Modified thrombopoietin

Abstract

The present invention relates to isolated and modified thrombopoietin polypeptides, uses thereof in expanding stem cells and producing platelets, and therapeutic applications of such modified thrombopoietin polypeptides, stem cells and platelets.

Description

[0001] MODIFIED THROMBOPOIETIN

[0002] SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 15, 2023, is named Sequence_Listing_st26_13893335. xml and is 39,105 bytes in size.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to isolated and modified thrombopoietin polypeptides, uses thereof in expanding stem cells and producing platelets, and therapeutic applications of such modified thrombopoietin polypeptides, stem cells and platelets.

[0006] BACKGROUND TO THE INVENTION

[0007] The class I cytokine thrombopoietin (TPO) is a master regulator of hematopoiesis, inextricably controlling several aspects thereof, including hematopoietic stem cell maintenance as well as megakaryocyte differentiation and platelet production. Loss-of-function mutations in either TPO or it’s receptor, TpoR (also called MPL), cause thrombocytopenia and bone marrow failure.

[0008] The human THPO gene encodes a secreted protein, expressed primarily in the liver, composed of a signal peptide followed by two functional domains: an N-terminal M PL-binding domain and a C-terminal, so-called glycan domain. The N-terminal domain of TPO consists of a classical 4-helix bundle, similar to erythropoietin and growth hormone, held together by two conserved disulfide bridges. TPO has two non-identical binding sites for its receptor: a high- affinity site (site 1) and a lower affinity site (site 2). These are located on opposite sides of the 4-helical bundle, such that the active signaling complex comprises one molecule of TPO bound to two molecules of TpoR.

[0009] Given its involvement in the pathology of several diseases, it is desirable to provide TPO as a treatment. However clinically, wild type (WT) TPO is immunogenic, and cannot be used as a direct therapy. Alternative TPO receptor agonists (i.e. agonists of TpoR) including the small molecule eltrombopag and the peptibody romiplostim have emerged as key therapies for chronic immune thrombocytopenia (ITP) and severe aplastic anemia. These therapies are useful to induce the production of platelets for therapeutic treatment of such disease, but the induction of platelet production comes at the cost of increased hematopoietic stem cells (HSC) proliferation, risk of bone marrow fibrosis and an increased incidence of thrombosis.

[0010] Aside from the medical uses of TPO as a therapy, TPO is also used extensively in both ex vivo platelet production and in vitro culturing of stem cells. Although TPO is a key cytokine used for ex vivo platelet production and is used in all current protocols, there are significant issues with the number of platelets per megakaryocyte produced using current methods. This significantly impacts the translation of these methods into clinical settings.

[0011] TPO is also one of three key cytokines that are essential for the expansion of functional HSCs in vitro, and is used in the majority of new clinical protocols aimed at increasing HSC expansion for transplantation across a range of malignant and non-malignant hematopoietic disorders. Unfortunately, issues exist with TPO-induced HSC proliferation with TPO dose being critical for optimum HSC output and excessive HSC expansion leading to loss of multi-lineage regenerative capacity, HSC exhaustion i.e. the a progressive decline in the number of functional HSCs throughout ex vivo culture, and poor cell viability. Thus, balancing TPO-TpoR signaling is a critical factor in HSC maintenance and expansion both in vivo and in vitro.

[0012] There is a critical need to decouple the various functionalities of TPO, namely its ability to induce platelet production, proliferate HSCs, and differentiate HSCs such that its different uses can be optimized.

[0013] The prior lack of any structural information regarding the complete TPO-TpoR complex has limited the ability to create modified TPO variants with altered signaling capabilities that may be more suited to induce platelet production for use for example in the treatment of disease, or which may be more suited to proliferate HSCs for use for example in biotechnological production methods such as those described above.

[0014] One or more aspects or embodiments of the present invention seek to address at least these problems or one or more alternative problems in the art.

[0015] SUMMARY OF THE INVENTION

[0016] The invention described herein provides in a first aspect an isolated and modified thrombopoietin polypeptide (TPO) having an amino acid sequence according to SEQ ID NO: 1 or at least 70% identity thereto, or a functional fragment thereof, the amino acid sequence comprising at least one amino acid modification of the amino acids in the site 2 region thereof, wherein the site 2 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with low affinity.

[0017] Suitably a low binding affinity is a binding affinity in the micromolar range, suitably a low binding affinity is a Kd in the micromolar range. Suitably the amino acids which bind to the TPO receptor with low affinity may be determined by a person skilled in the art from the information provided herein regarding the crystal structure of TPO bound to the TPO receptor as provided in the examples and figures. Suitably, binding affinity may be measured by any known technique and as described in the examples herein, for example it may be measured by the technique described in Feese et al. (2004). For example, it may be measured by surface plasmon resonance (SPR) experiments measuring the binding of TPO as analyte to biotinylated MPL (TpoR) extracellular domain as ligand. Suitably, the TPO receptor is the human TPO receptor. Suitably the TPO receptor is formed of a dimer. Suitably, the amino acids within the site 2 region bind to the second of the two TPO receptor monomers which form the TPO receptor dimer. Suitably, for completeness, the amino acids within the site 1 region bind to the first of the two TPO receptor monomers which form the TPO receptor dimer.

[0018] In certain embodiments, the at least one amino acid modification at a position in the site 2 region thereof is selected from: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 of SEQ ID NO:1 , or a position corresponding thereto. Suitably such positions are surface exposed amino acids. Suitably such positions bind to the TPO receptor with low affinity. Suitably said positions, or corresponding positions thereto, make up the site 2 region of the TPO polypeptide.

[0019] In certain embodiments, the at least one amino acid modification at a position in the site 2 region thereof is selected from: R31 , K35, R38, R99, and R119 of SEQ ID NO:1 , preferably K35 and R119 of SEQ ID NO:1 , or a position corresponding thereto. Suitably such positions are surface exposed amino acids. Suitably such positions bind to the TPO receptor with low affinity.

[0020] In certain embodiments, the at least one amino acid modification at position R31 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R31A, R31 E, R31 H, R31Q. In certain embodiments, the at least one amino acid modification at position K35 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: K35A, K35E, K35H, K35M, K35Q.

[0021] In certain embodiments, the at least one amino acid modification at position R38 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R38A, R38E, R38H, R38Q.

[0022] In certain embodiments, the at least one amino acid modification at position R99 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R99A, R99E, R99H, R99Q, R99W.

[0023] In certain embodiments, the at least one amino acid modification at position R119 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R119A, R119C, R119E, R119H, and R119Q.

[0024] In certain embodiments, the at least one amino acid modification in the site 2 region thereof is selected from K35A, K35Q, R119E, and R119H of SEQ ID NO:1 , or a corresponding modification thereto.

[0025] In certain embodiments, the isolated and modified thrombopoietin polypeptide comprises or consists of the amino acid sequence shown in any of SEQ ID NO: 2 to 24.

[0026] In a second aspect there is provided an isolated nucleic acid molecule encoding the modified thrombopoietin polypeptide or a functional fragment thereof of the first aspect.

[0027] In a third aspect there is provided an expression construct comprising the isolated nucleic acid molecule of the second aspect, operably linked to a promoter.

[0028] In a fourth aspect there is provided a vector comprising the nucleic acid molecule of the second aspect, or the expression construct of the third aspect.

[0029] In a fifth aspect there is provided a host cell transformed with the nucleic acid molecule of the second aspect, or the expression construct of the third aspect, or the vector of fourth aspect.

[0030] In a sixth aspect there is provided a method of manufacturing a modified thrombopoietin polypeptide or a functional fragment thereof of the first aspect: (a) Providing a host cell of fifth aspect;

[0031] (b) Culturing the host cell under suitable conditions for the cell to produce the modified thrombopoietin polypeptide or a functional fragment thereof;

[0032] (c) Isolating the modified thrombopoietin polypeptide or a functional fragment thereof from the host cell or from the cell culture thereof.

[0033] In a seventh aspect there is provided a method of expanding and / or maintaining stem cells, the method comprising:

[0034] (a) Providing one or more stem cells;

[0035] (b) Culturing the stem cells in the presence of a modified thrombopoietin polypeptide or a functional fragment thereof according to the first aspect, under suitable conditions for the stem cells to proliferate.

[0036] In certain embodiments, the stem cells are hematopoietic stem cells.

[0037] In certain embodiments, the modified thrombopoietin polypeptide or a functional fragment thereof comprises an amino acid modification selected from: K35A, K35Q, R119E, and R119H of SEQ ID NO:1 , or a corresponding modification thereto, preferably the modified thrombopoietin polypeptide or a functional fragment thereof comprises the amino acid modification R199H, of SEQ ID NO:1 , or a corresponding modification thereto.

[0038] In certain embodiments, the method does not substantially induce differentiation of the stem cells.

[0039] In an eighth aspect there is provided a stem cell or a population of stem cells produced by the method of the seventh aspect or a therapeutic composition thereof.

[0040] In a ninth aspect there is provided the stem cell or population thereof of the eighth aspect, for use in the prevention or treatment of a disease.

[0041] In a tenth aspect there is provided a vector according to the fourth aspect or a therapeutic composition thereof for use in a method of gene therapy or cell therapy.

[0042] In a eleventh aspect there is provided a host cell according to the fifth aspect or a therapeutic composition thereof for use in a method of gene therapy or cell therapy. In a twelfth aspect there is provided a stem cell or population thereof according to the eighth aspect or a therapeutic composition thereof for use in a method of gene therapy or cell therapy.

[0043] In a thirteenth aspect there is provided a method of gene therapy or cell therapy for preventing or treating a disease in a subject, the method comprising administering an effective amount of a vector according to the fourth aspect to the subject.

[0044] In a fourteenth aspect there is provided a method of gene therapy or cell therapy for preventing or treating a disease in a subject, the method comprising administering an effective amount of a host cell according to the fifth aspect to the subject.

[0045] In a fifteenth aspect there is provided a method of gene therapy or cell therapy for preventing or treating a disease in a subject, the method comprising administering an effective amount of a stem cell or population thereof according to the eighth aspect to the subject.

[0046] In certain embodiments, the gene therapy or cell therapy are for the treatment of an immunological disorder, a haematological disorder or cancer.

[0047] In certain embodiments, the gene therapy or cell therapy are for the treatment of aplastic anemia, bone marrow failure syndromes, Multiple Sclerosis, leukaemia, lymphoma, or myeloma.

[0048] In certain embodiments, the cell therapy is autologous or allogeneic stem cell transplantation.

[0049] In a sixteenth aspect there is provided a method of ex vivo platelet production, the method comprising:

[0050] (a) Providing one or more stem cells;

[0051] (b) Culturing the stem cells in the presence of a modified thrombopoietin polypeptide or a functional fragment thereof according to the first aspect under suitable conditions for the stem cells to differentiate into platelets; and

[0052] (c) Optionally isolating the platelets.

[0053] In certain embodiments, the modified thrombopoietin polypeptide or a functional fragment thereof comprises an amino acid modification selected from: K35A, K35Q, R119E and R119H of SEQ ID NO:1 , or a corresponding modification thereto. In certain embodiments, step (a) may comprise (i) culturing the stem cells under suitable conditions to differentiate into megakaryocytes and (ii) culturing the megakaryocytes under suitable conditions to differentiate into platelets.

[0054] In a seventeenth aspect there is provided one or more platelets produced by the method of the sixteenth aspect, or a therapeutic composition thereof.

[0055] In an eighteenth aspect there is provided, one or more platelets or a therapeutic composition thereof of the seventeenth aspect for use in the prevention or treatment of a disease.

[0056] In an nineteenth aspect there is provided, one or more platelets or a therapeutic composition thereof of the seventeenth aspect for use in the prevention or treatment of an immunological disorder, a haematological disorder or cancer.

[0057] In an twentieth aspect there is provided, a method of treating or preventing a disease in a subject, the method comprising: administering an effective amount of the one or more platelets of the seventeenth aspect to the subject in need thereof.

