CACAACGACTCGGCCACCGGGGAGCCCGAGGCGTCCTCGGGCTCGGGGCCGCCTTCCGGGGGCGCACTG GGGCCTCGGGCGCTGTTCGCCAGCCGCGTGCGCCCGGGCCAGCGCGTGTACGTGGTGGCCGAGCGTGAC GGGGACCGCCGGCTCCTGCCCGCCGCTGTGCACAGCGTGACCCTAAGCGAGGAGGCCGCGGGCGCCTAC GCGCCGCTCACGGCCCAGGGCACCATTCTCATCAACCGGGTGCTGGCCTCGTGCTACGCGGTCATCGAG GAGCACAGCTGGGCGCACCGGGCCTTCGCGCCCTTCCGCCTGGCGCACGCGCTCCTGGCTGCACTGGCG CCCGCGCGCACGGACCGCGGCGGGGACAGCGGCGGCGGGGACCGCGGGGGCGGCGGCGGCAGAGTAGCC CTAACCGCTCCAGGTGCTGCCGACGCTCCGGGTGCGGGGGCCACCGCGGGCATCCACTGGTACTCGCAG CTGCTCTACCAAATAGGCACCTGGCTCCTGGACAGCGAGGCCCTGCACCCGCTGGGCATGGCGGTCAAG TCCAGCTGA

[SEQ ID No:l]

The protein sequence (462 amino acids) of human Sonic Hh (Shh), having

ENST00000297261, is provided herein as SEQ ID No:2, as follows.

MLLLARCLLLVLVSSLLVCSGLACGPGRGFGKRRHPKKLTPLAYKQF IPNVAEKTLGASGRYEGKI SRN SERFKELTPNYNPDI IFKDEENTGADRLMTQRCKDKLNALAI SVMNQWPGVKLRVTEGWDEDGHHSEES LHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESKAHIHCSVKAENSVAAKSGGCFPGSATVHL

EQGGTKLVKDLSPGDRVLAADDQGRLLYSDFLTFLDRDDGAKKVFYVIETREPRERLLLTAAHLLFVAP HNDSATGEPEASSGSGPPSGGALGPRALFASRVRPGQRVYVVAERDGDRRLLPAAVHSVTLSEEAAGAY APLTAQGTILINRVLASCYAVIEEHSWAHRAFAPFRLAHALLAALAPARTDRGGDSGGGDRGGGGGRVA LTAPGAADAPGAGATAGIHWYSQLLYQIGTWLLDSEALHPLGMAVKSS

[SEQ ID No: 2]

The cDNA sequence (1236 nucleotides) of human Indian Hh(Ihh), having Transcript ID: CCDS33380.1, is provided herein as SEQ ID No:3, as follows.

ATGTCTCCCGCCCGGCTCCGGCCCCGACTGCACTTCTGCCTGGTCCTGTTGCTGCTGCTGGTGGTGCCG GCGGCATGGGGCTGCGGGCCGGGTCGGGTGGTGGGCAGCCGCCGGCGACCGCCACGCAAACTCGTGCCG CTCGCCTACAAGCAGTTCAGCCCCAATGTGCCCGAGAAGACCCTGGGCGCCAGCGGACGCTATGAAGGC AAGATCGCTCGCAGCTCCGAGCGCTTCAAGGAGCTCACCCCCAATTACAATCCAGACATCATCTTCAAG GACGAGGAGAACACAGGCGCCGACCGCCTCATGACCCAGCGCTGCAAGGACCGCCTGAACTCGCTGGCT ATCTCGGTGATGAACCAGTGGCCCGGTGTGAAGCTGCGGGTGACCGAGGGCTGGGACGAGGACGGCCAC CACTCAGAGGAGTCCCTGCATTATGAGGGCCGCGCGGTGGACATCACCACATCAGACCGCGACCGCAAT AAGTATGGACTGCTGGCGCGCTTGGCAGTGGAGGCCGGCTTTGACTGGGTGTATTACGAGTCAAAGGCC CACGTGCATTGCTCCGTCAAGTCCGAGCACTCGGCCGCAGCCAAGACGGGCGGCTGCTTCCCTGCCGGA GCCCAGGTACGCCTGGAGAGTGGGGCGCGTGTGGCCTTGTCAGCCGTGAGGCCGGGAGACCGTGTGCTG GCCATGGGGGAGGATGGGAGCCCCACCTTCAGCGATGTGCTCATTTTCCTGGACCGCGAGCCTCACAGG CTGAGAGCCTTCCAGGTCATCGAGACTCAGGACCCCCCACGCCGCCTGGCACTCACACCCGCTCACCTG CTCTTTACGGCTGACAATCACACGGAGCCGGCAGCCCGCTTCCGGGCCACATTTGCCAGCCACGTGCAG CCTGGCCAGTACGTGCTGGTGGCTGGGGTGCCAGGCCTGCAGCCTGCCCGCGTGGCAGCTGTCTCTACA CACGTGGCCCTCGGGGCCTACGCCCCGCTCACAAAGCATGGGACACTGGTGGTGGAGGATGTGGTGGCA TCCTGCTTCGCGGCCGTGGCTGACCACCACCTGGCTCAGTTGGCCTTCTGGCCCCTGAGACTCTTTCAC AGCTTGGCATGGGGCAGCTGGACTCCGGGGGAGGGTGTGCATTGGTACCCCCAGCTGCTCTACCGCCTG GGGCGTCTCCTGCTAGAAGAGGGCAGCTTCCACCCACTGGGCATGTCCGGGGCAGGGAGCTGA

[SEQ ID No: 3]

The protein sequence (411 amino acids) of human Indian Hh (Ihh), having

ENST00000295731, is provided herein as SEQ ID No:4, as follows. MSPARLRPRLHFCLVLLLLLVVPAAWGCGPGRVVGSRRRPPRKLVPLAYKQFSPNVPEKTLGASGRYEG KIARSSERFKELTPNYNPDI IFKDEENTGADRLMTQRCKDRLNSLAI SVMNQWPGVKLRVTEGWDEDGH HSEESLHYEGRAVDITTSDRDRNKYGLLARLAVEAGFDWVYYESKAHVHCSVKSEHSAAAKTGGCFPAG AQVRLESGARVALSAVRPGDRVLAMGEDGSPTFSDVLIFLDREPHRLRAFQVIETQDPPRRLALTPAHL LFTADNHTEPAARFRATFASHVQPGQYVLVAGVPGLQPARVAAVSTHVALGAYAPLTKHGTLVVEDVVA SCFAAVADHHLAQLAFWPLRLFHSLAWGSWTPGEGVHWYPQLLYRLGRLLLEEGSFHPLGMSGAGS

[SEQ ID No:4]

The cDNA sequence (1191 nucleotides) of human Desert Hh (Dhh), having Transcript ID: CCDS8779.1, is provided herein as SEQ ID No:5, as follows.

ATGGCTCTCCTGACCAATCTACTGCCCCTGTGCTGCTTGGCACTTCTGGCGCTGCCAGCCCAGAGCTGC GGGCCGGGCCGGGGGCCGGTTGGCCGGCGCCGCTATGCGCGCAAGCAGCTCGTGCCGCTACTCTACAAG CAATTTGTGCCCGGCGTGCCAGAGCGGACCCTGGGCGCCAGTGGGCCAGCGGAGGGGAGGGTGGCAAGG GGCTCCGAGCGCTTCCGGGACCTCGTGCCCAACTACAACCCCGACATCATCTTCAAGGATGAGGAGAAC AGTGGAGCCGACCGCCTGATGACCGAGCGTTGTAAGGAGCGGGTGAACGCTTTGGCCATTGCCGTGATG AACATGTGGCCCGGAGTGCGCCTACGAGTGACTGAGGGCTGGGACGAGGACGGCCACCACGCTCAGGAT TCACTCCACTACGAAGGCCGTGCTTTGGACATCACTACGTCTGACCGCGACCGCAACAAGTATGGGTTG CTGGCGCGCCTCGCAGTGGAAGCCGGCTTCGACTGGGTCTACTACGAGTCC CGCAACCACGTCCACGTG TCGGTCAAAGCTGATAACTCACTGGCGGTCCGGGCGGGCGGCTGCTTTCCGGGAAATGCAACTGTGCGC CTGTGGAGCGGCGAGCGGAAAGGGCTGCGGGAACTGCACCGCGGAGACTGGGTTTTGGCGGCCGATGCG TCAGGCCGGGTGGTGCCCACGCCGGTGCTGCTCTTCCTGGACCGGGACTTGCAGCGCCGGGCTTCATTT GTGGCTGTGGAGACCGAGTGGCCTCCACGCAAACTGTTGCTCACGCCCTGGCACCTGGTGTTTGCCGCT CGAGGGCCGGCGCCCGCGCCAGGCGACTTTGCACCGGTGTTCGCGCGCCGGCTACGCGCTGGGGACTCG GTGCTGGCGCCCGGCGGGGATGCGCTTCGGCCAGCGCGCGTGGCCCGTGTGGCGCGGGAGGAAGCCGTG GGCGTGTTCGCGCCGCTCACCGCGCACGGGACGCTGCTGGTGAACGATGTC CTGGCCTCTTGCTACGCG GTTCTGGAGAGTCACCAGTGGGCGCACCGCGCTTTTGCCCCCTTGAGACTGCTGCACGCGCTAGGGGCG CTGCTCCCCGGCGGGGCCGTCCAGCCGACTGGCATGCATTGGTACTCTCGGCTCCTCTACCGCTTAGCG GAGGAGCTACTGGGCTGA

