WO2013132272A1 - Erythropoïèse - Google Patents

Erythropoïèse Download PDF

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Publication number
WO2013132272A1
WO2013132272A1 PCT/GB2013/050586 GB2013050586W WO2013132272A1 WO 2013132272 A1 WO2013132272 A1 WO 2013132272A1 GB 2013050586 W GB2013050586 W GB 2013050586W WO 2013132272 A1 WO2013132272 A1 WO 2013132272A1
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Prior art keywords
dhh
modulator
signalling
erythropoiesis
spleen
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PCT/GB2013/050586
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English (en)
Inventor
Tessa Crompton
Ching-In LAU
Susan OUTRAM
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Ucl Business Plc
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Priority to EP13709525.3A priority Critical patent/EP2822570A1/fr
Publication of WO2013132272A1 publication Critical patent/WO2013132272A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids

Definitions

  • the present invention relates to erythropoiesis, and particularly, although not exclusively, to the treatment of conditions characterised by inappropriate
  • 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.
  • Hh 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.
  • Hh proteins 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
  • 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.
  • HSC hematopoietic stem cell
  • Hh pathway activation caused cycling and expansion of bone marrow (BM) hematopoietic cells, leading to HSC exhaustion.
  • BM bone marrow
  • 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.
  • HSCs 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).
  • CMP common myeloid progenitor cells
  • GMP granulocyte/macrophage progenitor cells
  • MEP megakaryocyte/erythroid progenitor cells
  • 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.
  • Terii9 hi CD7i- (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.
  • a modulator of Hedgehog (Hh) signalling for use in the treatment, amelioration or prevention of a disease characterised by inappropriate erythropoiesis.
  • a method of treating, ameliorating or preventing a disease characterised by inappropriate erythropoiesis in a subject comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a modulator of Hedgehog (Hh) signalling.
  • Hh Hedgehog
  • 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.
  • Hh Hedgehog
  • the modulator maybe capable of modulating Shh, Ihh and/or Dhh signalling.
  • 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.
  • ENST00000297261 is provided herein as SEQ ID No:2, as follows.
  • 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.
  • 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.
  • ENST00000266991 is provided herein as SEQ ID No:6, as follows.
  • 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
  • Hh signalling e.g. Hh protein per se
  • modulators which can inhibit Hh signalling e.g. Hh inhibitors and anti-Hh antibodies
  • Erythropoietin 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.
  • EPO Erythropoietin
  • 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.
  • 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.
  • malaria is another condition which is characterised by depleted erythrocytes, and so could also be treated by a compound which reduces Hh signalling.
  • the modulator maybe a negative modulator of Hedgehog (Hh) signalling (for example an antagonist), which is capable of increasing erythropoiesis.
  • Hh Hedgehog
  • the negative modulator may be capable of:-
  • 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).
  • Ptch cell- surface receptor Patched
  • Smo Smoothened
  • 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.
  • anti-Hh antibodies and suitable Hh inhibitors are well- known to the skilled person.
  • suitable anti-Hh antibodies are examples of suitable anti-Hh antibodies.
  • anti-Shh and anti-Ihh antibodies are described in Ericson, J. et a!.. (1996), Cell 87, 661-673.
  • suitable Hh inhibitors include cyclopamine (Chen et al, Genes and Development 16, 2743; 2002); and SMO antagonist BMS 833923.
  • 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.
  • 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.
  • the modulator may be a positive modulator of Hedgehog (Hh) signalling (for example an agonist), which is capable of decreasing erythropoiesis.
  • Hh Hedgehog
  • the positive modulator may be capable of:-
  • Hh signalling 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;
  • transducer Smo through which Hh signalling is achieved, or agonists thereof;
  • 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.
  • 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.
  • 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
  • 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.
  • the modulator of Hh signalling may be used to treat a condition characterised by inappropriate myeloid leukopoiesis.
  • 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.
  • 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.
  • 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.
  • modulators of the invention may be used in
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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
  • 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).
  • the modulators may require administration twice or more times during a day.
  • 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.
  • 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.
  • an erythropoiesis- treatment composition comprising a modulator of Hedgehog (Hh) signalling and a pharmaceutically acceptable vehicle.
  • Hh Hedgehog
  • 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.
  • 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 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.
  • the modulator may comprise Shh, Ihh or Dhh, or a functional variant or fragment thereof.
  • the modulator comprises Dhh.
  • a "subject” maybe a vertebrate, mammal, or domestic animal.
  • 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 procedures.
  • 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.
  • 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.
  • 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.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • 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.