[0058] In a twenty-first aspect there is provided, a method of treating or preventing an immunological disorder, haematological disorder or cancer in a subject, the method comprising administering an effective amount of the one or more platelets of the seventeenth aspect to the subject in need thereof.

[0059] In a twenty-second aspect there is provided a peptibody comprising one or more fragments of the modified thrombopoietin polypeptide according to the first aspect.

[0060] In one embodiment, the peptibody comprises the structure: (Fc1)-(L1)-P1-(L2)-P2-(L3)- (Fc2), wherein:

[0061] Fc is an Fc region of an immunoglobulin protein; at least one of Fc1 and Fc2 is present;

[0062] L1 , L2 and L3 are optional linkers;

[0063] P1 is a peptide fragment between 10 to 20 amino acids in length, derived from the site 1 region of the modified thrombopoietin polypeptide of the first aspect, wherein the site 1 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with high affinity;

[0064] P2 is a peptide fragment of between 10 to 20 amino acids in length, derived from the site 2 region of the modified thrombopoietin polypeptide of the first aspect. Suitably wherein the site 2 region consists of amino acids within the range of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with low affinity. Suitably wherein the site 2 region has been modified according to the first aspect. Suitably wherein the site 2 region comprises at least one amino acid modification. Suitably therefore the P2 peptide fragment is a fragment of 10 to 20 amino acids in length derived from within the region of positions 21 to 184 of SEQ ID NO: 1 , or a corresponding region thereto, and having at least one amino acid modification of a position therein which binds to the TPO receptor with low affinity.

[0065] In an twenty third aspect there is provided, a peptibody or a therapeutic composition thereof of the twenty second aspect for use in the prevention or treatment of a disease.

[0066] In an twenty fourth aspect there is provided, a peptibody or a therapeutic composition thereof of the twenty second aspect for use in the prevention or treatment of an immunological disorder, a haematological disorder, or cancer.

[0067] In an twenty fifth aspect there is provided, a method of treating or preventing a disease in a subject, the method comprising: administering an effective amount of the peptibody or a therapeutic composition thereof of the twenty second aspect to the subject in need thereof.

[0068] In a twenty sixth aspect there is provided, a method of treating or preventing an immunological disorder, haematological disorder or cancer in a subject, the method comprising administering an effective amount of peptibody or a therapeutic composition thereof of the twenty second aspect to the subject in need thereof.

[0069] In an twenty seventh aspect there is provided, an isolated and modified TPO polypeptide, a functional fragment thereof, or a therapeutic composition thereof of the first aspect for use in the prevention or treatment of a disease.

[0070] In an twenty eighth aspect there is provided, an isolated and modified TPO polypeptide, a functional fragment thereof, or a therapeutic composition thereof of the first aspect for use in the prevention or treatment of an immunological disorder, a haematological disorder or cancer.

[0071] In an twenty ninth aspect there is provided, a method of treating or preventing a disease in a subject, the method comprising: administering an effective amount of an isolated and modified TPO polypeptide, a functional fragment thereof, or a therapeutic composition thereof of the first aspect to the subject in need thereof.

[0072] In a thirtieth aspect there is provided, a method of treating or preventing an immunological disorder, haematological disorder or cancer in a subject, the method comprising administering an effective amount of an isolated and modified TPO polypeptide, a functional fragment thereof, or a therapeutic composition thereof of the first aspect to the subject in need thereof.

[0073] In certain embodiments, the immunological disorder, haematological disorder or cancer comprise thrombocytopenia and / or anemia.

[0074] In certain embodiments, the platelets are for use in preventing or treating thrombocytopenia or anemia in or caused by an immunological disorder, haematological disorder or cancer.

[0075] In certain embodiments, the thrombocytopenia comprises immune thrombocytopenia, chronic thrombocytopenia, or chronic immune thrombocytopenia.

[0076] In certain embodiments, the anemia comprises aplastic anemia, severe aplastic anemia, hypoplastic anemia, or severe hypoplastic anemia.

[0077] In a thirty-first aspect there is provided a pharmaceutical composition comprising the isolated and modified TPO polypeptide, or a functional fragment thereof, peptibody, stem cell or population thereof, host cell, or platelet as described herein.

[0078] DETAILED DESCRIPTION OF THE INVENTION

[0079] Features and embodiments further to the above aspects will now be described under the following headed sections, however any feature, embodiment or definition described herein is not limited to any particular aspect, and may be combined with any aspect in any workable combination. Workable combinations may be determined using techniques described in the Examples.

[0080] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0081] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0082] Identity” or “percent identity” refers to the degree of sequence variation between two given nucleic acid or amino acid sequences. For sequence comparison, typically, one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by visual inspection. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the world wide web at ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al., J. Mol. Biol.215: 403-410 (1990)). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1.

[0083] As used herein, "homologue" refers to a protein that is functionally equivalent to the referenced protein, but may have a limited number of amino acid substitutions, deletions, insertions or additions in the amino acid sequence. In order to maintain the function of the protein, the substitutions may be conservative substitutions, replacing an amino acid with one having similar properties. A homologue may refer to a protein which has an identity of at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% with the amino acid sequence of referred to. Algorithms for determining sequence identity are publicly available and include, e.g. BLAST, available through the National Center for Biotechnology Information (NCBI). One skilled in the art can determine if the sequences are similar to a degree that indicates homology and thus similar or identical function.

[0084] A person skilled in the art can obtain a polynucleotide encoding a homologue of each protein by appropriately introducing substitution, deletion, insertion, and / or addition to the DNA encoding the protein, using methods such as site-specific mutagenesis (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRL Press pp. 200 (1991)). Any reference to amino acid positions in respect of the modified TPO polypeptides described herein relate to the corresponding amino acid positions of wild-type human TPO as represented in SEQ ID NO: 1.

[0085] The term ‘TpoR’ ‘TPO receptor’ and ‘MPL’ may be used interchangeably herein to refer to the thrombopoietin (TPR) receptor or signalling complex, either in monomeric or homodimeric form.

[0086] The inventors have surprisingly shown that amino acid modifications of the TPO polypeptide, which were made possible and guided by their elucidation of the crystal structure of the TPO molecule bound to its receptor TpoR, can alter the stability of the active TPO-TpoR receptor dimers and thus display a range of altered functionalities compared to wild type (WT) TPO. These surprising effects include the following.

[0087] Modified TPO polypeptides disclosed herein may support increased cell growth compared to wild type TPO depending on the modification made. Modified TPO polypeptides disclosed herein may also activate downstream pathway signaling in target cells to a greater extent than wild type TPO, once bound to their receptor TpoR. Supporting increased cell growth and / or over-activating target cell downstream signaling are particularly advantageous properties for in vitro (i.e. ex vivo) stem cell expansion protocols such as HSC expansion protocols, wherein these effects may lead to increased stem cell product. Increased cell growth and / or overactivation of target cell TPO-induced signaling are also particularly advantageous for ex vivo platelet production protocols wherein the increase may lead to both increased stem cells as well as increased levels of platelets being produced by those stem cells, thus making such processes overall more efficient, cost-effective and economically viable.

[0088] Modified TPO polypeptides disclosed herein can also improve the preservation of HSCs i.e. HSC identity throughout in vitro culturing, or in other words, limit HSC exhaustion, without affecting their differentiation potential. Modified TPO polypeptides disclosed herein may also be capable of expanding and / or maintaining HSCs without significant loss of their multi-lineage regenerative capacity i.e. differentiation potential. Modified TPO polypeptides disclosed herein may also increase the viability of in vitro cultured, expanded, and / or maintained HSCs. These surprising effects are particularly advantageous for ex vivo stem cell expansion protocols employing TPO as a chemokine. HSC cell populations obtained through ex vivo cell expansion wherein a higher proportion of cells are HSCs, wherein cells including the HSCs are more viable, and / or wherein HSCs retain their ability for differentiation, may be more clinically useful. Modified TPO polypeptides disclosed herein may be capable of driving platelet production in HSCs without causing significant HSC expansion or differentiation in vitro and / or in vivo. This is particularly advantageous for in vivo, in vitro or ex vivo platelet production protocols employing TPO as a chemokine. Modified TPO polypeptides of the invention appear to enhance platelet production ~5-fold, and could therefore be used as a direct replacement for wild type TPO to increase platelet output.

[0089] Modified thrombopoietin (TPO) polypeptides

[0090] Aspects of the present invention relate to isolated and modified thrombopoietin (TPO) polypeptides or a functional fragment thereof. Suitably the TPO has been modified, suitably such that it contains one or more modifications relative to a wild type sequence, which may be defined as SEQ ID NO: 1. Suitably said modifications do not exist in nature. Suitably the TPO is also isolated, such that it is isolated from nature, suitably the TPO is isolated from a cell, or organism in which it is expressed.

[0091] TPO polypeptides disclosed herein may be modified versions of any TPO polypeptide, such as any TPO polypeptides that may be found in a public databases that are annotated as TPO and are believed to fulfil the same function as the TPO found in humans. TPO polypeptides disclosed herein may be modified versions of TPO found in any organism, suitably any animal, suitably any primate, suitably any human. TPO polypeptides disclosed herein may be modified versions of human TPO. In one embodiment, TPO polypeptides disclosed herein are a modified versions of SEQ ID NO: 1 (i.e. human TPO UniProt ID P40225).

[0092] Suitably, isolated and modified TPO polypeptides disclosed herein may have at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity to any TPO polypeptide in a public database that is annotated as TPO and is believed to fulfil the same function as the TPO found in humans, suitably to human TPO, suitably to SEQ ID NO: 1.

[0093] Suitably, an amino acid modification may be any modification that is possible to make to an amino acid. For example, deletions, insertions and / or substitutions of amino acid residues. Additionally or alternatively, modification may include post-translational modifications such as phosphorylation, methylation, acetylation, amidation, formation of pyrrolidone carboxylic acid, isomerization, hydroxylation, sulfation, flavin-binding, cysteine oxidation and nitrosylation. Suitably amino acid modification includes amino acid substitutions, such as substitutions to any amino acids, for example, substitution of an amino acid residue to alanine, arginine, asparagine, aspartic acid (aspartate), cysteine, glutamine, glutamic acid (glutamate), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, serine, selenocysteine, threonine, tryptophan, tyrosine, valine. Modification may include substitution or insertion of either L- or D-stereoisomers thereof. Modification may include substitution or insertion of any known synthetic amino acid analogs. For example, any modified amino acids including, 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine, beta-Aminoproprionic acid, 2-Aminobutyric acid, 4-Aminobutyric acid, piperidinic acid, 6-Aminocaproic acid, 2- Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid, 2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine, 2,2’-Diaminopimelic acid, 2,3-Diaminoproprionic acid, N- Ethylglycine, N-Ethylasparagine, Hydroxylysine, allo-Hydroxylysine, 3-Hydroxyproline, 4- Hydroxyproline, Isodesmosine, allo-lsoleucine, N-Methylglycine, sarcosine, N- Methylisoleucine, 6-N-Methyllysine, N-Methylvaline, Norvaline, Norleucine, Ornithine. Modification may include also include insertion of any other entity that may be incorporated into a polypeptide via peptide bonds.

[0094] Fragments of the isolated and modified TPO polypeptides disclosed herein may include fragments of a full-length TPO. The term "fragment" in reference to a polypeptide refers to a portion (i.e. , a subsequence) of a polypeptide. Suitably, isolated and modified TPO polypeptide fragments may include truncations of a full-length TPO, such as N-terminal truncations, C- terminal truncations, or both N-terminal and C-terminal truncations. Suitably, isolated and modified TPO polypeptide fragments may include any stretch of amino acids that is shorter than a full-length TPO polypeptide. Suitably, isolated and modified TPO polypeptide fragments may include any stretch of amino acids of the site 2 region of a full-length TPO (site 2 being defined elsewhere herein). Not wishing to be bound by theory, isolated and modified TPO polypeptide fragments may confer the effects of a full-length TPO or a full-length modified TPO disclosed herein so long as a sufficient number of active-site residues thereof are included in said fragments to confer such effects when bound to the TPO receptor (TpoR, a.k.a., MPL). As such, provided herein are functional fragments of a modified TPO as described here. A "functional fragment" refers to a protein fragment that retains function and / or activity as the full length protein. Suitably, isolated and modified TPO polypeptide fragments conferring the effects of a full-length TPO or a full-length modified TPO polypeptides as disclosed herein may be ascertained using the techniques described in the examples.