[SEQ ID No:5] The protein sequence (396 amino acids) of human Desert Hh (Dhh), having

ENST00000266991, is provided herein as SEQ ID No:6, as follows.

MALLTNLLPLCCLALLALPAQS CGPGRGPVGRRRYARKQLVPLLYKQFVPGVPERTLGAS GPAE GRVAR GSERFRDLVPNYNPD I I FKDEENS GADRLMTERCKERVNALAIAVMNMWPGVRLRVTE GWDE DGHHAQD SLHYE GRALD I TT S DRDRNKYGLLARLAVEAGF DWVYYE SRNHVHVSVKADNS LAVRAGGCFPGNATVR LWS GERKGLRE LHRGDWVLAADAS GRVVPTPVLLF LDRDLQRRASFVAVE TEWPPRKLLLTPWHLVFAA RGPAPAPGDFAPVFARRLRAGD SVLAPGGDALRPARVARVAREEAVGVFAP LTAHGTLLVNDVLAS CYA VLE SHQWAHRAFAPLRLLHALGALLPGGAVQPTGMHWYSRLLYRLAEE LLG

[SEQ ID No: 6]

Underlined nucleotides and amino acids denote alternate exons, and bold residue indicates amino acid encoded across a splice junction.

As described herein, the inventors have shown experimentally that Dhh surprisingly negatively regulates erythropoiesis, and that erythropoiesis is accelerated in Dhh-/- mice. It follows, therefore, that this work is a strong indication that modulators which can trigger Hh signalling (e.g. Hh protein per se) may be used to reduce erythropoiesis, and, conversely, that modulators which can inhibit Hh signalling (e.g. Hh inhibitors and anti-Hh antibodies) may be used to stimulate erythropoiesis. As illustrated in figure 4b, the inventors have shown that Dhh surprisingly influences lineage choice of CMP, favouring differentiation to GMP, and inhibiting

differentiation to MEP, and this information is very useful in tailoring therapies for cancers of myeloid and erythroid lineages, and to the treatment of anaemia in these diseases.

Erythropoietin (EPO) is mostly produced by the kidneys, in response to hypoxia. It is therefore important in the treatment of the anaemia that results from kidney failure (i.e. conditions of low EPO concentrations), and it can also be used to boost erythropoiesis in 'normal' situations (e.g. to increase stamina in an athlete), post- surgery, and post-chemotherapy etc. However, if there is anaemia, but kidney function and EPO levels are already normal, then, for some reason, EPO is not working, and therefore increasing EPO to above physiological levels may have little effect. It is under such circumstances that other ways of increasing erythropoiesis (e.g. by inhibiting Dhh signalling) would have great utility. Examples of this include anaemia as a result of bone marrow failure, anaemia of chronic inflammation and some blood cancers. Also, many medical organisations (including the military) are interested in being able to produce artificial blood, and also to be able to differentiate erythrocytes quicker in vitro and in vivo.

Figure 4b shows that Dhh influences the lineage choice between GMP and MEP, as well as inhibiting differentiation along the erythroid lineage. Inhibiting Dhh or rDhh may therefore be used to treat pre-cancerous syndromes, such as myelodysplastic syndrome, in which erythropoiesis and myeloid differentiation are dysregulated. The choice of treatment would depend on the nature of the dysregulation. In addition, malaria is another condition which is characterised by depleted erythrocytes, and so could also be treated by a compound which reduces Hh signalling.

Thus, in one embodiment, the modulator maybe a negative modulator of Hedgehog (Hh) signalling (for example an antagonist), which is capable of increasing erythropoiesis. The negative modulator may be capable of:-

(i) altering the conformational state of the receptor or signal transduction molecule through which Hh signalling is achieved, for example by

destabilizing the active conformation of that receptor and/or maintaining the receptor in its inactive conformation to thereby prevent it from binding its natural ligand;

(ii) binding to the receptor through which Hh signalling is achieved, and preventing, decreasing or attenuating transmission at that receptor;

(iii) down-regulating or de-activating the downstream signalling pathways activated by the modulator binding to the receptor through which Hh signalling is achieved, for example by inhibiting or blocking Smo activity or Gli activity;

(iv) decreasing, preventing or attenuating transcription, translation or expression of the signal transduction molecule Smo;

(v) inhibiting synthesis or release, from intracellular stores, of the signal transduction molecule Smo; and/or (vi) increasing the rate of degradation of Smo; and/or

(vii) increasing transcription, translation or expression of the receptor Ptch through which Hh signalling is achieved.

It will be appreciated that each of mechanisms (i) to (vii) results in altering transmission at the receptor/signal transduction molecule through which Hh signalling is directed, and the activity thereof, to thereby negatively modulate the Hh signalling. The receptor through which Hh signalling is achieved may be the cell- surface receptor Patched (Ptch), which inhibits activity of the Hh-signal transduction molecule Smoothened (Smo). When Hh binds Ptch, the inhibition of Smo is relieved, and Smo signals into the cell.

In this embodiment, the modulator may comprise an anti-Hh antibody or an Hh inhibitor, which is capable of altering receptor/signal transduction molecule conformation/stability, or blocking the receptor's activity. The modulator may comprise an anti-Shh, anti-Ihh or anti-Dhh antibody, or a Shh, Ihh or Dhh inhibitor. It will be appreciated that anti-Hh antibodies and suitable Hh inhibitors are well- known to the skilled person. For example, suitable anti-Hh antibodies are

commercially available (e.g. anti-Shh from R&D systems, Catalog number:MAB464; anti-Ihh from R&D systems, Catalog number:MABi705; anti-Dhh from Creative Biomart, Catalog number: 14753MH), and anti-Shh and anti-Ihh antibodies are described in Ericson, J. et a!.. (1996), Cell 87, 661-673. Examples of suitable Hh inhibitors include cyclopamine (Chen et al, Genes and Development 16, 2743; 2002); and SMO antagonist BMS 833923.

Preferably, the modulator comprises an anti-Dhh antibody or a Dhh inhibitor.

Such negative modulators may be suitable for use in treating any condition where it is desired to increase or promote erythropoiesis, for example malaria, anaemia, or blood. The anaemic condition which may be treated may be a result of bone marrow failure, or of chronic inflammation. Anaemia may also be the result of chemotherapy or radiotherapy, or following blood loss, particularly in groups of patients who cannot receive blood transfusions, such as some religious groups (e.g. Jehovas witnesses). One example of a blood disorder which can be treated includes myelodysplastic syndrome. The blood disorder, therefore, may be cancer of the blood. The inventors have found that the invention enables the modulation of

erythropoiesis, whereas EPO can only be used to stimulate erythropoiesis. The ability to reduce erythropoiesis would also be important, for example in the treatment of polycythemia, which is a disorder in which too many erythrocytes are produced. As this condition is currently treated by bleeding patients, it would be very useful to have a drug that could be used to specifically reduce erythrocyte production. Furthermore, acute leukaemias of erythroid lineage are rare, but very aggressive with a poor prognosis, and such conditions may also be treated with compounds which increase Hh signalling.