  • the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
  • 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
  • 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.
  • 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.
  • 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.
  • solutes or suspending agents for example, enough saline or glucose to make the solution isotonic
  • bile salts for example, enough saline or glucose to make the solution isotonic
  • acacia gelatin
  • sorbitan monoleate sorbitan monoleate
  • polysorbate 8o oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide
  • compositions 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ClustalW 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • BM was cultured for 18 hours, control or treated with rDhh, anti-Hh mab (5E1), rDhh and 5E1 together, and isotype control mab.
  • 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,
  • Photograph shows typical spleen from WT (left-hand) and Dhh-/- (right-hand).
  • 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).
  • (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.
  • MEP 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).
  • Lower bar chart shows the mean progenitor ratio in WT (filled bars) and Dhh-/- (open bars).
  • 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).
  • 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,
  • Photograph shows typical spleen from WT (left-hand) and Dhh-/- (right- hand) at 14 days after irradiation,
  • 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
  • RBC red blood cell
  • 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.
  • 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.
  • Bar charts show the mean fold change in expression in Dhh-/- compared to WT.
  • 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.
  • mice were irradiated with 4 Gy from a 6o Co 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.
  • 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).
  • 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.
  • Biotechnology as isotype control, in 5 ⁇ 1 of PBS for 60 minutes and washed with PBS and 0.5%BSA. Cells were then incubated with 2 ⁇ g of Biotinconjugated F(ab')2 fragment of donkey anti-goat IgG in 5 ⁇ 1 of PBS and 1.5% donkey serum for 30 minutes and washed with PBS and 0.5%BSA. Cells were finally incubated with:
  • CD16/CD32 in 5 ⁇ 1 of PBS for 5 minutes, then incubated with 2 ⁇ g anti-Ptch (R&D systems) or rat IgG (Santa Cruz Biotechnology), as isotype control, in 5 ⁇ 1 of PBS for 60 minutes and washed with PBS and 0.5%BSA. Cells were then incubated with 2 ⁇ g of Biotin-conjugated anti-rat IgG in 5 ⁇ 1 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 - c 3 ⁇ 4'5-5. For assessment of reticulocytes in blood, reticulocytes were counted from Giemsa-stained blood films.
  • 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 /).
  • Colony forming assays were performed using methocult methylcellulose based medium (StemCell Technologies). 2 x 10 6 BM cells and 2 x 10 7 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.
  • 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.
  • 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.
  • HPRT and Glii primers were as described Rowbotham et al., Blood. 20075109:3757- 3766.
  • Example 1 - Dhh and components of the Hh signalling pathway are expressed in adult spleen and bone marrow
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • Example 4 - Dhh is a negative regulator of erythrocyte differentiation during recovery following irradiation
  • 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).
  • 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
  • MEP cells defined as Sca-i-ckit+ progenitors, that are CD34- and FcyRII/III-
  • 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
  • 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 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.
  • 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).
  • RP:WP ratio was reduced to 2.5 in Dhh-/- spleen, and 3.7 in WT spleen.
  • Example 7 - Dhh modulates expression of Lm.02
  • 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.
  • Lmo2 is essential for erythrocyte differentiation, and is involved in the
  • 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.
  • 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).
  • 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.
  • 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
  • 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 is one of three Hh family members, and both Ihh and Shh are also expressed in the spleen.
  • Ihh and Shh are also expressed in the spleen.
  • Hh proteins can function as morphogens, signalling for distinct outcomes dependent on strength and duration of signal received.
  • Hh signal by Dhh-deficiency
  • Smo-deficiency which resulted in reduced stress-induced erythropoiesis.
  • Hh signal accelerated pre-TCR induced differentiation, although some Hh signal transduction was still required for differentiation.
  • Ihh has been shown to promote the earliest stages of haematopoiesis and vasculogenesis and to support definitive erythropoiesis in the mouse embryo.
  • 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.

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Abstract

L'invention concerne des méthodes de traitement d'états pathologiques caractérisés par une mauvaise érythropoïèse. Elle concerne également le traitement d'états pathologiques impliquant une mauvaise leucopoïèse myéloïde. L'invention concerne également des compositions pharmaceutiques destinées à être utilisées dans le traitement de tels états, et des méthodes de traitement.
PCT/GB2013/050586 2012-03-09 2013-03-08 Erythropoïèse WO2013132272A1 (fr)

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Citations (3)

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WO1998035020A2 (fr) * 1997-02-10 1998-08-13 The Presidents And Fellows Of Harvard College Procedes destines a moduler l'hematopoiese et la croissance vasculaire
WO2000074706A1 (fr) * 1999-06-08 2000-12-14 Lorantis Limited Utilisation therapeutique d'un inhibiteur d'un trajet de signalisation du herisson ou apparente au herisson
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