[0095] Suitably, the isolated and modified TPO polypeptide fragment may include any stretch of amino acids of SEQ ID NO: 1. Suitably, amino acids 10-50, 51-100, 101-150, 151-200, 201- 250, 251-300 or 301-353. Suitably, amino acids 21-184 or any subsequences thereof such as amino acids 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121- 130, 131-140, 141-150, 151-160, 161-170 or 171-184. For example, the modified TPO polypeptides described herein may comprise or consist of amino acids 21-184 of SEQ ID NO: 1 . Suitably which may be regarded as a functional fragment thereof.

[0096] The term "polypeptide" refers to a polymer in which the monomers are amino acid residues which are joined together through peptide bonds, covering all such sequences regardless of post-translational modification (e.g., phosphorylation or glycosylation) and / or complexation with additional polypeptides, and covering both naturally occurring polypeptides as well as recombinantly or synthetically produced ones. The term polypeptide encompasses peptides, which generally consist of on the order of a few tens of amino acids, to proteins, which can be of any length but are generally longer than peptides. As used herein the term ‘polypeptide’ may be substituted with the term ‘protein’.

[0097] Isolated and modified TPO polypeptides disclosed herein comprise at least one amino acid modification. Suitably at least one amino acid modification in the site 2 region, defined as amino acids within positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with low affinity. Suitably at least one amino acid modification of the surface-exposed amino acids thereof.

[0098] Suitably isolated and modified TPO polypeptides disclosed herein having at least one amino acid modification may have at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acid modifications. Suitably at least 1 amino acid modifications. Suitably at least 2 amino acid modifications. Suitably at least 3 amino acid modifications. Suitably at least 4 amino acid modifications. Suitably at least 5 amino acid modifications. Suitably at least 6 amino acid modifications. Suitably at least 7 amino acid modifications. Suitably at least 8 amino acid modifications. Suitably at least 9 amino acid modifications. Suitably at least 10 amino acid modifications. Suitably at least 11 amino acid modifications.

[0099] A skilled person will be aware of the concept of surface-exposed amino acids, however, for completeness, various non-limiting definitions are provided as follows. Suitably, surface- exposed amino acids are amino acids located on the surface of a protein, in particular, amino acids which have sidechains located on the surface of a protein. Suitably, surface-exposed amino acids are amino acids that are solvent-accessible in solution, in particular amino acids which have sidechains that are solvent-accessible in solution. Suitably, surface-exposed amino acids are amino acids that are available for interactions with other molecules, in particular amino acids which have sidechains that are available for interactions with other molecules, suitably in the case of TPO such other molecules include the TPO receptor (TpoR a.k.a., MPL). Suitably, surface-exposed amino acids are amino acids which may be comprised within the active site or sites of polypeptides. Suitably, surface-exposed amino acids are amino acids which are not buried within the internal 3D structure of the polypeptide, therefore surface-exposed amino acids are amino acids which are available for interactions with other molecules such as solvents, receptors and binding proteins.

[0100] Suitably, the isolated and modified TPO polypeptides disclosed herein may have at least one amino acid modification of the amino acids which bind to the TPO receptor with low affinity, suitably the surface-exposed amino acids thereof which bind to the TPO receptor with low affinity. Suitably at least one amino acid modification in the site 2 region thereof. Suitably, at least one amino acid modification of the surface-exposed amino acids of the site 2 region thereof. Suitably at least one amino acid modification of the surface-exposed amino acids of the site 2 region of the TPO according to SEQ ID NO: 1 , or a corresponding region thereof.

[0101] Suitably the site 2 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with low affinity. Suitably, the site 2 region of TPO is located on the opposite side of the 4-helical bundle structure of TPO compared to the surface-exposed amino acids in site 1 (site 1 described hereinbelow). Suitably, the site 2 region of TPO has a low binding affinity to TpoR, suitably a lower binding affinity than site 1 , suitably a binding affinity in the micromolar range (10-6) as measured by the technique described in Feese et al. (2004). Suitably, of the two TpoR receptor monomers with which TPO interacts, site 2 interacts with the second T poR monomer. Suitable amino acid positions within the site 2 region, and which make up the 2 site region, are described extensively herein.

[0102] Suitably the site 1 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with high affinity. Suitably, the site 1 region of TPO is located on the opposite side of the 4-helical bundle structure of TPO compared to the surface-exposed amino acids in site 2 (site 2 described elsewhere herein). Suitably, the site 1 region of TPO has a high binding affinity to TpoR, suitably a higher binding affinity than site 2, suitably a binding affinity in the nanomolar range (10-9) as measured by the technique described in Feese et al. (2004). Suitably a binding affinity (Kd) in the range of 3nM to 10nM. Suitably, of the two TpoR receptor monomers with which TPO interacts, site 1 interacts with the first TpoR monomer. Suitable amino acid positions within the site 1 region, and which make up the 1 site region, may be selected from one or more of the following: F162, R161 , R157, L37, G158, D66, F67, R138, L69, S68, and H154 of SEQ ID NO:1 or corresponding positions thereto. Suitably any such amino acid positions of the site 1 region may be modified as described herein, suitably in addition to one or more amino acid modifications in the site 2 region of the TPO polypeptide.

[0103] Suitably, the at least one amino acid modification in the site 2 region is of at least one amino acid which interacts with a TpoR, suitably with low affinity. Suitably, the isolated and modified TPO polypeptides disclosed herein may have an amino acid sequence according to SEQ ID NO: 1, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or may be a functional fragment thereof, and may comprise at least one amino acid modification, suitably at least one amino acid modification of the amino acids which bind to the TpoR with low affinity, suitably surface- exposed amino acids thereof, and / or suitably at least one amino acid modification in the site 2 region thereof. Suitably wherein the at least one amino acid modification is of an amino acid which interacts with TpoR.

[0104] Suitably, the site 2 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto, which bind to the TPO receptor with low affinity. Suitably, the isolated and modified TPO polypeptides disclosed herein may comprise at least one amino acid modification in the site 2 region thereof. Suitably, the isolated and modified TPO polypeptides disclosed herein may comprise at least one amino acid modification of the surface-exposed amino acids in the site 2 region thereof. Suitably the or each amino acid modification may be of a basic amino acid, suitably a basic amino acid which is surface exposed and / or which is located in the site 2 region thereof. Suitably, the isolated and modified TPO polypeptides disclosed herein may comprise at least one amino acid modification of the basic amino acids in the site 2 region thereof. Suitably, the isolated and modified TPO polypeptides disclosed herein may comprise at least one amino acid modification of the basic, surface-exposed amino acids in the site 2 region thereof. Suitably, wherein the site 2 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto, which bind to the TPO receptor with low affinity.

[0105] The terms “region corresponding thereto” or “position corresponding thereto” as used herein refer to the equivalent positions or regions in another TPO polypeptide, which is suitably a homologous polypeptide from a different organism (i.e., a paralogous polypeptide). Suitably, a corresponding region or position thereto may include the equivalent region or position in a TPO polypeptide found in any organism, suitably any animal, suitably any primate, suitably any human. It is possible to compare TPO polypeptides by sequence comparison and locating conserved regions that correspond to the amino acid positions or regions listed. The term “corresponding position thereto” refers to amino acids in a sequence of interest, which correspond to those amino acids of an identified reference sequence, typically herein the reference sequence is SEQ ID NO:1. Corresponding positions or regions may be determined by aligning the amino acid sequences of the proteins from the different species, using an alignment program such as BLAST® or ClustalW. Note that the corresponding positions or regions in a sequence of interest should be determined by comparison with a like for like reference sequence.

[0106] Unless otherwise stated, a “corresponding” amino acid position or region with reference to a given SEQ ID NO is determined using Geneious as a global alignment with free end gaps having the following parameters: cost matrix Blossum 62, gap open penalty 12, gap extension penalty 3, refinement iterations 2; or an equivalent program thereof. The term “equivalent program” as used herein refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical corresponding nucleotide or amino acid residue matches when compared to the corresponding alignment generated by the program provided above.

[0107] Corresponding regions or positions of other TPO proteins, such as homologues, may be determined by sequence alignment with SEQ ID NO:1 , or other methods known in the art. Suitably alignment techniques for determining corresponding positions to those described herein in relation to SEQ ID NO:1 are known to the skilled person and provided elsewhere herein.

[0108] Suitably the or each amino acid in the site 2 region which is modified may comprise a surface exposed amino acid, suitably selected from any of the lists or statements herein.

[0109] Suitably, the site 2 region, i.e. amino acids within positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with low affinity, may comprise amino acid positions selected from one or more of: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 of SEQ ID NO: 1 , or positions corresponding thereto. Suitably, the site 2 region, i.e. the amino acids within positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with low affinity may comprise amino acid positions P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 of SEQ ID NO: 1 , or positions corresponding thereto. Suitably said positions, or corresponding positions thereto, make up the site 2 region of the TPO polypeptide. Suitably, the site 2 region may comprise a modification at an amino acid position selected from one or more of: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 of SEQ ID NO: 1 or at a position corresponding thereto. Suitably, the site 2 region may comprise a modification at an amino acid positions selected from one or more of: R31 , K35, R38, R99, and R119 of SEQ ID NO: 1 , or at a position corresponding thereto.

[0110] Suitably, the isolated and modified TPO polypeptides disclosed herein may comprise at least one amino acid modification at a position in the site 2 region thereof selected from one or more of: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 of SEQ ID NO: 1 , or at a position corresponding thereto.

[0111] Suitably, the isolated and modified TPO polypeptides disclosed herein may comprise at least one amino acid modification at a position in the site 2 region thereof, or at least one amino acid modification of a basic amino acid at a position in the site 2 region thereof, selected from one or more of: R31 , K35, R38, R99, and R119 of SEQ ID NO: 1 or at a position corresponding thereto.

[0112] Suitably in any of the statements herein, the at least one amino acid modification may be of a basic amino acid, suitably a surface-exposed basic amino acid, suitably present at a position in the site 2 region of SEQ ID NO: 1 , or in a corresponding region thereto.

[0113] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of a modification at position R31 of SEQ ID NO: 1 or at a position corresponding thereto.

[0114] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of a modification at position K35, of SEQ ID NO: 1 or at a position corresponding thereto.

[0115] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of a modification at position R38 of SEQ ID NO: 1 or at a position corresponding thereto.

[0116] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of a modification at position R99 of SEQ ID NO: 1 or at a position corresponding thereto.

[0117] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of a modification at position R119 of SEQ ID NO: 1 or at a position corresponding thereto. Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , K35, R38 and R99 of SEQ ID NO: 1 or at positions corresponding thereto.

[0118] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , K35, R38 and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0119] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , K35, R99, and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0120] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , R38, R99, and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0121] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions K35, R38, R99, and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0122] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , K35, and R38 of SEQ ID NO: 1 or at positions corresponding thereto.

[0123] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , K35, and R99 of SEQ ID NO: 1 or at positions corresponding thereto.

[0124] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , K35, and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0125] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , R38, and R99 of SEQ I D NO: 1 or at positions corresponding thereto. Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , R38, and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0126] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 , R99, and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0127] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions K35, R38, and R99 of SEQ ID NO: 1 or at positions corresponding thereto.

[0128] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions K35, R38, and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0129] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R38, R99, and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0130] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 and K35 of SEQ ID NO: 1 or at positions corresponding thereto.

[0131] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 and R38 of SEQ ID NO: 1 or at positions corresponding thereto.

[0132] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 and R99 of SEQ ID NO: 1 or at positions corresponding thereto.

[0133] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R31 and R119 of SEQ ID NO: 1 or at positions corresponding thereto. Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions K35 and R38 of SEQ ID NO: 1 or at positions corresponding thereto.

[0134] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions K35 and R99 of SEQ ID NO: 1 or at positions corresponding thereto.