Hence, in another embodiment, the modulator may be a positive modulator of Hedgehog (Hh) signalling (for example an agonist), which is capable of decreasing erythropoiesis. The positive modulator may be capable of:-

(i) altering the conformational state of the receptor or signal transduction

molecule through which Hh signalling is achieved, for example by stabilizing the active conformation of that receptor and/ or maintaining the receptor in its active conformation to thereby increase its binding to its natural ligand;

(ii) binding to the receptor through which Hh signalling is achieved, and

increasing, promoting or augmenting transmission at that receptor;

(iii) promoting or activating the downstream signalling pathways activated by the modulator binding to the receptor through which Hh signalling is achieved, for example by increasing Smo signal transduction or Gli activity;

(iv) increasing, promoting or augmenting transcription, translation or

expression of the signal transducer Smo through which Hh signalling is achieved;

(v) increasing synthesis or release, from intracellular stores, of the signal

transducer Smo through which Hh signalling is achieved, or agonists thereof;

(vi) decreasing the rate of degradation of the signal transducer Smo through which Hh signalling is achieved, or agonists thereof; and/or

(vii) decreasing, inhibiting or preventing transcription, translation or expression of the receptor Ptch through which Hh signalling is achieved.

It will be appreciated that each of mechanisms (i) to (vii) results in altering transmission at the receptor/signal transduction complex through which Hh signalling is directed, and the activity thereof, to thereby positively modulate the Hh signalling.

In this embodiment, the modulator may comprise Shh, Ihh or Dhh, or a functional variant or fragment thereof. The modulator may comprise a protein comprising an amino acid sequence substantially as set out in SEQ ID No: 2, 4 or 6, or a functional variant or fragment thereof. The protein may be recombinant, i.e. produced using recombinant DNA technology, known to the skilled person. The protein may be encoded by a nucleic acid sequence substantially as set out in SEQ ID No: 1, 3 or 5, or a functional variant or fragment thereof.

Preferably, however, the modulator comprises Dhh, which may comprise an amino acid sequence substantially as set out in SEQ ID No: 6, or a functional variant or fragment thereof. The protein may be encoded by a nucleic acid sequence

substantially as set out in SEQ ID No: 5, or a functional variant or fragment thereof.

Such positive modulators may be suitable for use in treating any condition where it is desired to reduce or prevent erythropoiesis, for example polycythemia or leukaemia The leukaemia may be acute, for example leukaemia of erythroid lineage.

It will be appreciated that a disease which is characterised by inappropriate erythropoiesis may also be characterised by inappropriate myeloid leukopoiesis, i.e. production of (non-lymphoid) white blood cells. Furthermore, as illustrated in Figure 4b, the inventors have shown that Dhh surprisingly influences lineage choice of CMP, favouring differentiation to GMP, and inhibiting differentiation to MEP. Therefore, in some embodiments, the modulator of Hh signalling may be used to treat a condition characterised by inappropriate myeloid leukopoiesis. For example, the modulator maybe used to treat a condition in which a subject suffers from too much or too little myeloid leukopoiesis. For example, in acute, and chronic myeloid leukaemia, acute promyelocytic leukaemia, in myodysplastic syndrome, and following chemotherapy, radiotherapy and bone marrow transplant.

It will be appreciated that modulators according to the invention may be used in a medicament, which maybe used in a monotherapy, i.e. use of only a positive modulator of Hedgehog signalling, which decreases erythropoiesis, for treating, ameliorating, or preventing a disease condition characterised by excessive erythropoiesis, or the use of only a negative modulator of Hedgehog signalling, which increases erythropoiesis, for treating, ameliorating, or preventing a disease condition characterised by insufficient erythropoiesis. Alternatively, modulators according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing diseases characterised by inappropriate erythropoiesis. For example, modulators of the invention may be used in

combination with known agents for treating anaemia, such as EPO. Similarly, modulators of the invention may be used in combination with known techniques for treating leukaemia, such as radiotherapy.

The modulators according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition maybe in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given. Medicaments comprising modulators according to the invention may be used in a number of ways. For instance, oral administration may be required, in which case the modulators maybe contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising modulators of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin, for example, adjacent the treatment site.

Modulators according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with modulators used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection). In a preferred embodiment, modulators and compositions according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

It will be appreciated that the amount of the modulators that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the modulator and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the modulators within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular modulators in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease being treated. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Generally, a daily dose of between o.o^g/kg of body weight and o.5g/kg of body weight of the modulators according to the invention may be used for treating, ameliorating, or preventing the disease characterised by inappropriate

erythropoiesis, depending upon which modulators is used. More preferably, the daily dose of modulator is between o.oimg/kg of body weight and 500mg/kg of body weight, more preferably between o.img/kg and 200mg/kg body weight, and most preferably between approximately lmg/kg and loomg/kg body weight.

The modulators may be administered before, during or after onset of the disease characterised by inappropriate erythropoiesis. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the modulators may require administration twice or more times during a day. As an example, modulators may be administered as two (or more depending upon the severity of the disease being treated) daily doses of between 25mg and 7000 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device maybe used to provide optimal doses of modulators according to the invention to a patient without the need to administer repeated doses. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations comprising the modulators according to the invention and precise therapeutic regimes (such as daily doses of the modulators and the frequency of administration). The inventors believe that they are the first to describe a composition for treating diseases characterised by inappropriate erythropoiesis, based on the use of the modulators of the invention. Hence, in a third aspect of the invention, there is provided an erythropoiesis- treatment composition, comprising a modulator of Hedgehog (Hh) signalling and a pharmaceutically acceptable vehicle.

The term "erythropoiesis-treatment composition" can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of any disease condition characterised by inappropriate (i.e. too much or too little) erythropoiesis in a subject. Similarly, the composition can also be used to treat a condition characterised by inappropriate myeloid leukopoiesis. The invention also provides in a fourth aspect, a process for making the

erythropoiesis-treatment composition according to the third aspect, the process comprising contacting a therapeutically effective amount of a modulator of Hedgehog (Hh) signalling and a pharmaceutically acceptable vehicle. The modulator may comprise anti-Hh antibody or an Hh inhibitor.

Alternatively, the modulator may comprise Shh, Ihh or Dhh, or a functional variant or fragment thereof. Preferably, the modulator comprises Dhh. A "subject" maybe a vertebrate, mammal, or domestic animal. Hence, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other veterinary

applications. Most preferably, however, the subject is a human being. A "therapeutically effective amount" of the modulator is any amount which, when administered to a subject, is the amount of medicament or drug that is needed to treat the condition characterised by inappropriate erythropoiesis, or produce the desired effect.

For example, the therapeutically effective amount of modulator used may be from about o.oi mg to about 8oo mg, and preferably from about o.oi mg to about 500 mg. It is preferred that the amount of modulator is an amount from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg.

A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (e.g. the modulator) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The modulator according to the invention may be dissolved or suspended in a

pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The modulator may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

The modulators and pharmaceutical compositions of the invention maybe administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The modulators according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral

administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms "substantially the amino acid/ nucleotide/ peptide sequence", "functional variant" and "functional fragment", can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleotide sequence identified as SEQ ID No:5 (i.e. Dhh cDNA) or the protein identified as SEQ ID No: 6 (i.e. Dhh protein), or 40% identity with the nucleotide identified as SEQ ID No: i (i.e. Shh gene) or the protein identified as SEQ ID No:2 (i.e. Shh protein), and so on.

Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein. The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g.

functional form and constants.

Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al, 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al, 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino

acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N/T)*ioo.

Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No's: 1, 3 or 5 or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/ sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No:2, 4 or 6.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non- polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which: -

Figure l shows that components of the Hh pathway are expressed in erythroblasts in spleen and BM. (a) Quantitative RT-PCR of Dhh expression in WT and Dhh-/- spleen and bone marrow, (b) Histograms show anti-Smo staining (left-hand column) and anti-Ptch staining (right-hand column), gated on erythroblast populations I-IV, as defined by Ten.19 and CD71 expression (dot plots). Upper panel shows WT BM and lower panel shows WT spleen, (c) QRT-PCR analysis in sorted erythroblast population I-III from WT BM. Dot plots show sorting strategy. Cells were first magnetic bead depleted to remove lymphocyte, macrophage and granulocyte populations, and the negative populations (negative for CD3, CD4, CD8, B220, Mac-i and Gr-i, left-hand dot plot) were then stained with anti-CD7i and anti-Teriii9. Gates used for sorting are shown for BM (middle dot plot) and spleen (right hand dot plot). Bar charts show Smo, Ptch and Glii expression in BM (left-hand) and spleen (right-hand), (d) QRT-PCR of Dhh expression in WT and Dhh-/- splenic stroma; Figure 2 shows that erythroblasts are Hh responsive in vitro and in vivo, (a) BM was cultured for 18 hours, control or treated with rDhh, anti-Hh mab (5E1), rDhh and 5E1 together, and isotype control mab. After culture, cells were stained with anti- CD71 and anti-Terii9 to confirm that the short treatment had not changed the relative distribution of the subsets (upper panel, dot plots), and with PI to confirm that survival/cell cycle status was not affected (middle panel, histograms) in erythroblasts. Erythroblasts (II to IV) were purified from each culture by magnetic bead purification for Terii9+ cells, and RNA prepared for qRT-PCR analysis of Glii expression (bar chart), (b) Erythroblast population II was sorted from magnetic bead lymphocyte-depleted cells from BM (left26— hand dot plot) and spleen (right-hand dot plot). Gates used for sorting are shown. Bar chart shows Glii expression, measured by qRT-PCR, on RNA prepared from population II sorted from WT (filled bars) and Dhh-/- (open bars) BM and spleen; Figure 3 shows abnormal erythropoiesis in Dhh-/- mice, (a) Photograph shows typical spleen from WT (left-hand) and Dhh-/- (right-hand). Histogram Bar chart shows mean spleen cell number from WT and Dhh-/-. The difference in mean between WT and Dhh-/- is statistically significant (p=0.003). (b) Bar chart shows the mean red blood cell count in WT (shaded bar, 8.1 x

and Dhh-/- (open bar, 8.8 x io⁹/ml) blood, (c) Bar chart shows mean percentage of reticulocytes in red blood cells from WT (shaded bar,) and Dhh-/- (open bar) blood. The difference between the mean percentage of reticulocytes of Dhh-/- and WT was statistically significant (p = 0.014) (d) Spleen cells (upper panel) and BM (lower panel) from WT and Dhh-/- were stained with antibodies against CD71 and Ten.19. Dot plots show the percentage of cells in the four erythroblast subsets (I-rV), defined by CD71 and Terii9 expression, as shown in the regions indicated, in WT (left-hand) and Dhh-/- (right hand). For spleen, bar charts show the mean of the number of cells in each erythroblast subset in Dhh-/-, divided by the number of cells in their counterpart subset in WT spleen, for each erythroblast population. The difference between the mean of Dhh-/- and WT was statistically significant for population II (p=o.oi2), population III (p=o.oi5) and population Γν (p=o.oo6). For BM, bar chart shows the mean relative percentage of erythroblast populations I-IV, in Dhh-/- BM, relative to WT littermate. The difference between the means of Dhh-/- and WT was statistically significant for population III (p=o.022) and population IV (p=0.02). (e) Cell cycle analysis of erythroblasts from WT and Dhh-/- spleen and BM. Bar charts show mean percentage of cells in Go/ Gi and S+G2/M, as assessed by PI staining and flow cytometry in magnetic-bead purified Terii9+ cells from WT (filled bars) and Dhh-/- (open bars) BM (left-hand side) and spleen (right-hand side). There were no significant differences between WT and Dhh-/- in either BM or spleen; Figure 4 shows that Dhh influences early erythropoiesis and progenitor

differentiation, (a) Scatter plot shows BFU-assays, carried out on BM and spleen, from WT (filled squares) and Dhh-/- (open squares). The mean for each group is indicated with a line. The increase in mean number of colonies in Dhh-/- compared to WT is statistically significant for both BM (p=o.050) and spleen (p =0.032). (b) Dot plots show analysis of Lin- Sca-i+ ckit+ (LSK) stem cells and Sca-i-ckit+ progenitors in WT (left-hand) and Dhh-/- (right hand) BM, staining with antibodies against Sca-i and c-kit, after exclusion of Lin+ cells. The bold region shows the gating used to analyse the Sca-i- ckit+ progenitor population. Contour plots show the subdivision of the progenitor population from WT (left-hand) and Dhh-/- (right- hand), staining against CD34 and FcyRII/III, into CMP, GMP and MEP. The percentage of progenitors in each subset and the regions used for their definition are shown. Upper bar chart shows the mean percentage of Lin- cells that are LSK (Sca-i+ ckit+) and Progenitor (Sca-i-ckit+) in WT (filled bars) and Dhh-/- (open bars) BM. The increase in the mean percentage of progenitors in the Dhh-/- compared to WT is statistically significant (p =0.019). Middle bar chart shows the percentage of the progenitor population that is CMP, GMP or MEP, as defined by CD34 and FcyRII/III expression, in WT (filled bars) and Dhh-/- (open bars). The difference in mean proportion is statistically significant between WT and Dhh-/- for CMP (p =0.05), GMP (p=o.ooo6) and MEP (p =0.005). Lower bar chart shows the mean progenitor ratio in WT (filled bars) and Dhh-/- (open bars). The difference in mean ratios between WT and Dhh-/- is statistically significant for MEP:GMP (p =0.009) and CMP:GMP (p =0.024), but not for CMP:MEP. The cartoon shows a summary of the effects of Dhh-deficency at this stage of differentiation. Absence of Dhh, reduced differentiation from CMP to GMP, and the GMP population is reduced. The CMP population is increased, and differentiation to MEP is favoured, (c) Dot plot shows staining against Gr-i and Mac-i on BM cells from WT (left-hand) and Dhh-/- (right- hand). The percentage of cells in each quadrant is shown. Bar chart shows the mean percentage of BM cells that are macrophages (Mac- i+Gr-i-) and granulocytes (Mac- i-Gr-i+) in WT (filled bars) and Dhh-/- (open bars). The reduction in the mean percentage of granulocytes in Dhh-/- compared to WT is statistically significant (p =0.04); Figure 5 shows acceleration of differentiation and recovery following irradiation in Dhh-/- mice, (a) Kinetics of recovery of the erythroblast populations following non- lethal irradiation was measured in the spleen (left-hand panel) and BM (right-hand panel) at 7 (upper row), 9 (2nd row), 14 (3rd row) and 21 (4th row) days after induction. Dot plots show erythroblast populations I-IV, defined by CD71 and Terii9 expression, and the regions used and percentage of cells in each population are shown, (b) Photograph shows typical spleen from WT (left-hand) and Dhh-/- (right- hand) at 14 days after irradiation, (c) Bar chart shows the mean spleen cell number following irradiation in WT (filled bars and Dhh-/ - (open bars). The increase in mean cell number in Dhh-/- compared to WT on day 14 is statistically significant

(p=o.046). (d) Bar chart shows the mean number of cells in each erythroblast subset in WT (filled bars) and Dhh-/- (open bars) in the spleen on day 14. The difference in mean between WT and Dhh-/- was statistically significant for population I (p = 0.003) and population II (p = 0.044). (e) Dot plot shows staining against Mac-i and Gr-i on day 14 after irradiation from WT (left hand plot) and Dhh-/- (right-hand plot) BM. Bar chart shows the mean relative percentage of Maci+ cells in WT (filled bars) and Dhh-/- (open bars) BM, 14 days after irradiation. The difference in mean relative percentage is statistically significant (p <o.ooi);

Figure 6 shows Dhh-/- mice recover more quickly during stress-induced anaemia that WT. Anaemia was induced in Dhh-/- and WT mice by PHZ treatment and the kinetics of stress-induced erythropoiesis and recovery was monitored over time, (a) Photographs show typical WT and Dhh spleen size at 5, 9 and 14 days after PHZ- treatment. Upper bar chart shows time course of red blood cell (RBC) concentration in WT (filled bars) and Dhh-/- (open bars) blood, following PHZ-treatment. The difference in mean RBC concentration between WT and Dhh-/- was statistically significant on day 7 (p = 0.011). Lower bar chart shows percentage of reticulocytes in red blood cells in blood, as above. The difference in mean percentage between WT and Dhh-/- was statistically significant before treatment (day o, p = 0.015) and on day 9 (p = 0.045). (b) Kinetics of recovery of the erythroblast populations following PHZ-treatment was measured in the spleen (left hand panel) and BM (right-hand panel) at 5 (upper row), 9 (2nd row), 14 (3rd row) days after treatment. Dot plots show erythroblast populations I-IV, defined by CD71 and Terii9 expression, and regions used and percentage of cells in each population are shown, (c) The relative percentage of MEP (measured by Facs, staining Lin- cells with antibodies against CD34 and FcyRII/III) in spleen (left-hand side) and BM (right-hand side) in WT (filled bars) and Dhh-/- (open bars), 7 days after PHZ-treatment. The difference in mean was statistically significant for spleen (p = 0.017) and BM (p = 0.033). (d) The relative percentage of neutrophils (measured by Facs, staining with antibodies against Ly6g) in BM in WT (filled bars) and Dhh-/- (open bars), 7 days after PHZ- treatment. The difference in means was statistically significant (p < 0.001);