[0135] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions K35 and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0136] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R38 and R99 of SEQ ID NO: 1 or at positions corresponding thereto.

[0137] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R38 and R119 of SEQ ID NO: 1 or at positions corresponding thereto.

[0138] Suitably, the at least one amino acid modification in the site 2 region may comprise or consist of modifications at positions R99 and R119, of SEQ ID NO: 1 or at positions corresponding thereto.

[0139] Suitably in any of the statements above, the site 2 region of the TPO polypeptide is defined as the amino acids within positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with low affinity.

[0140] Suitably the modifications at the positions described herein are amino acid substitutions. Suitably the modifications at the positions described herein are non-conservative amino acid substitutions. Suitably the modifications at the positions described herein are amino acid substitutions to an amino acid selected from: A, Q, H, M, W, or E.

[0141] “Non-conservative substitution” refers to substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and may affect (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0142] Suitably, the modification at position R31 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R31A (SEQ ID NO: 2), R31 E (SEQ ID NO: 3), R31 H (SEQ ID NO: 4), and R31Q (SEQ ID NO: 5).

[0143] Suitably, the modification at position K35 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: K35A (SEQ ID NO: 6), K35E (SEQ ID NO: 7), K35H (SEQ ID NO: 9), K35M (SEQ ID NO: 10), and K35Q (SEQ ID NO: 8).

[0144] Suitably, the modification at position R38 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R38A (SEQ ID NO: 11), R38E (SEQ ID NO: 12), R38H (SEQ ID NO: 14), and R38Q (SEQ ID NO: 13).

[0145] Suitably, the modification at position R99 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R99A (SEQ ID NO: 15), R99E (SEQ ID NO: 16), R99H (SEQ ID NO: 18), R99Q (SEQ ID NO: 17), and R99W (SEQ ID NO: 19).

[0146] Suitably, the modification at position R119 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R119A (SEQ ID NO: 20), R119C (SEQ ID NO: 24), R119E (SEQ ID NO: 21), R119H (SEQ ID NO: 23), and R119Q (SEQ ID NO: 22).

[0147] In one embodiment, the isolated and modified TPO polypeptides disclosed herein may comprise at least one amino acid modification of the amino acids in the site 2 region thereof, at position R31 of SEQ ID NO:1 , or at a position corresponding thereto, selected from: R31A, R31 E, R31 H, and R31Q; at position K35 of SEQ ID NO:1 , or at a position corresponding thereto, selected from: K35A, K35E, K35H, K35M, and K35Q; at position R38 of SEQ ID NO:1 , or at a position corresponding thereto, selected from: R38A, R38E, R38H, and R38Q; at position R99 of SEQ ID NO:1 , or at a position corresponding thereto, selected from: R99A, R99E, R99H, R99Q, and R99W; and / or at position R119 of SEQ ID NO:1 , or at a position corresponding thereto, selected from: R119A, R119C, R119E, R119H, and R119Q; or any combination of the above. The term “corresponding modification” as used herein refers to the same amino acid modification at a corresponding position in a homologous TPO polypeptide from another organism (i.e., a paralogous polypeptide). Suitably, as defined hereinabove, corresponding position thereto may include an equivalent position in a TPO polypeptide such as a TPO homologue found in any organism, suitably any animal, suitably any primate, suitably any human, as defined hereinabove. Methods of determining homologous polypeptides and determining corresponding amino acid positions will be well known by those skilled in the art. For example, corresponding amino acid positions may be determined by methods such as sequence alignment.

[0148] Suitably, the at least one amino acid modification in the site 2 region thereof of SEQ ID NO:1 is selected from: K35A or K35Q, and / or R119E or R119H, or a corresponding modification thereto. Suitably, the at least one amino acid modification in the site 2 region thereof of SEQ ID NO:1 is selected from: K35A or K35Q, or a corresponding modification thereto. Suitably, the at least one amino acid modification in the site 2 region thereof of SEQ ID NO:1 is selected from: R119E or R119H, or a corresponding modification thereto. Suitably, the amino acid modifications in the site 2 region thereof, of SEQ ID NO:1 are selected from: K35A, K35Q, R119E, R119H, K35A and R119H; K35A and R119E; K35Q and R119E; or K35Q and R119H; or corresponding modifications thereto.

[0149] For example, the isolated and modified TPO polypeptide disclosed herein may comprise an amino acid sequence according to SEQ ID NO: 1 or having at least 70% identity thereto, or a functional fragment thereof, having the amino acid modification K35A, or a corresponding modification thereto. For example, the isolated and modified TPO polypeptide disclosed herein may comprise or consist of an amino acid sequence according to SEQ ID NO: 6.

[0150] For example, the isolated and modified TPO polypeptide disclosed herein may comprise an amino acid sequence according to SEQ ID NO: 1 or having at least 70% identity thereto, or a functional fragment thereof, having the amino acid modification K35Q, or a corresponding modification thereto. For example, the isolated and modified TPO polypeptide disclosed herein may comprise or consist of an amino acid sequence according to SEQ ID NO: 8.

[0151] For example, the isolated and modified TPO polypeptide disclosed herein may comprise an amino acid sequence according to SEQ ID NO: 1 or having at least 70% identity thereto, or a functional fragment thereof, having the amino acid modification R119E, or a corresponding modification thereto. For example, the isolated and modified TPO polypeptides disclosed herein may comprise or consist of an amino acid sequence according to SEQ ID NO: 21. For example, the isolated and modified TPO polypeptide disclosed herein may comprise an amino acid sequence according to SEQ ID NO: 1 or having at least 70% identity thereto, or a functional fragment thereof, having the amino acid modification R119H, or a corresponding modification thereto. For example, the isolated and modified TPO polypeptide disclosed herein may comprise or consist of an amino acid sequence according to SEQ ID NO: 23.

[0152] For example, the isolated and modified TPO polypeptide disclosed herein may comprise an amino acid sequence according to SEQ ID NO: 1 or having at least 70% identity thereto, or a functional fragment thereof, having the amino acid modifications K35A and R119H, or corresponding modifications thereto.

[0153] For example, the isolated and modified TPO polypeptide disclosed herein may comprise an amino acid sequence according to SEQ ID NO: 1 or having at least 70% identity thereto, or a functional fragment thereof, having the amino acid modifications K35A and R119E, or corresponding modifications thereto.

[0154] For example, the isolated and modified TPO polypeptide disclosed herein may comprise an amino acid sequence according to SEQ ID NO: 1 or having at least 70% identity thereto, or a functional fragment thereof, having the amino acid modifications K35Q and R119E, or corresponding modifications thereto.

[0155] For example, the isolated and modified TPO polypeptide disclosed herein may comprise an amino acid sequence according to SEQ ID NO: 1 or having at least 70% identity thereto, or a functional fragment thereof, having the amino acid modifications K35Q and R119H, or corresponding modifications thereto.

[0156] Isolated and modified TPO polypeptides as described herein alone are likely to be removed in vivo either by renal filtration, by cellular clearance mechanisms in the reticuloendothelial system, or by proteolytic degradation. Attachment to a vehicle may improve the therapeutic value of an isolated and modified TPO polypeptide as described herein by reducing degradation of the binding agent and / or increasing half-life, reducing toxicity, reducing immunogenicity, and / or increasing the biological activity of the binding agent.

[0157] In further aspect of the invention there is provided a conjugate comprising an isolated and modified TPO polypeptide, or functional fragment thereof, as described herein in association with a vehicle. Any uses or methods described herein in relation to the modified TPO polypeptide or functional fragment thereof may equally apply to the conjugate.

[0158] Exemplary vehicles include Fc domains; linear polymers such as polyethylene glycol (PEG), polylysine, dextran; a branched chain polymer (see for example U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981 ; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20, 1993; WO 93 / 21259 by Frechet et al., published 28 Oct. 1993); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor.

[0159] An isolated and modified TPO polypeptide may also be covalently or noncovalently associated with a carrier molecule, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see, for example, U.S. Patent 4,289,872 to Denkenwalter et al, issued September 15, 1981 ; 5,229,490 to Tarn, issued July 20, 1993; WO 93 / 21259 by Frechet et al, published 28 October 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide. Other possible carriers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Patent Nos: 4,640,835, 4,496,689, 4,301 ,144, 4,670,417, 4,791 ,192 and 4,179,337. Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)- polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide / ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers. A preferred such carrier is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be straight chain or branched. The average molecular weight of the PEG will preferably range from about 2 kDa to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kDa to about 10 kDa. The PEG groups will generally be attached to the compounds of the invention via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation / ligation methods through a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group) to a reactive group on the target compound (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group

[0160] Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligo saccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligo saccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser / Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids not counting proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N- acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.

[0161] Suitably the isolated and modified TPO polypeptides, or functional fragments thereof, disclosed herein may act as neutral antagonists, partial agonists, or super agonists of the TPO receptor TpoR (MPL).

[0162] Neutral antagonists

[0163] Isolated and modified TPO polypeptides or a functional fragment thereof disclosed herein may act as neutral antagonists of TpoR. Suitably, any isolated and modified TPO polypeptide or a functional fragment thereof disclosed herein may act as a neutral antagonist of TpoR which is measurable using the techniques described in the Examples. Suitably such an activity profile may be useful in certain applications of TPO polypeptides or a functional fragment thereof.

[0164] Suitably, an isolated and modified TPO polypeptide or a functional fragment thereof as described herein, may act as a neutral antagonist of TpoR. In particular, an isolated and modified TPO polypeptide comprising an amino acid sequence according to SEQ ID NO: 1 , or having at least 70% identity thereto, or a functional fragment thereof, having the modification K35E, or a corresponding modification thereto, may act as a neutral antagonist of TpoR.

[0165] Suitably, neutral antagonists may fail to activate downstream signalling pathways after binding to TpoR, said downstream signalling pathways suitably comprising any one or more of the proteins STAT1 , AKT, MEK1 , STAT3, ERK, STAT5, JAK2, TOR, CREB and IRF-1. Suitably, isolated and modified TPO polypeptides disclosed herein may act as neutral antagonists of TpoR. Suitably, any isolated and modified TPO polypeptide disclosed herein may fail to activate downstream signalling pathways after binding to TpoR. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a functional fragment thereof, and comprising at least one amino acid modification in the site 2 region thereof, selected from: K35E, or a corresponding modification thereto may fail to activate downstream signalling pathways after binding to TpoR. Activation of downstream signalling pathways upon exposure to a TPO polypeptide or a functional fragment thereof, may be determined and measured using the techniques described in the Examples or other well-known methods in the art such as RNA analysis methods or florescent microscopy based methods. Such as phospho-flow cytometry.

[0166] Suitably, neutral antagonists may not affect cell growth (i.e. expansion, proliferation, or increase in cell number over time of culturing) compared to a wild type TPO polypeptide. Suitably, neutral antagonists may not affect the cell growth of TPO-dependent cells compared to a wild type TPO polypeptide. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a functional fragment thereof, and comprising at least one amino acid modification in the site 2 region thereof selected from: K35E or a corresponding modification thereto, may not affect cell growth compared to wild type TPO polypeptide, suitably it may not affect the cell growth of TPO-dependent cells compared to wild type TPO polypeptide. Cell growth, suitably of TPO-dependent cells, upon exposure to wild type or isolated and modified TPO polypeptides or a functional fragment thereof may be determined and measured using the techniques described in the Examples or other well know methods in the art, such as optical density measurement methods or cell counting (such as FACs or flow cytometry).

[0167] As such, an isolated and modified TPO polypeptide comprising or consisting of an amino acid sequence according to SEQ ID NO: 7 may act as a neutral antagonist of the TPO receptor (TpoR / MPL).

[0168] Partial agonists

[0169] Alternatively, isolated and modified TPO polypeptides or a functional fragment thereof disclosed herein may act as partial agonists of TpoR. Suitably, any isolated and modified TPO polypeptide disclosed herein may act as a partial agonist of TpoR which is measurable using the techniques described in the Examples. Suitably such an activity profile may be useful in certain applications of TPO polypeptides or a functional fragment thereof. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a functional fragment thereof, and comprising a modification in the site 2 region thereof at a position selected from: K35 or R119, or a corresponding position thereto, may act as a partial agonist of TpoR.

[0170] Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a functional fragment thereof, and comprising a modification in the site 2 region thereof selected from K35A, K35Q, and / or R119E of SEQ ID NO: 1 or a corresponding modification thereto, may act as a partial agonist of TpoR.

[0171] Suitably, partial agonists may activate downstream signalling pathways to a lesser extent after binding to TpoR compared to a wild type TPO polypeptide, said downstream signalling pathways comprising any of proteins STAT1 , AKT, MEK1 , STAT3, ERK, STAT5, JAK2, TOR, CREB and IRF-1. Suitably, isolated and modified TPO polypeptides disclosed herein may activate downstream signalling pathways to a lesser extent after binding to TpoR compared to a wild type TPO polypeptide. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a fragment thereof, and comprising at least one amino acid modification in the site 2 region thereof, selected from: K35A, K35Q, R119E or a corresponding modification thereto, may activate downstream signaling pathways to a lesser extent after binding to TpoR compared to a wild type TPO polypeptide. Activation of downstream signalling pathways upon exposure to wild type or isolated and modified TPO polypeptides, suitably comprising any of proteins STAT1 , AKT, MEK1 , STAT3, ERK, STAT5, JAK2, TOR, CREB and IRF-1 , may be determined and measured using the techniques described in the Examples or as described herein. Suitably such as phospho-flow cytometry.

[0172] Suitably, partial agonists may support less cell growth compared to a wild-type TPO polypeptide, suitably partial agonists may support less cell growth of TPO-dependent cells compared to a wild-type TPO polypeptide. Suitably, isolated and modified TPO polypeptides or a functional fragment thereof disclosed herein may support less cell growth compared to a wild-type TPO polypeptide, suitably the less cell growth of TPO-dependent cells. Cell growth, suitably of TPO-dependent cells, upon exposure to wild type or isolated and modified TPO polypeptides or a functional fragment thereof may be determined and measured using the techniques described in the Examples or other well know methods in the art, such as optical density measurement methods or cell counting (such as FACs or flow cytometry).

[0173] Suitably, partial agonists may provide reduced activation of the TPO receptor (TpoR) compared to a wild type TPO polypeptide. Suitably reduced activation of the TPO receptor may comprise a reduction in dimerization of the TPO receptor in the presence of a TPO polypeptide. Suitably isolated and modified TPO polypeptides or a functional fragment thereof disclosed herein may provide a reduction in dimerization of TpoR compared to a wild type TPO polypeptide of 30%, 40%, 50%, 60%, 70% less TpoR dimerization, preferably 70% less TpoR dimerization than in the presence of a wild type TPO polypeptide. Suitably, isolated and modified TPO polypeptides or a functional fragment thereof disclosed herein may provide 30%, 40%, 50%, 60%, 70% less TpoR dimerization than a wild type TPO polypeptide, preferably 70% less TpoR dimerization than a wild type TPO polypeptide. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a fragment thereof, and comprising at least one amino acid modification in the site 2 region thereof, selected from: K35A, K35Q, and / or R119E of SEQ ID NO 1 , or corresponding modifications thereto, may may provide reduced dimerization of TpoR compare to a wild type TPO polypeptide, suitably 30%, 40%, 50%, 60%, 70% less TpoR dimerization than a wild type TPO polypeptide, preferably 70% less TpoR dimerization than a wild type TPO polypeptide . Activation and / or dimerization of TPO receptor in the presence of a TPO polypeptide or a functional fragment thereof may be measured by the techniques used in the examples, such as single molecule Forster resonance energy transfer (smFRET).

[0174] As such, an isolated and modified TPO polypeptide comprising or consisting of an amino acid sequence according to SEQ ID NOs: 6, 8 or 21 may act as a partial agonist of the TPO receptor (TpoR / MPL).

[0175] Super agonists

[0176] Alternatively, isolated and modified TPO polypeptides or a functional fragment thereof disclosed herein may act as super agonists of TpoR. Suitably, any isolated and modified TPO polypeptide disclosed herein may act as a super agonist of TpoR which is measurable using the techniques described in the Examples. Suitably such an activity profile may be useful in certain applications of TPO polypeptides or a functional fragment thereof. Suitably, super agonists may activate downstream signaling pathways to a greater extent after binding to TpoR, said downstream signaling pathways suitably comprising any of proteins STAT1 , AKT, MEK1 , STAT3, ERK, STAT5, JAK2, TOR, CREB and IRF-1 , in particular JAK2, STAT1 STAT3, STAT5. Suitably, isolated and modified TPO polypeptides or a functional fragment thereof disclosed herein may activate downstream signaling pathways to a greater extent after binding to TpoR, compared to a wild type TPO polypeptide. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a fragment thereof, and comprising a modification at a position selected from: R119 or a position corresponding thereto, may activate downstream signaling pathways to a greater extent after binding to TpoR than a wild type TPO polypeptide. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a fragment thereof, and comprising at least one amino acid modification in the site 2 region thereof, selected from R119H, or a corresponding modification thereto, may activate downstream signaling pathways to a greater extent after binding to T poR than a wild type TPO polypeptide.

[0177] Activation of downstream signaling pathways upon exposure to a TPOs polypeptide, suitably comprising any of proteins STAT1 , AKT, MEK1 , STAT3, ERK, STAT5, JAK2, TOR, CREB and IRF-1 , may be determined and measured using the techniques described in the Examples. Such as phospho-flow cytometry.

[0178] Suitably, super agonists may provide or stimulate increased cell growth (i.e. expansion, proliferation or increase in number of cells over time of culturing) compared to a wild-type TPO polypeptide, suitably super agonists may provide or stimulate increased cell growth of TPO- dependent cells compared to a wild-type TPO polypeptide. Suitably, isolated and modified TPO polypeptides or a functional fragment thereof disclosed herein may provide increased cell growth compared to a wild-type TPO polypeptide. Suitably, isolated and modified TPO polypeptides disclosed herein may provide increased cell growth of TPO-dependent cells compared to a wild-type TPO polypeptide. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a fragment thereof, and comprising a modification at a position selected from: R119, or a position corresponding thereto, may support greater cell growth compared to wild-type TPO. Suitably, an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a fragment thereof, and comprising at least one amino acid modification in the site 2 region thereof, selected from R119H, or a corresponding modification thereto, may provide increased cell growth compared to wild-type TPO. Cell growth, suitably of TPO-dependent cells, upon exposure to wild type or isolated and modified TPO polypeptides or a functional fragment thereof may be determined and measured using the techniques described in the Examples or other well know methods in the art, such as optical density measurement methods or cell counting (such as FACs or flow cytometry).

[0179] As such, an isolated and modified TPO polypeptide comprising or consisting of an amino acid sequence according to SEQ ID NO: 23 may act as a super agonist of the TPO receptor (TpoR / MPL).

[0180] Nucleic acid molecules

[0181] Also provided herein are nucleic acid molecules encoding an isolated and modified TPO polypeptide, or a functional fragment thereof, as disclosed herein. Preferably, said nucleic acid molecule is an isolated nucleic acid molecule.

[0182] An “isolated” biological molecule is substantially separated away from other biological molecules with which the molecule is normally associated, such as, from the proteins, or chromosomal or extrachromosomal DNA of a cell in which the biological molecule naturally occurs. A nucleic acid molecule may be an isolated nucleic acid molecule when it comprises a transgene or part of a transgene present in the genome of another organism. The term also embraces biological molecules that are biochemically purified so as to substantially remove contaminating biological molecules and other cellular components. Isolated nucleic acid molecules are substantially free of sequences (such as protein encoding sequences) that naturally flank the nucleic acid (i.e. , sequences located at the 5' and 3' ends of the nucleic acid molecule) in the genomic DNA of the organism from which the nucleic acid molecule is derived. For example, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid molecule is derived. The isolated nucleic acid molecule may be flanked by its native genomic sequences that control its expression in the cell, for example, the native promoter, or native 3 ' untranslated region. Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a functional fragment thereof, and comprising at least one amino acid modification at a position in the site 2 region thereof, wherein the site 2 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto which bind to the TPO receptor with low affinity.

[0183] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0184] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , K35, R38, R99, and R119 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0185] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , K35, R38 and R99 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0186] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , K35, R38 and R119; of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof. Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , K35, R99, and R119 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0187] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , R38, R99, and R119 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0188] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from K35, R38, R99, and R119 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0189] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , K35, and R38 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0190] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , K35, and R99 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0191] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , K35, and R119 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0192] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , R38, and R99 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0193] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , R38, and R119 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0194] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 , R99, and R119 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0195] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from K35, R38, and R99 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0196] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from K35, R38, and R119 of SEQ ID NO: 1 or at a position corresponding thereto, or any combination thereof.

[0197] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R38, R99, and R119 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof.

[0198] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 and K35 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof.

[0199] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 and R38 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof.

[0200] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 and R99 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof.

[0201] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R31 and R119 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof. Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from K35 and R38 of SEQ ID NO: 1 or positions corresponding thereto.

[0202] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from K35 and R99 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof.

[0203] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from K35 and R119 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof.

[0204] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R38 and R99 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof.

[0205] Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R38 and R119 of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof. Suitably, there is provided herein a nucleic acid molecule encoding an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1 , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto preferably at least 70% identity thereto, or a functional fragment thereof, comprising at least one amino acid modification at a position selected from R99 and R119, of SEQ ID NO: 1 or positions corresponding thereto, or any combination thereof.

[0206] Suitably, the nucleic acid encodes an isolated and modified TPO polypeptide disclosed herein wherein the modification at position R31 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R31A, R31 E, R31 H, and R31Q.

[0207] Suitably, the nucleic acid encodes an isolated and modified TPO polypeptide disclosed herein wherein the modification at position K35 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: K35A, K35E, K35H, K35M, and K35Q.

[0208] Suitably, the nucleic acid encodes an isolated and modified TPO polypeptide disclosed herein wherein the modification at position R38 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R38A, R38E, R38H, and R38Q.

[0209] Suitably, the nucleic acid encodes an isolated and modified TPO polypeptide disclosed herein wherein the modification at position R99 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R99A, R99E, R99H, R99Q, and R99W.

[0210] Suitably, the nucleic acid encodes an isolated and modified TPO polypeptide disclosed herein wherein the modification at position R119 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R119A, R119C, R119E, R119H, and R119Q.

[0211] Suitably the nucleic acid of the invention may encode any modified TPO polypeptide described hereinabove or a functional fragment thereof, having any amino acid modification or combination of amino acid modifications described herein in relation to the TPO polypeptide.

[0212] Once a DNA sequence encoding a native sequence of the TPO polypeptide is isolated, it can be modified to encode a modified TPO polypeptide or functional fragment thereof as described. The modifications to the nucleic acid sequence can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, and PCR mutagenesis. Site- directed mutagenesis [Carter et al., Nucl. Acids Res. 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the nucleic acid molecules to produce a nucleic acid molecule encoding a modified TPO polypeptide or functional fragment thereof as described.

[0213] Expression constructs & Vectors

[0214] Also provided herein are expression constructs and vectors comprising the nucleic acid molecules encoding an isolated and modified TPO polypeptide, or a functional fragment thereof, as described herein. The expression constructs and vectors, which include at least one nucleic acid molecule of the present invention inserted therein may be any construct or vector capable of delivering the nucleic acid molecule into a host or host cell and allowing expression of the nucleic acid molecule to provide a functional modified TPO polypeptide as described herein or functional fragment thereof.

[0215] Such constructs or vectors may contain heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that may be derived from a species other than the species from which the nucleic acid molecule molecule(s) are derived. The construct or vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically the vector is a virus or a plasmid.