Figure 7 shows histology showing red and white pulp areas in Dhh-/- and WT spleens, prior to and following induction of stress-induced erythropoiesis. Parafin embedded spleen sections were stained with hematoxilin-eosin, to identify white pulp areas, which stained deeper purple, and red pulp areas, which stained pink. Typical white pulp (WP) and red pulp (RP) are illustrated in A. Left-hand column (a,c,e,g,i) shows typical histology in WT spleens and right-hand column (b,d,f,h,j) shows typical histology in Dhh-/- spleen, without treatment (a-b) and during a time course following induction of stress-erythropoiesis following PHZ-treatment, on day 5 (c-d), 7 (e-f), 9 (g-h), and 14 (ij). Scale is shown; and

Figure 8 shows modulation of LMO2 expression by Dhh. (a) QRT-PCR analysis of relative expression of Gatai, Lm.02 and Runxi in sorted erythroblast population II (left-hand chart) and (b) magnetic bead purified Terii9+ cells (right-hand chart) from WT and Dhh-/- BM. Bar charts show the mean fold change in expression in Dhh-/- compared to WT. The difference in mean fold change was statistically significant for Lm.02 in sorted population II (p= 0.033) and in magnetic bead purified Terii9+ (p=o.05) but not for Gatai or Runxi in either population, (c) Dhh-/- BM treated with rDhh, 5E1, rDhh+5Ei, or control, and WT control BM were cultured for 18 hours, and Terii9+ cells were separated with magnetic beads for RNA preparation and qRT-PCT analysis. Bar chart shows the mean Lm.02 expression. The difference in mean Lmo2 expression was statistically significant between control WT and Dhh-/- (p=o.oi7), and between control Dhh-/- and rDhh-treated Dhh-/- (p=o.oi2).

Example 1

Materials and methods

Mice

C57BL/6 mice (B & K Universal Ltd, UK), Dhh+/- mice (Bitgood et al. Curr Biol. 1996;6:298-304), a gift from Andrew McMahon, backcrossed onto C57BL/ 6 mice for >8 generations, were bred and maintained at University College London. In some experiments mice were irradiated with 4 Gy from a ^6oCo gamma-ray source, or anaemia was induced by intraperitoneal injection of phenylhydrazine (6omg/kg body weight, Sigma Chemical, St Louis, MO). All animal work was carried out under UK Home Office regulations.

Flow cytometry, histology, staining and antibodies

BM was isolated from femur. Cell suspensions from spleen and BM were prepared, stained and analyzed as described (Shah et al., J Immunol. 2004;172:2296-2306; Hager-Theodorides et al., Eur J Immunol. 2007;37:487-500), using directly conjugated antibodies from BD Pharmingen and eBioscience. Data are representative of >three experiments. Statistical analysis was unpaired Students-t test (equal or unequal variance depending on data). For some experiments, BM cells and splenocytes were isolated and sorted using MoFlo XDP Sorter (Beckman Coulter) to obtain populations of Terii9+ and CD71+ erythroblast populations, following magnetic bead depletion for cells positive for CD3, CD4, CD8, B220, Mac-i and Gr-i. Staining with CD7i^FITC and TERii9^PE allowed sorting of early stage (I) to late stage (IV) erythroblast populations. Propidium iodide (PI) staining was carried out on magnetic-bead purified populations as described and examined using FACSCalibur (Becton Dickinson), (see Hager-Theodorides AL et al., Blood. 2005; 106: 1296-1304). For anti-Smo staining, cells were first incubated for 30 minutes in PBS with 1.5% donkey serum (Santa Cruz Biotechnology). After washing with PBS, cells were incubated with o^g anti-CD 16/CD32 in 5θμ1 of PBS for 5 minutes, then incubated with 2μg anti-Smo N-19 (Santa Cruz Biotechnology) or goat IgG (Santa Cruz

Biotechnology), as isotype control, in 5θμ1 of PBS for 60 minutes and washed with PBS and 0.5%BSA. Cells were then incubated with 2μg of Biotinconjugated F(ab')2 fragment of donkey anti-goat IgG in 5θμ1 of PBS and 1.5% donkey serum for 30 minutes and washed with PBS and 0.5%BSA. Cells were finally incubated with:

streptavidincychrome, anti-CD7i^FITC, anti-TERii9^PE as above. For anti-Ptch staining, cells were first incubated for 30 minutes in PBS with 1.5% rat serum (Santa Cruz Biotechnology). After washing with PBS, cells were incubated with o^g anti-

CD16/CD32 in 5θμ1 of PBS for 5 minutes, then incubated with 2μg anti-Ptch (R&D systems) or rat IgG (Santa Cruz Biotechnology), as isotype control, in 5θμ1 of PBS for 60 minutes and washed with PBS and 0.5%BSA. Cells were then incubated with 2μg of Biotin-conjugated anti-rat IgG in 5θμ1 of PBS and 1.5% rat serum for 30 minutes and washed with PBS and 0.5%BSA. Cells were finally incubated with: streptavidin^PE, anti-CD7i^FITC, anti-TERii9^PerCP-^c¾'5-5. For assessment of reticulocytes in blood, reticulocytes were counted from Giemsa-stained blood films.

For histology, spleens were fixed in phosphate buffered formalin (io%vol/vol), paraffin embedded and sectioned for hematoxilin-eosin staining, by standard protocols. Quantification of red pulp and white pulp surface area on hematoxilin- eosin stained sections was carried out using ImageJ software (Rasband, W.S., ImageJ, http:/ /imagej.nih.gov/ij /).

Hematopoietic Colony Assay

Colony forming assays were performed using methocult methylcellulose based medium (StemCell Technologies). 2 x 10⁶ BM cells and 2 x 10⁷ spleen cells were plated in 1 ml of methylcellulose medium (M3334 and M3434) in a 35mm culture dish (StemCell Technologies). Cultures were incubated at 370C in 5% CO2. BFU-E on methylcellulose medium M3434 were counted after 7 days.

Cell culture and purification

BM cells and splenocytes were isolated and cultured at a concentration of 5x106 cells/ml in AIM-V medium at 37°C and 5% CO2 . Cells were harvested at 18 hours and Terii9+ erythroblast populations purified by magnetic bead separation using the EasySep Biotin positive selection kit (StemCell Technologies, UK) according to the manufacturer's instructions.

Genotyping and polymerase chain reactions (PCR) analysis

Animals were genotyped by PCR. DNA extraction and PCR analysis were as described (Shah DK et al., 2004;172:2296-2306), using ~ o^g genomic DNA as template.

Primers: For mutated Dhh allele,

Dhh/neoForward: GGCATGCTGGGGATGCGGTG [SEQ ID No: 7];

Reverse: CCAGGAAGACGAGCACTGGCGTG [SEQ ID No: 8];

WT Dhh Forward: ATCCACGTATCGGTCAAAGC [SEQ ID No: 9]; and

Reverse: GGTCCAGGAAGAGCAGCAC [SEQ ID No: 10]. Quantitative RT-PCR

RNA extraction and cDNA synthesis were as described (Hager-Theodorides AL et al., Blood. 2005;106:1296-1304). One primer for each pair was designed to span exon- exon boundaries to avoid amplification of genomic DNA.

The following primers were used:

Gapdh Forward: CCTGGAGAAACCTGCCAAGTATG [SEQ ID No: 11];

Reverse AGAGTGGGAGTTGCTGTTGAAGTC [SEQ ID No: 12];

Smo : Forward TTCTTCAACCAGGCTGAGTG [SEQ ID No: 13];

Reverse CGTATGGCTTCTCATTGGAGTG [SEQ ID No: 14];

Ptch : Forward TGCTCTCCAGTTCTCAGACTC [SEQ ID No: 15]; and

Reverse CCACAACCTTGGGTTTGG [SEQ ID No: 16].

HPRT and Glii primers were as described Rowbotham et al., Blood. 20075109:3757- 3766.

Results

Example 1 - Dhh and components of the Hh signalling pathway are expressed in adult spleen and bone marrow

Given that the involvement of Hh proteins in erythropoiesis is controversial, the inventors investigated the possible function of Dhh in erythropoiesis. They therefore first asked if Dhh is expressed in the adult spleen and BM, comparing expression between WT and Dhh-/-, as negative control. They found Dhh transcription, by quantitative (q) RT-PCR, in WT spleen, but not Dhh-/- spleen, and they were also able to detect Dhh transcription in WT BM (Figure la). This is consistent with recent reports that Dhh is expressed in stromal cells in the BM, and by non-hematopoetic cells of the spleen stroma (Perry et al., Blood. 2009;113:911-918; Hegde GV et al., Mol Cancer Res. 2008;6:1928-1936).

As Dhh is expressed in tissues where erythropoiesis occurs, the inventors tested if developing erythrocyte-lineage cells express components of the Hh signalling pathway. They stained the four erythrocyte-committed erythroblast populations, defined by Ten.19 and CD71 expression, with antibodies directed against the Hh- signal transduction molecule Smo and the cell surface Hh-receptor Ptch (Figure lb). In cells isolated from both spleen and BM, they found highest Smo expression on the most immature erythroblast population I (Terii9medCD7ihi), with gradual reduction in cell surface expression in each subsequent population, so that population IV did not express detectable cell-surface Smo. In contrast, cell surface expression of Ptch was detectable on all erythroblast populations, consistent with its potential function as a Hh-sequestering protein. To assess expression of the Hh target genes/signalling genes in erythroblast populations, the inventors sorted populations I to III from BM and spleen and performed qRT-PCR for analysis of transcription of Ptch, Smo and Glii (Figure lc). They did not carry out this analysis on population IV because the RNA isolated from these cells was of poor quality, as the nucleus is shrinking and will be shed. Transcription of Smo was highest in population I, and down-regulated in populations II and III, in cells sorted from both BM and spleen. Transcription of Ptch, the cell surface receptor for Hh proteins, mirrored that of Smo, with highest expression in population I, and reduced but detectable expression in populations II and III. Interestingly, the inventors found highest expression of the Hh-responsive target gene Glii in the later population III. The fact that Smo and Ptch transcription are highest in population I suggests that these cells transduce the strongest Hh signal. Glii is not required to initiate the Hh signal, but is up-regulated in response to it, consistent with the fact that the highest level Glii expression was observed in the later population III.

This pattern of expression is similar to that observed during thymocyte development, where the earliest progenitors express highest levels of Smo, but Glii expression peaks at a later stage. The inventors did not detect Dhh expression in erythroblast populations I to III from either spleen or BM (data not shown), and a previous study has located Dhh protein expression to spleen stroma by immunohistochemistry. To confirm this, they prepared RNA from splenic rudiment (stroma) and carried out qRT-PCR. Transcription of Dhh was detected in WT spleen stroma, but not stroma from Dhh-/- spleen (Figure id).

Example 2 - Erythroblasts are Dhh-responsive in vitro and ex vivo

In order to verify that erythroblasts are capable of transducing a Dhh signal, the inventors treated WT BM for 18 hours with recombinant(r) Dhh, neutralizing anti- Hh mab 5E1, both treatments together, or isotype control mab, and purified Terii9+ cells from the cultures by magnetic bead separation, for RNA preparation. To test if the treatments caused changes in the cellular composition of the Terii9+ population (subsets II-IV) during the short culture period, they analyzed the cultures for CD71 and Terii9 expression, and cell cycle/survival status. Viability was good, and the inventors found no difference between the conditions in relative composition of the erythroblast subsets, cell cycle or survival status after culture (Figure 2a), confirming that it was reasonable to carry out the Glii expression studies on the Terii9+ population. Treatment with rDhh increased expression of the Hh-target gene Glii, whereas neutralization of Hh by 5Ei-treatment downregulated Glii transcription. Addition of rDhh to 5Ei-treatment partially recovered Glii transcription, confirming specificity of the reagents, whereas treatment with an isotype-control antibody did not significantly effect Glii transcription.

To assess the impact of Dhh on Hh signal transduction in erythroblasts ex vivo, the inventors sorted erythroblast population II from WT and Dhh-/- BM and spleen and again measured transcription of the Hh-target gene Glii. Expression of Glii was ~6- fold higher in WT erythroblasts compared to their Dhh-/- counterparts in spleen, and ~2.25-fold higher in WT erythroblasts compared to Dhh-/- in BM, indicating that Dhh signal transduction is active in developing WT erythroblasts and accounts for most Hh-dependent transcription in these cells. The fact that some Glii expression was detectable in the Dhh-/- cells indicated that erythroblasts were also transducing signals from either Ihh or Shh in both BM and spleen, hence the remaining Glii expression. Taken together, these experiments show that developing erythroblasts undergo active Dhh signalling during their differentiation in the spleen and BM. Example 3 - Abnormal erythropoiesis in Dhh-/ - mice

Given that Dhh is expressed at sites of erythropoiesis and differentiating cells of the erythroid lineage transduce Hh signals, the inventors asked if Dhh plays a role in the regulation of erythropoiesis, by analysis of Dhh-/- mice. They found that the spleen of Dhh-/- mice was larger than that of WT littermates (Figure 3a and b). There was no significant difference in total red blood cell counts in Dhh-/- blood compared to WT (Figure 3b), but the number of reticulocytes in the blood was significantly increased (Figure 3c). In the spleen, there was an increase in each population of erythroblasts (populations I to IV) (Figure 3d). In the BM the inventors found a statistically significant increase in the proportions of erythroblast populations III and IV (Figure 3d). The increase in reticulocytes in the Dhh-/- blood and in erythroblast populations in the Dhh-/- BM and spleen indicated that Dhh is a negative regulator of erythropoiesis. There are several possible mechanisms that might contribute to, or account for, the increased erythropoiesis in Dhh-/- mice. First, absence of Dhh might increase the proliferation of the erythroblast subsets. Second, it might influence the frequency of the precursors that give rise to them. Third, absence of Dhh might increase the rate of erythroblast differentiation between the different stages to produce more reticulocytes.

To distinguish between these possibilities, the inventors first assessed the cell-cycle status of the erythroblast populations by PI staining of Terii9+ cells isolated from Dhh-/- and WT spleen and BM. There was no significant difference in the proportion of cells in S+G2/M between Dhh-/- and WT in either spleen or BM (Figure 3e). These experiments indicated that Dhh does not act as a negative regulator of proliferation of erythroblast cells, but more likely regulates an earlier precursor and/or the rate of differentiation along the erythrocyte lineage.

The inventors therefore tested the ability of progenitors from Dhh-/- and WT spleen and BM to differentiate along the erythroid lineage in vitro, by assessment of their ability to form BFU. They found a statistically significant increase in BFU-Es in both BM and spleen from Dhh-/- compared to WT (Figure 4a). These data show that Dhh is a negative regulator of erythropoiesis, so they examined its influence on earlier hematopoietic populations. The inventors found no difference in the relative proportions of Lin- Sca-i+ ckit+ (LSK) stem cells, but the population of Sca-i-ckit+ progenitors was increased in Dhh-/- BM compared to WT (Figure 4b). When the inventors gated on the progenitor population and sub-divided it by expression of CD34 and FcyRII/III, they found that the proportion of the earlier CMP was increased in Dhh-/- compared to WT. There was also a statistically significant increase in the proportion of MEPs in the Dhh-/- compared to WT, and a

concomitant decrease in the proportion of GMP. Thus, the ratio of MEP to GMP was statistically increased in the Dhh-/- BM compared to WT (Figure 4b).