[0216] Expression constructs of the invention may include one or more regulatory sequences operably linked to the nucleic acid molecule of interest, suitably encoding an isolated and modified TPO polypeptide, or a functional fragment thereof, as described herein. For example, the expression vectors can contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal. Such a portion of a vector may be referred to as an expression construct. Suitably therefore vectors of the present invention may comprise an expression construct of the invention. The expression construct may include one or more regulatory sequences that are functional in host cells as described herein. Thus allowing expression of the nucleic acid molecule encoding a modified TPO polypeptide of the invention in a host cell.

[0217] "Expression construct" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operably linked to the nucleic acid molecule of interest, which is optionally further operably linked to termination signal sequences. It also typically comprises sequences required for proper translation of the nucleic acid molecule sequence. The expression construct comprising the nucleic acid molecule of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression construct may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression construct is heterologous with respect to the host, i.e. , the particular nucleic acid molecule of the expression construct does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleic acid molecule sequence in the expression construct may be under the control of, for example, a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus.

[0218] An expression construct may include transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region of the nucleic acid of interest is operably linked under the transcriptional control of the transcriptional initiation region and a transcriptional and translational termination region. These control regions may be native to a wild type nucleic acid encoding a TPO polypeptide or may be derived from exogenous sources, including species-specific endogenous promoters. In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In addition to constitutive and inducible promoters, strong promoters (e.g., T7, CMV, and the like) find use in the expression constructs described herein, particularly where high expression levels are desired in an in vivo (cell-based) or in an in vitro expression system. Other exemplary promoters include mouse mammary tumor virus (MMTV) promoters, Rous sarcoma virus (RSV) promoters, adenovirus promoters, the promoter from the immediate early gene of human CMV, and the promoter from the long terminal repeat (LTR) of RSV. Alternatively, the promoter can also be provided by, for example, a 5'IITR of a retrovirus.

[0219] For example, the nucleic acid of interest may be under the control of an inducible promoter, such as tetracycline-controlled transcriptional activation where transcription is reversibly turned on (Tet-On) or off (Tet-Off) in the presence of the antibiotic tetracycline or a derivative thereof, such as doxycycline. Other inducible promoters include a promoter capable of inducing expression of the downstream nucleic acid of interest by binding to a complex including a tetracycline antibiotic (tetracycline, doxycycline, or the like) and a tetracycline transactivator in a case where the external stimulus is the presence of the tetracycline antibiotic; a promoter capable of inducing expression of the downstream nucleic acid of interest by release of a tetracycline repressor in a case where the external stimulus is the absence of a tetracycline antibiotic; a promoter capable of inducing expression of the downstream nucleic acid of interest by binding of an ecdysteroid (ecdysone, muristerone A, ponasterone A, or the like) to an ecdysone receptor-retinoid receptor complex in a case where the external stimulus is the presence of the ecdysteroid; and a promoter capable of inducing expression of the downstream nucleic acid of interest by binding of FKCsA to a complex including a Gal4 DNA binding domain fused to FKBP12 and a VP16 activator domain fused to cyclophilin in a case where the external stimulus is the presence of FKCsA.

[0220] The expression construct may comprise, as necessary, an enhancer, a silencer, a selection marker gene (for example, a drug resistance gene such as a neomycin resistance gene), an SV40 replication origin, and the like. Further, those skilled in the art could construct an expression construct capable of inducing expression of a modified TPO polypeptide as described herein at a desired expression level by appropriately selecting a combination of known enhancers, silencers, selection marker genes, terminators, and so forth in consideration of the type of the promoter utilized and so on. In addition, as necessary, an expression construct may also be introduced into a host cell that is capable of constantly expressing in the nucleus a factor (for example, tetracycline transactivator, a tetracycline repressor, an ecdysone receptor-retinoid receptor complex, a complex including a Gal4 DNA binding domain fused to FKBP12 and aVP16 activator domain fused to cyclophilin) for inducing expression of the modified TPO polypeptides described herein in response to an external stimulus.

[0221] Expression constructs generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid molecules (such as, in the present invention, a nucleic acid molecule encoding a modified TPO polypeptide as described herein). A selectable marker operative in the host cell may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems; thus allowing it to be maintained in different organisms, for example in mammalian cells for expression and in a prokaryotic host for cloning and amplification. In addition the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

[0222] The expression constructs may also comprise transcription termination regions. Where transcription terminations regions are used, any termination region may be used in the preparation of the expression constructs. For example, the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleic acid molecule of interest, may be native to the host cell, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleic acid molecule of interest encoding modified TPO polypeptide, the host cell, or any combination thereof).

[0223] In addition, other sequence modifications can be made to the nucleic acid molecules of the invention. For example, additional sequence modifications that are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon / intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression.

[0224] A “vector” is a replicon, such as plasmid, phage, viral construct, cosmid, bacterial artificial chromosome, derived artificial chromosome or yeast artificial chromosome to which another heterologous DNA segment may be inserted. In some instances a vector may be a chromosome such as in the case of an arm exchange from one endogenous chromosome engineered to comprise a recombination site to a synthetic chromosome. Vectors are used to transduce and express a DNA segment, such as a nucleic acid or an expression construct comprising a nucleic acid encoding a modified TPO polypeptide or functional fragment thereof as described herein in a host cell.

[0225] One of skill in the art would be well-equipped to construct a vector through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference). Exemplary vectors that may be used include but are not limited to those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. For example, plasmid vectors such as pZEM-hTHPO, pD649, pBR322, plIC 19 / 18, plIC 118, 119 and the M13 mp series of vectors may be used. Bacteriophage vectors may include Agt10, Agt11 , Agt18-23, AZAP / R and the EM BL series of bacteriophage vectors. Cosmid vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors. Additional vectors include bacterial artificial chromosomes (BACs) based on a functional fertility plasmid (F-plasmid), yeast artificial chromosomes (YACs), and P1-derived artificial chromosomes, DNA constructs derived from the DNA of P1 bacteriophage (PACS). Alternatively and preferably, recombinant virus vectors may be engineered, including but not limited to those derived from viruses such as herpes virus, retroviruses, vaccinia virus, poxviruses, adenoviruses, lentiviruses, adeno-associated viruses or bovine papilloma virus. In one embodiment, the vector of the present invention is pZEM- hTHPO (Zymogenetics). . In one embodiment, the vector of the present invention is pD649 (ATLIM).

[0226] Methods for introducing vectors into host cells are known and include, as non-limiting examples stable transformation methods, transient transformation methods, and virus mediated methods. The vectors described herein may be introduced into the host cell by for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes and the like. For example, transient transformation methods include microinjection, electroporation, or particle bombardment. Suitable means of transformation are described in more detail below.

[0227] Vectors may also include selectable markers. The phrase “selectable marker” refers to a protein that enables the separation of host cells expressing the marker from those that lack or do not express it. The selectable marker may be a fluorescent marker, for instance.

[0228] Expression of the marker by host cells having successfully integrated the nucleic acid molecule allows the isolation of these cells using methods such as, for example, FACS (fluorescent activated cell sorting). Alternatively, expression of a selectable marker may confer an advantageous property to the host cell that allows survival of only those cells carrying the gene. For example, the marker protein may allow for the selection of the host cell by conferring an antibiotic resistance to the host cell. Consequently, when host cells are cultured in medium containing said antibiotic, only cell clones expressing the marker protein that mediates antibiotic resistance are capable of propagating, indicating the successful integration of the intended nucleic acid, expression construct, or vector.

[0229] Host cells & Methods of Manufacturing Modified TPO polypeptides

[0230] Also provided herein are host cells comprising nucleic acid molecules, expression constructs and / or vectors as described herein.

[0231] The host cell may be any cell that is suitable for expression of the nucleic acid molecules as described herein to produce modified TPO polypeptides, or functional fragments thereof, as described herein either in vitro or in vivo.

[0232] Suitably the host cell can be any animal cell, a vertebrate cell, a mammalian cell, a human cell, a plant cell, a nematodal cell, an invertebrate cell such as an insect cell or a mollusc cell, a stem cell derived of any of the foregoing, or a fungal cell or a yeast cell or a bacterial cell. Suitably the host cell is a mammalian cell. Suitably such as a human or rodent or bovine cell, cell line or cell strain. Examples of specific mammalian cells suitable as host cells described herein are mouse myeloma (NSO)-cell lines, Chinese hamster ovary (CHO)-cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, MDCK, NIH3T3, PC12, BHK (baby hamster kidney cell), VERO, SP2 / 0, YB2 / 0, YO, C127, L cell, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EBI, EB2, EB3, oncolytic or hybridoma-cell lines. Eukaryotic cells also include avian cells, cell lines or cell strains, such as for example, EBx® cells, EB14, EB24, EB26, EB66, or EBvl3, an insect cell (e.g., Sf9, Mimic Sf9, Sf21 , High Five™ (BT1-TN-5B1-4), or BT1-Ea88 cells), an algae cell (e.g., of the genus Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, or Ochromonas), or a plant cell ((e.g., cells from monocotyledonous plants (e.g., maize, rice, wheat, or Setaria), or from a dicotyledonous plants (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens or Arabidopsis)).

[0233] Suitable host cells are commercially available, for example, from culture collections such as the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0234] In particular a host cell may be an Expi293F cell, UT-7 / TPO cell, HEK-293T cell, HEK293S GnTI- cell (e.g., ATCC CRL-3022) or a Spodoptera frugiperda (Sf9) ovarian cell (e.g., ATCC CRL-1711).

[0235] In one embodiment, the host cell may be a an Expi293F cell or a HEK-293T cell. Suitably such cells are capable of being transformed with an expression construct or vector described herein and cultured under suitable conditions to produce the modified TPO polypeptides of the invention. Advantageously, the use of mammalian host cells provides the innate ability to glycosylate the TPO polypeptide as required during production, suitably during translation or post-translationally.

[0236] In some other examples, the host cell may be a stem cell. For example, the host cell may be a haematopoietic stem cell (HSC). Suitably HSCs are described elsewhere herein. Suitably the HSC may have been expanded using the methods described herein (or alternatively expanded by methods known in the art) and is capable of expressing a modified TPO polypeptide or functional fragment thereof as described herein (for example, the HSC may have been transformed to comprise a nucleic acid molecule, expression construct or vector as described herein). In some embodiments, the host cell is capable of expressing a modified TPO polypeptide, or functional fragments thereof, as described herein in a subject in need thereof.

[0237] Such host cells, in particular stem cells, capable of expressing a modified TPO polypeptide or functional fragment thereof as described herein may be for use in methods of gene therapy or cell therapy described elsewhere herein.

[0238] The nucleic acid molecules, expression constructs and vectors described herein can be delivered to the host cell by any method known in the art. The terms transfection and transformation refer to the taking up of exogenous nucleic acid, e.g., an expression construct or recombinant vector, by a host cell. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, by Agrobacterium-mediated transformation, protoplast transformation (including polyethylene glycol (PEG)-mediated transformation, electroporation, protoplast fusion, and microcell fusion), lipid-mediated delivery, liposomes, electroporation, sonoporation, microinjection, particle bombardment and silicon carbide whisker-mediated transformation and combinations thereof (see, e.g., Paszkowski, et al., EMBO J., 3:2717-2722 (1984); Potrykus, et al., Mol. Gen. Genet., 199:169-177 (1985); Reich, et al., Biotechnology, 4:1001-1004 (1986); Klein, et al., Nature, 327:70-73 (1987); U.S. Pat. No. 6,143,949; Paszkowski, et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, (Schell and Vasil, eds., Academic Publishers 1989); and Frame, et al., Plant J., 6:941-948 (1994)); direct uptake using calcium phosphate (Wigler, et al., PNAS U.S.A., 76:1373-1376 (1979)); polyethylene glycol (PEG)-mediated DNA uptake; lipofection (see, e.g., Strauss, Meth. Mol. Biol., 54:307-327 (1996)); microcell fusion (Lambert, PNAS U.S.A., 88:5907-5911 (1991); U.S. Pat. No. 5,396,767; Sawford, et al., Somatic Cell Mol. Genet., 13:279-284 (1987); Dhar, et al., Somatic Cell Mol. Genet., 10:547-559 (1984); and McNeill-Killary, et al., Meth. Enzymol., 254:133-152 (1995)); lipid-mediated carrier systems (see, e.g., Teifel, et al., Biotechniques, 19:79-80 (1995); Albrecht, et al., Ann. Hematol., 72:73-79 (1996); Holmen, et al., In Vitro Cell Dev. Biol. Anim., 31 :347-351 (1995); Remy, et al., Bioconjug. Chem., 5:647-654 (1994); Le Bolch, et al., Tetrahedron Lett., 36:6681- 6684 (1995); and Loeffler, et al., Meth. Enzymol., 217:599-618 (1993)); or other suitable methods. Methods for production of synthetic chromosomes are described in U.S. application Ser. No. 09 / 815,979. Successful transfection is generally recognized by detection of the presence of nucleic acid molecules encoding the modified TPO polypeptide, or modified TPO polypeptides or functional fragments thereof themselves as described herein within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid, expression of a selectable marker or any indication of the operation of a vector within the host cell. For a description of delivery methods, see U.S. Pat. No. 5,011 ,776; U.S. Pat. No. 5,747,308; U.S. Pat. No. 4,966,843; U.S. Pat. No. 5,627,059; U.S. Pat. No. 5,681 ,713; Kim and Eberwine, Anal. Bioanal. Chem. 397(8): 3173-3178 (2010).