When the inventors compared the ratio of the precursor CMP population to each of its progeny populations, they found that while the ratio of CMP:MEP was not different between Dhh-/- and WT, the ratio of CMP:GMP was statistically

significantly increased. These data therefore indicate that Dhh acts on the CMP population: in the absence of Dhh, differentiation from CMP to GMP is decreased, whereas differentiation to the erythrocyte lineage (from CMP to MEP) continues at its normal rate, but given the increase in the progenitor CMP population, this results in an overall increase in differentiation to the erythroid lineage (Figure 4b). The proportion of granulocytes was significantly reduced in the Dhh-/- BM compared to WT (Figure 4c). Thus, Dhh negatively regulates early erythroid differentiation, but is required for normal granulocyte production.

Example 4 - Dhh is a negative regulator of erythrocyte differentiation during recovery following irradiation

In order to test if Dhh reduces the rate of differentiation along the erythrocyte lineage, the inventors examined erythropoiesis under conditions in which

haematopoiesis and erythropoiesis take place in a more-or-less synchronized wave, following recovery from sub-lethal irradiation. They depleted the haematopoetic system in Dhh-/- and WT littermates by sublethal irradiation. They then followed the regeneration of erythroblast populations in the spleen and BM for three weeks following irradiation. Erythropoiesis occurred more rapidly in both spleen and BM in Dhh-/- compared to WT, and both returned to pre-irradiation values by day 21 (Figure 5a). Population II was already present on day 7 on Dhh-/- spleen and BM, but not in WT. On day 14 after irradiation, the Dhh-/- spleen was significantly larger and contained more cells in subsets I and II than WT (Figure 5a-d).

Staining with antibodies against Gr-i and Mac-i showed that the proportion of cells that were committed to the macrophage lineage (Mac-i+) was significantly reduced in the Dhh-/- BM compared to WT. Thus, as haematopoiesis recovers following irradiation, differentiation along the erythroid lineage was accelerated in the Dhh-/- BM and spleen compared to WT, whereas macrophage differentiation in the BM was reduced.

Example 5 - Dhh is a negative regulator of stress-induced erythropoiesis in the spleen Under conditions of erythropoietic stress, the production of erythrocytes is increased, and the major site of erythropoiesis moves to the spleen (Socolovsky Curr Opin

Hematol. 2007;14:215-224). To investigate the function of Dhh specifically in stress- induced erythropoiesis in the spleen, the inventors treated Dhh-/- and WT mice with phenylhydrazine (PHZ) to cause anaemia, and followed the kinetics of recovery. The Dhh-/- mice recovered more quickly than WT, and red blood cell counts

had returned to normal in both by day 14 (Figure 6). Five days after treatment, the Dhh-/- spleen was larger than WT (Figure 6a). Erythropoiesis was strongly induced in both Dhh-/- and WT spleen, with an increase in erythroblast population II from <io% in untreated animals (Figure 3) to more than 50% at day 5 after PHZ-treatment (Figure 6b). Interestingly, the inventors also observed an increase in erythropoiesis in the BM following PHZ treatment in the Dhh-/- mice. At day 5 following treatment the proportion of erythroblast population II was increased to 47% in the Dhh-/-BM, from ~27% in untreated Dhh-/- controls (Figure 3d and 6b). Seven days after treatment the red blood cell count and reticulocyte count were significantly higher in the Dhh-/- bloodcompared to WT, although the proportion of reticulocytes was equivalent. Nine days after treatment, the Dhh-/- spleen was smaller than WT, and the proportion of blood reticulocytes was significantly lower (Figure 6b), consistent with accelerated differentiation in the Dhh-/-, so that by day 9 its proportion of reticulocytes was already in decline, while in the WT it was still rising.

To ask if Dhh-deficiency influenced earlier stages of erythropoiesis following induction of anaemia, the inventors assessed the proportion of MEP cells (defined as Sca-i-ckit+ progenitors, that are CD34- and FcyRII/III-) in BM and spleen seven days after PHZ-treatment. In the BM the proportion of MEP was significantly reduced in the Dhh-/- relative to WT. Interestingly, the proportion of MEP was significantly increased in the Dhh-/- spleen, compared to WT spleen, consistent with increased mobilization of progenitors to the Dhh-/- spleen and the increase in erythropoiesis observed in the Dhh-/- spleen compared to WT. The proportion of Ly6g+ cells (neutrophils) was significantly reduced in the Dhh-/- BM compared to WT.

Example 6 - Dhh-/- spleen contains more red pulp areas than WT, and histology returns to normal more quickly on recovery from stress-induced erythropoiesis

Given that erythropoiesis was increased in Dhh-/- spleen compared to WT, both in normal conditions and after induction of anaemia, the inventors tested if Dhh-/- spleen showed abnormal histology or a difference in the proportion of red pulp area, compared to WT. Hematoxilin-eosin staining on paraffin-embedded longitudinal spleen sections revealed normal histology, and red pulp (RP) and white pulp (WP) areas in both WT and Dhh-/- spleen (Figure 7a-b). The inventors measured RP and WP surface area across the entire surface area of the central longitudinal spleen section, and calculated RP:WP ratio and the percentage of RP (see Table 1). Table 1 - Red pul /White pulp ratio and percentage of Red pulp in PHZ-treated mice spleen

Table 1. Red pulp/White pulp ratio and percentage of

Red pulp in PHZ-treated mice spleen

Red pulp/White

pulp Red pulp %

WT Dhh-/- WT Dhh-/-

Day 0 1.4 2.1 59 68

Day 5 5.2 4.8 84 83

Day 7 4.8 6.2 83 86

Day 9 5.0 4.9 83 83

Day 14 3.7 2.5 79 71

Table l: Red pulp area, white pulp area and entire surface area of longitudinal sections of paraffin embedded spleen from WT and Dhh-/- mice untreated (day o) and at time points after PHZ-treatment, were quantified using ImageJ software. Ratio of red pulp : white pulp and percentage of red pulp were calculated.

Consistent with the increased erythropoiesis in Dhh-/- spleen, the ratio of RP:WP was increased from 1.4 in WT to 2.1 in Dhh-/- spleen, and the percentage of RP increased from 59% in WT to 68% in Dhh-/- spleen. The inventors then compared the induction of red pulp during stress-induced erythropoiesis caused by anaemia following PHZ-treatment (Figure 7C-j). In both WT and Dhh-/- RP area was increased at 5 days after treatment to 84% and 83% respectively (Figure 7c-d, Table 1). RP area remained high at days 7 and 9 following treatment, but resolved more quickly in the Dhh-/- than in the WT (Figure 7 i-j), consistent with the faster resolution of the proportion of reticulocytes in the blood in the Dhh-/- (Figure 6a). Thus, on day 14 after treatment, RP:WP ratio was reduced to 2.5 in Dhh-/- spleen, and 3.7 in WT spleen.

The dynamics of RP induction and resolution therefore mirror the kinetics of erythrocyte differentiation in Dhh-/- and WT spleen, both under normal conditions (Figure 7a-b) and during stress-induced anaemia (Figure 7C-j). Example 7 - Dhh modulates expression of Lm.02 In order to investigate the mechanism of action of Dhh on erythropoiesis, the inventors investigated transcription of genes known to be involved in the regulation of erythropoiesis in erythroblasts. They sorted erythroblast population II from spleen and BM from Dhh-/- and WT and prepared RNA for qRT-PCR. The inventors chose subset II for this experiment because it is actively responding to the Hh signal, it is abundant enough to accurately sort for preparation of RNA (Fig. l), and because the rate of differentiation from population II to III is increased in the Dhh-/- BM.

They then compared expression of genes known to be involved in the transcriptional regulation of erythropoiesis: Gatai, Lmo2, and 1171x138,45. Gatai and Runxi were not differentially expressed (Figure 8a). Interestingly, Lm.02 was statistically significantly upregulated more than 2.5-fold in Dhh-/- population II compared to WT (p=0.033; Figure 8a).

Lmo2 is essential for erythrocyte differentiation, and is involved in the

transcriptional regulation of erythrocyte differentiation. It is believed to provide scaffolding on which complexes of multiple transcription factors and co-factors, including Gatai, come together to regulate transcription. Given that Lm.02 is essential for erythropoiesis and is upregulated in Dhh-/- erythroblasts, which differentiate more efficiently than WT (Figure 3-5), the inventors decided to test if Lm.02 is a direct functional target of negative transcriptional regulation by Dhh by treatment of Dhh-/- BM with rDhh in our 18-hour culture system. To confirm that Terii9+ erythroblasts show the same pattern of Lmo2 expression as sorted population II, the inventors first compared Lmo2 transcription by qRT-PCR in Terii9+ BM cells prepared from freshly isolated BM from WT and Dhh-/-. Lmo2 expression was 2-3 fold higher in Dhh-/- compared to WT, as seen in sorted population II, whereas expression of Gatai was equivalent in both (Figure 8a-b).

The inventors then treated Dhh-/- BM for 18 hours with rDhh, neutralizing anti-Hh mab 5E1, or both treatments together and purified Terii9+ cells from the cultures by magnetic bead separation, for RNA preparation and qRT-PCR analysis of Lmo2 expression (Figure 8c). In the Dhh-/- cultures, rDhh-treatment down-regulated Lmo2 more than two-fold, to levels equivalent to that found in Terii9+ cells prepared in control WT BM 18 hour cultures, indicating that Dhh signalling negatively regulates Lmo2 transcription. Addition of the Hh-neutralizing antibody 5E1 to the rDhh-treatment counteracted the effect of rDhh alone, demonstrating that the impact of rDhh on Lmo2 expression was specifically due to Hh pathway activation, rather than an effect due to non-specific toxicity of the reagent. Interestingly, treatment with 5E1 alone did not significantly affect Lm.02 expression in the Dhh-/- cultures, indicating that where Dhh is not present, neutralization of Hh does not influence Lm.02 expression, and therefore it is Dhh, and not Ihh or Shh, that functions to down- regulate Lm.02 in the BM cultures, and consistent with the ex-vivo analysis showing that Dhh-/- erythroblasts express more Lm.02 than WT (Figure 8a). Taken together, these data show that Lmo2 is a functional target of physiological Dhh signalling in Terii9+ erythroblasts. Discussion

This analysis of Dhh mutant mice showed that Dhh is a negative regulator of differentiation of erythroid progenitors, as multiple stages of their development. Differentiation from CMP to GMP was decreased in Dhh-/- BM, indicating that Dhh is required for granulocyte/macrophage lineage differentiation. Interestingly, in the Dhh-/- BM, although the proportion of GMP was decreased, there was an increase in both CMP and MEP populations, so that in the absence of Dhh, erythroid

differentiation was favored. Analysis of subsequent BFU-e and erythroblast populations revealed that Dhh negatively regulated multiple stages of erythroid lineage differentiation. In addition, following recovery from non-lethal radiation, and on induction of stress-induced anaemia by PHZ-treatment, differentiation in both spleen and BM were accelerated. The inventors did not find evidence that absence of Dhh increased proliferation in the erythroblast populations, so the increased number of erythroblasts in the spleen and BM seemed to be the result of both the increase in early progenitors (CMP and MEP) and increased rate of differentiation.

The reduction in differentiation from CMP to GMP, mature granulocyte numbers, proportion of macrophages following irradiation, and of neutrophils following PHZtreatment, in the Dhh-/- BM is consistent with a recent study showing that Glii plays a role in myeloid differentiation. In that study, the influence of Glii-deficiency on erythroblast differentiation was not investigated. It is, however, in contrast to recent reports that conditional deletion of Smo from hematopoietic lineage cells has no impact on any stage of hematopoiesis. The reason for this difference is not clear. The impact of conditional deletion of Smo from hematopoietic cells has also been examined in stress-induced hematopoiesis in the spleen. Smo-deficient cells were found to be unable to respond to BMP4 and to recover normally following stress- induction. In contrast, the data described here indicate that recovery and

erythropoiesis in spleen and BM is accelerated in Dhh-/- mice. The reason for this discrepancy may lie in the fact that Smo is believed to be the essential non-redundant signal transduction component of the Hh signalling pathway, so Smo-deficient cells should be unable to transduce a Hh signal.

Dhh, however, is one of three Hh family members, and both Ihh and Shh are also expressed in the spleen. Thus, although erythroblasts undergoing differentiation in the Dhh-/- spleen would have reduced Hh pathway activation, some Hh signal transduction was present (Fig. 2), whereas Smo-deficient erythroblasts would be assumed to have none. Hh proteins can function as morphogens, signalling for distinct outcomes dependent on strength and duration of signal received. Thus, it is possible that reduction in Hh signal (by Dhh-deficiency) could produce the distinct outcome of accelerating stress-induced erythropoiesis in the spleen, compared to Smo-deficiency, which resulted in reduced stress-induced erythropoiesis. Indeed, in the thymus, several experimental systems have shown that reduction of Hh signal accelerated pre-TCR induced differentiation, although some Hh signal transduction was still required for differentiation. In contrast to the inventor's finding that Dhh negatively regulates erythrocyte differentiation, Ihh has been shown to promote the earliest stages of haematopoiesis and vasculogenesis and to support definitive erythropoiesis in the mouse embryo. These opposing functions for Ihh and Dhh are accountable by the different spatial and temporal expression patterns of the two genes, strength of signal transduced by each protein, and stage of differentiation on which they act. In this study, Dhh acted at multiple stages of erythrocyte differentiation from the Lin-Sca-i-ckit+ stage onwards, but the inventors did not find an influence of Dhh-deficiency on the LSK stem cells. In conclusion, the inventors have shown that Dhh signalling is a negative regulator of erythropoiesis, thus adding erythropoiesis to the very few functions currently ascribed to Dhh. This finding is of general importance to an understanding of erythropoiesis, and will have relevance to the treatment of human hematological disease, including blood cancers and anaemia.

Claims

Claims

1. A modulator of Hedgehog (Hh) signalling, for use in the treatment, amelioration or prevention of a disease characterised by inappropriate

erythropoiesis.

2. A modulator according to claim l, wherein the modulator is capable of modulating Sonic Hh (Shh), Indian Hh (Ihh) and/or Desert Hh (Dhh) signalling.

3. A modulator according to either claim 1 or claim 2, wherein the modulator is capable of modulating Dhh signalling.

4. A modulator according to any preceding claim, wherein the modulator is a negative modulator of Hedgehog (Hh) signalling, which is capable of increasing erythropoiesis.

5. A modulator according to claim 4, wherein the modulator comprises an anti- Hh antibody or an Hh inhibitor, which is capable of altering conformation/stability of the receptor through which Hh signalling is directed, or blocking the receptor's activity.

6. A modulator according to either claim 4 or claim 5, wherein the modulator comprises an anti-Shh, anti-Ihh or anti-Dhh antibody, or a Shh, Ihh or Dhh inhibitor.

7. A modulator according to any one of claims 4-6, wherein the modulator comprises an anti-Dhh antibody or a Dhh inhibitor.

8. A modulator according to any one of claims 4-7, for use in treating malaria, anaemia, or blood disorder.

9. A modulator according to any one of claims 1-3, wherein the modulator is a positive modulator of Hedgehog (Hh) signalling, which is capable of decreasing erythropoiesis.

10. A modulator according to claim 9, wherein the modulator comprises Shh, Ihh or Dhh, or a functional variant or fragment thereof.

11. A modulator according to either claim 9 or claim 10, wherein the modulator comprises a protein comprising an amino acid sequence substantially as set out in SEQ ID No: 2, 4 or 6, or a functional variant or fragment thereof.

12. A modulator according to claim 11, wherein the protein is recombinant.

13. A modulator according to either claim 11 or claim 12, wherein the protein is encoded by a nucleic acid sequence substantially as set out in SEQ ID No: 1, 3 or 5, or a functional variant or fragment thereof.

14 A modulator according to any one of claims 9-13, wherein the modulator comprises Dhh.

15. A modulator according to any one of claims 9-14, for use in treating polycythemia or leukaemia.

16. A modulator according to any preceding claim, wherein the modulator of Hh signalling is used to treat a condition characterised by inappropriate myeloid leukopoiesis.

17. An erythropoiesis-treatment composition, comprising a modulator of Hedgehog (Hh) signalling and a pharmaceutically acceptable vehicle.

18. A process for making the erythropoiesis-treatment composition according to claim 17, the process comprising contacting a therapeutically effective amount of a modulator of Hedgehog (Hh) signalling and a pharmaceutically acceptable vehicle.

19. A composition according to claim 17, or a process according to claim 18, wherein the modulator is as defined in any one of claims 1-16.

20. A method of treating, ameliorating or preventing a disease characterised by inappropriate erythropoiesis in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a modulator of Hedgehog (Hh) signalling.