[0239] In addition to vector-based delivery of modified TPO polypeptides or functional fragments thereof and nucleic acid molecules encoding said modified TPO polypeptides or functional fragments thereof described herein, it is also contemplated that cells, such as MK cells, blood stem cells or human pluripotent stem cells of choice can be engineered to produce human synthetic chromosomes that express modified TPO polypeptides or functional fragments thereof as described herein. Fully-functional human synthetic chromosomes offer several advantages over viral-based delivery systems including increased payload size, the fact that extrachromosomal maintenance avoids host-cell disruption, and transcriptional silencing of introduced genes and possible immunological complications are avoided. Currently, there are several methods for engineering human synthetic chromosomes, including the “top down” method, the “bottom up” method, creating minichromosomes, and induced de novo chromosome generation. The “bottom up” approach of synthetic chromosome formation relies on cell-mediated de novo chromosome formation following transfection of a permissive cell line with cloned a-satellite sequences, which comprise typical host cell-appropriate centromeres and selectable marker gene(s), with or without telomeric and genomic DNA. (For protocols and a detailed description of these methods see, e.g., Harrington, et al., Nat. Genet., 15:345-55 (1997); Ikeno, et al., Nat. Biotechnol., 16:431-39 (1998); Masumoto, et al., Chromosoma, 107:406-16 (1998), Ebersole, et al., Hum. Mol. Gene., 9:1623-31 (2000); Henning, et al., PNAS USA, 96:592-97 (1999); Grimes, et al., EMBO Rep. 2:910-14 (2001); Mejia, et al., Genomics, 79:297-304 (2002); and Grimes, et al., Mol. Then, 5:798-805 (2002).) The “top down” approach of producing synthetic chromosomes involves sequential rounds of random and / or targeted truncation of pre-existing chromosome arms to result in a pared down synthetic chromosome comprising a centromere, telomeres, and DNA replication origins. (For protocols and a detailed description of these methods see, e.g., Heller, et al., PNAS USA, 93:7125-30 (1996); Saffery, et al., PNAS USA, 98:5705-10 (2001); Choo, Trends Mol. Med., 7:235-37 (2001); Barnett, et al., Nuc. Ac. Res., 21 :27-36 (1993); Farr, et al., PNAS USA, 88:7006-10 (1991); and Katoh, et al., Biochem. Biophys. Res. Commun., 321 :280-90 (2004).) “Top down” synthetic chromosomes are constructed optimally to be devoid of naturally- occuring expressed genes and are engineered to contain DNA sequences that permit sitespecific integration of target DNA sequences onto the truncated chromosome, mediated, e.g., by site-specific DNA integrases. Other methods for producing synthetic chromosomes includes those described in , Auriche, et al., EMBO Rep. 2:102-07 (2001); Moralli, et al., Cytogenet. Cell Genet., 94:113-20 (2001); Carine, et al., Somat. Cell Mol. Genet., 15:445-460 (1989); Csonka, et al., J. Cell Sci 113:3207-16 (2002); Hadlaczky, et al., Curr. Opini. Mol. Ther., 3:125-32 (2001); and Lindenbaum and Perkins, et al., Nuc. Ac. Res., 32(21):e172 (2004).

[0240] Alternatively, modified TPO polypeptides or functional fragments thereof, described herein can be introduced into a host cell by insertion of nucleic acid molecules encoding said modified TPO polypeptides or functional fragments thereof as described herein into endogenous chromosomal sites by site-specific recombination. Site-specific recombination requires specialized recombinases to recognize specific recombination sites and catalyze recombination at these sites. A number of bacteriophage- and yeast-derived site-specific recombination systems, each comprising a recombinase and specific cognate sites, have been shown to work in eukaryotic cells for the purpose of DNA integration and are therefore applicable for use in engineering cells to express mutant TPO polypeptides described herein. Site-specific recombination systems include but are not limited to the bacteriophage P1 Cre / lox system, yeast FLP-FRT system, and the Dre system of the tyrosine family of sitespecific recombinases. Such systems and methods of use are described, for example, in U.S. Pat. Nos. 7,422,889; 7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and 4,959,317, which are incorporated herein by reference to teach methods for using such recombinases. Other systems of the tyrosine family such as bacteriophage lambda Int integrase, HK2022 integrase, and systems belonging to a separate serine family of recombinases such as bacteriophage phiC31 , R4Tp901 integrases are known to work in mammalian cells are also applicable for use in the present invention.

[0241] In one embodiment the host cell is transformed with the vector or expression construct described herein using jetPRIME transfection reagent (Polyplus Transfections), suitably according to the protocol described in the examples herein. In one embodiment the host cell is transformed with the vector or expression construct described herein using ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific), suitably according to the protocol described in the examples herein.

[0242] Host cells of the invention may be used to produce modified TPO polypeptides or functional fragments thereof, described herein. The invention provides a method of manufacturing a modified TPO polypeptide or functional fragments thereof, according to the first aspect of the invention comprising culturing a host cell transformed with a nucleic molecule, an expression construct, or a vector encoding the modified TPO polypeptide under suitable conditions to produce the TPO polypeptide or functional fragments thereof,. For example, by culturing the host cell under conditions suitable for expression and production of the modified TPO polypeptides or functional fragments thereof, described herein. Culturing conditions may be selected depending on the host cell used. Specific culture conditions for cells for the production of modified and recombinant proteins are well known in the art.

[0243] Culture medium that may be used for culturing the host cell may be any suitable cell culture medium and may be selected depending on the types of cells being cultured. Examples of culture medium that may be used include minimal essential medium (MEM, Sigma, St. Louis, Mo); Dulbecco's modified Eagle medium (DMEM, Sigma); Ham F10 medium (Sigma); Cell culture media (HyClone, Logan, Utah); RPMI-1640 culture media (Sigma); and chemical- defined (CD) culture media (which are formulated for individual cell types), such as CD-CHO culture media (Invitrogen, Carlsbad, Calif).

[0244] The culture solution described above can be supplemented with auxiliary components or contents as needed. The culture medium may include one or more additives such as antibiotics, proteins, amino acids and / or sugars.

[0245] “Medium” and “cell culture medium” refer to a nutrient source used for growing or maintaining cells. As is understood by a person of skill in the art, the nutrient source may contain components required by the cell for growth and / or survival or may contain components that aid in cell growth and / or survival. Vitamins, essential or non-essential amino acids, trace elements, and surfactants (e.g., poloxamers) are examples of medium components. Any media provided herein may also be supplemented with any one or more of insulin, plant hydrolysates and animal hydrolysates.

[0246] In one particular example, the culture media may be Freestyle 293 Expression Medium (Thermo Fisher) optionally supplemented with about 1 % (v / v) FBS (Sigma-Aldrich), Sf-900 III medium (Thermo Fisher) optionally supplemented with about 10% (v / v) FBS (Sigma-Aldrich) and about 10% GlutaMAX (Thermo Fisher).

[0247] In one particular example, the culture media may be Roswell Park Memorial Institute (RPMI) 1640 medium optionally supplemented with 2 mM L-glutamine, 10% (v / v) FBS, 1 * P / S and 10 ng / mL recombinant human TPO (rhTPO, Zymogenetics).

[0248] In one particular example, the culture media may be Dulbecco’s Modified Eagle’s Medium (DMEM) optionally supplemented with 2 mM L-glutamine, 10% (v / v) fetal bovine serum (FBS), 1 x penicillin / streptomycin (P / S; all Gibco). In one particular example, the culture media may be Sf-900 III medium (Thermo Fisher) optionally supplemented with 10% (v / v) FBS (Sigma-Aldrich) and GlutaMAX (Thermo Fisher.

[0249] In one particular example, the culture media may be Expi293 Expression Medium (Thermo Fisher Scientific).

[0250] Suitable culturing conditions comprise suitable conditions of temperature, culture media, aeration, and / or mixing. Specific conditions may depend on the host cells used.

[0251] Suitably, the host cell may be cultured at a temperature of about 15°C to 40°C. Suitably, the host cell may be cultured at a temperature of about 20°C to 40°C. Suitably, the host cell may be cultured at a temperature of about 25°C to 37°C. Suitably, the host cell may be cultured at a temperature of about 37°C. Suitably, the host cell may be cultured at a temperature of about 22°C. Suitably, the host cell may be cultured at a temperature of about 30°C. Suitably, the host cell may be cultured at a temperature of about 27°C.

[0252] Suitably, the host cell may be cultured under ambient CO2 concentration. Suitably, the host cell may be cultured at 5% CO2 concentration. The CO2 concentration may be selected based on the culturing temperature used. For example, culturing at 30°C with 5% CO2 may provide for higher yields of modified TPO polypeptides.

[0253] Suitably, host cells may be cultured with agitation or without agitation. In some examples, culturing may include agitation for part of the culturing. Agitation may be achieved by any suitable means such as using shakers or stirrers.

[0254] Host cells may be cultured for any suitable time period. For example, for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 ,15, 20, 24, 30, 32, 46, 58 or 72 hours.

[0255] After culturing of the host cell, the modified TPO polypeptide or functional fragments thereof, may be isolated and / or purified from the host cell or from the culture media. Methods of purifying recombinantly expressed proteins are well known in the art. The terms “purification” and “isolation” in reference to proteins refers to a process of purifying a protein and may employ any technique used to separate and isolate a protein of interest to a satisfactory level of purity. Protein purification exploits a protein's various properties such as size, charge, binding affinity, and biological activity. Suitably the method may comprise a step of lysing the host cell to release the expressed protein, to form a cell lysate comprising the expressed protein. Suitably this takes place prior to the step of isolating and / or purifying the protein. Alternatively, the host cell may secrete the expression protein into the culture medium, to form a culture medium comprising the expressed protein, suitably from which the expressed protein is isolated and / or purified.

[0256] Suitably therefore in a further aspect of the invention there is provided a culture medium comprising a modified TPO polypeptide, or functional fragments thereof, according to the first aspect.

[0257] Suitably the method may further comprise a step of concentrating the cell lysate, suitably concentrating the modified TPO polypeptide product, or functional fragments thereof. Suitable means of concentration may be centrifugation, optionally using spin filters.

[0258] Exemplary protein purification methods include: solubility-based purification methods such as salt precipitation and solvent precipitation; purification methods based on molecular weight difference, such as dialysis, ultrafiltration, gel filtration and SDS-PAGE; charge-based purification methods such as ion exchange chromatography and hydroxylapatite chromatography; specific affinity- based purification methods such as affinity chromatography; purification methods based on hydrophobicity difference, such as reverse-phase high- performance liquid chromatography; and purification methods based on isoelectric point difference, such as isoelectric focusing.

[0259] For example, Liquid column chromatography is commonly used in protein purification where a cell lysate containing an expressed protein is passed over a “resin” with particular binding affinity for the protein of interest. A resin is a compound or a polymer with chemical properties that supports the purification of proteins via ion exchange, hydrophobic interaction, size exclusion, reverse phase, or affinity tag chromatography. In one example, the modified TPO polypeptides or functional fragments thereof are isolated by passing the cell lysate over a Ni- NTA agarose resin, suitably in such embodiments, the TPO polypeptide comprises a His tag, such as an octahistidine tag, suitably at the C terminus thereof. Suitably the His tag is capable of binding to the Ni-NTA resin. A protein may also be purified by non-chromatographic techniques such as through the electroporation of protein from an excised piece of a polyacrylamide gel that contained a protein sample of interest, or alternatively by using a binding molecule specifically targeted to bind to the expressed protein such as an antibody or binding fragment thereof, suitably which may be immobilised. Suitably therefore the modified TPO polypeptides are isolated by contacting the cell lysate with anti-TPO antibodies, suitably which may be immobilised. Suitably, the isolation and purification of modified TPO polypeptides or functional fragments thereof, may also take place by affinity chromatography. For example, immobilized metal affinity chromatography (IMAC). Suitably, when affinity chromatography is used to recover the modified TPOs, the modified TPOs or functional fragments thereof, may comprise an affinity tag which is capable of binding to affinity agents used for the affinity chromatography. For example, when immobilized metal affinity chromatography (IMAC) is used the modified TPO polypeptide or functional fragments thereof, may comprise a metal binding affinity tag, suitably fused to the N or C terminus thereof.

[0260] Suitably therefore in some examples of the invention, the isolated and modified TPO polypeptide of the first aspect may further comprise a tag, suitably at the N or C terminus. Suitably such a tag may be used to recover the polypeptide from cell cultures.

[0261] Suitably the method may comprise further steps of purification of the modified TPO polypeptide product or functional fragments thereof, optionally by filtering, passing through columns, etc. Suitably to remove toxins and cellular debris.

[0262] Methods of expanding stem cells

[0263] Aspects of the invention disclosed herein relate to a method of expanding stem cells, suitably HSCs.

[0264] Methods of expanding stem cells are known in the art, as described for example in US5409825A, W02010065546A2, W02007028079A2, WO2010138873A1 , and such methods and their details will therefore be known by the skilled person. Multipotent hematopoietic stem cell (HSC) transplantation is currently the most popular stem cell therapy (Zakrzewski et al. 2019). Details of such procedures are therefore well known. Described herein below is the use of the isolated and modified TPOs or functional fragments thereof, disclosed herein in such stem cell expansion methods. For example, any method of expanding HSCs known in the art that comprises the addition of wild type or other modified TPO polypeptides not of this invention may be adapted by the replacement of the TPO therein with an isolated and modified TPO polypeptide or functional fragments thereof, as described herein, or by the addition of an isolated and modified TPO polypeptide as described herein.

[0265] Suitably therefore in a further aspect of the invention there is provided the use of a modified thrombopoietin polypeptide, or functional fragments thereof, according to the first aspect of the invention in a method of expanding and / or maintaining stem cells. In accordance with an aspect of the invention there is provided is a method of expanding and / or maintaining stem cells, the method comprising:

[0266] (a) Providing one or more stem cells;

[0267] (b) Culturing the stem cells in the presence of a modified thrombopoietin polypeptide, or functional fragments thereof, according to the first aspect of the invention, under suitable conditions for the stem cells to proliferate.

[0268] Suitably the method comprises a step of providing one or more stem cells, in one embodiment providing one or more hematopoietic stem cells (HSCs).

[0269] As used herein “hematopoietic stem cells (HSCs)” and “blood stem cells” means stem cells having no differentiation potential to cells other than blood cells but having a differentiation potential to various types of blood cells. HSCs are known to be abundantly included in cell populations separated and collected from certain tissues, such as umbilical cord blood, peripheral blood, bone marrow, and foetal liver by, for example, flow cytometry or the like using an antibody that binds specifically to a cell surface antigen such as, CD34 on HSCs. Suitably HSCs are CD34+ cells, and may be identified as such. Other markers which may identify an HSC include: CD59+, CD90 / Thy1+, CD38lowA, c-Kit / low, and Lin" Suitably an HSC as used herein may comprise one or more of these markers.

[0270] Suitably the HSCs used in the method are derived from certain tissues, such as umbilical cord blood, peripheral blood, bone marrow, and foetal liver. Suitably the HSCs used in the method are isolated from umbilical cord blood, peripheral blood, bone marrow, and foetal liver, suitably by flow cytometry. Suitably such cells may be obtained from the Anthony Nolan Trust.

[0271] Alternatively the HSCs of the present invention can be prepared by inducing differentiation of human pluripotent stem cells. “Human pluripotent stem cells” as used herein may be any human cells that renew and can be induced to differentiate into blood stem cells. Examples of human pluripotent stem cells include human embryonic stem cells (ES cells); human embryonal carcinoma cells (EC cells); human embryonic germ cells (EG cells); human multipotent germline stem cells (mGS cells); human mesodermal stem cells; human mesenchymal stem cells. HSCs that can be induced to produce megakaryocytes include human colony forming units-granulocytes, erythrocytes, monocytes and megakaryocytes (CPU-GEMMs); burst forming units-megakaryocytes (BFU-MKs); colony forming units- megakaryocytes (CFU-MKs); promegakaryoblasts, megakaryoblasts, and promegakaryocytes (collectively, “blood stem cells”). In addition, an example of human pluripotent stem cells includes cells artificially prepared in such a manner as to have differentiation pluripotency, such as induced pluripotent stem cells (iPSCs).

[0272] A “megakaryocyte” (“MK) is a cell having a differentiation potential to produce platelets and no other cell. An “MK cell line” means immortalized MKs, which can be maintained in culture through many passages.

[0273] The methods of HSC cell expansion described herein require provision of or one or more stem cells, as described above. In the context of aspects of the present invention where HSCs (which may be expanded by the methods of the invention) or therapeutic compositions of said HSCs, are used in the treatment or prevention of disease, suitably the HSCs may be autologous cells or allogeneic cells. As described above, HSCs may be identified using commonly known markers in the art such as CD34+. “Autologous cells” refers to cells which are intended for subsequent therapeutic use in the same subject from which the HSCs were originally derived. “Allogeneic cells” refers to cells which are intended for subsequent therapeutic use in a different subject from which the HSCs were originally derived.

[0274] By way of example, the stem cells used in the method of expansion may be stem cells derived from umbilical cord blood or from bone marrow, in one embodiment, the stem cells used in the method of expansion may be HSCs derived from umbilical cord blood or from bone marrow.

[0275] Culturing, maintenance, and / or expansion of stem cells, such as HSCs, requires suitable conditions for the stem cells to proliferate. Such conditions are known in the art including in the documents cited above.

[0276] In general, HSCs can be maintained and expanded in culture medium that is available to and well-known in the art. Such media include, but are not limited to, Dulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 medium®, Eagle's Minimum Essential Medium®, F-12K medium®, Iscove's Modified Dulbecco's Medium®, RPMI-1640 medium®, and serum- free medium for culture and expansion of hematopoietic cells SFEM® (STEMCELL Technologies). Many media are also available as low-glucose formulations, with or without sodium pyruvate.

[0277] The media used may be referred to as an expansion or proliferation media, suitably which enables the stem cells to grow. Proliferation medium may be a medium comprising a source of nutrients, such as vitamins, minerals, carbon and energy sources, and other beneficial compounds that facilitate the biochemical and physiological processes occurring during expansion or proliferation of cells. The proliferation medium may comprise one or more carbon sources, vitamins, amino acids, and inorganic nutrients. Representative carbon sources include monosaccharides, disaccharides, and / or starches. For example, the proliferation medium may contain one or more carbohydrates such as sucrose, fructose, maltose, galactose, mannose, and lactose. The proliferation medium may also comprise amino acids. Suitable amino acids may include amino acids commonly found incorporated into proteins as well as amino acids not commonly found incorporated into proteins, such as argininosuccinate, citrulline, canavanine, ornithine, and D-steroisomers. The proliferation medium may also comprise proteins such as foetal bovine serum albumin. The proliferation medium may also comprise antibiotics.

[0278] In one embodiment, the stem cells are cultured in StemSpan SFEM II (STEMCELL Technologies) media.

[0279] Also contemplated is supplementation of cell culture medium with mammalian sera. Sera often contain cellular factors and components for viability and expansion. Examples of sera include fetal bovine serum (FBS), bovine serum (BS), calf serum (CS), foetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), human serum, chicken serum, porcine serum, sheep serum, rabbit serum, serum replacements and bovine embryonic fluid. It is understood that sera can be heat-inactivated at 55-65°C if deemed necessary to inactivate components of the complement cascade. Additional supplements also can be used advantageously to supply the cells with the trace elements for optimal growth and expansion. Such supplements include insulin, transferrin, sodium selenium and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution® (HBSS), Earle's Salt Solution®, antioxidant supplements, MCDB- 201® supplements, phosphate buffered saline (PBS), ascorbic acid and ascorbic acid-2- phosphate, as well as additional amino acids. Many cell culture media already contain amino acids, however, some require supplementation prior to culturing cells. Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L- glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. It is well within the skill of one in the art to determine the proper concentrations of these supplements.

[0280] Hormones also can be advantageously used in the cell cultures of the present invention and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, |3- estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine and L-thyronine.

[0281] Lipids and lipid carriers also can be used to supplement cell culture media, depending on the type of cell. Such lipids and carriers can include, but are not limited to, cyclodextrin (a, p, y), cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin and oleic acid unconjugated and conjugated to albumin, among others.

[0282] Cells may be cultured in low-serum or serum- free culture medium. Serum- free medium used to culture cells is described in, for example, U.S. Patent 7,015,037. Many cells have been grown in serum-free or low-serum medium. For example, the medium can be supplemented with one or more growth factors. Commonly used growth factors include, but are not limited to, bone morphogenic protein, basis fibroblast growth factor, platelet-derived growth factor and epidermal growth factor, haematopoietic growth factor (FLT3L), and Stem cell factor (SCF). See, for example, U.S. Patent Nos. 7,169,610; 7,109,032; 7,037,721 ; 6,617,161 ; 6,617,159; 6,372,210;6, 224, 860; 6,037,174; 5,908,782; 5,766,951 ; 5,397,706; and 4,657,866; all incorporated by reference herein for teaching growing cells in serum- free medium.

[0283] Cells in culture can be maintained either in suspension or attached to a solid support, such as extracellular matrix components. Stem cells often require additional factors that encourage their attachment to a solid support, such as type I and type II collagen, chondroitin sulfate, fibronectin, "superfibronectin" and fibronectin-like polymers, gelatin, poly-D and poly-L-lysine, thrombospondin and vitronectin. Hematopoietic stem cells can also be cultured in low attachment flasks such as but not limited to Corning Low attachment plates.

[0284] In one embodiment, the stem cells are cultured in culture media may be StemSpan SFEM II (STEMCELL Technologies) optionally supplemented with 150 ng / mL stem cell factor and 150 ng / mL FltSL (both Biolegend).

[0285] Suitably, the stem cells may be cultured at a temperature of about 15°C to 40°C. Suitably, the stem cells may be cultured at a temperature of about 20°C to 40°C. Suitably, the stem cells may be cultured at a temperature of about 25°C to 37°C. Suitably, the stem cells may be cultured at a temperature of about 37°C. Suitably, the stem cells may be cultured at a temperature of about 22°C. Suitably, the stem cells may be cultured at a temperature of about 30°C. Suitably, the stem cells may be cultured at a temperature of about 27°C. Suitably, the stem cells may be cultured under ambient CO2 concentration. Suitably, the stem cells may be cultured at 5% CO2 concentration. The CO2 concentration may be selected based on the culturing temperature used.

[0286] Suitably, stem cells may be cultured with agitation or without agitation. In some examples, culturing may include agitation for part of the culturing. Agitation may be achieved by any suitable means such as using shakers or stirrers.

[0287] Stem cells may be cultured for any suitable time period. For example, for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 ,15, 20, 24, 30, 32, 46, 58 or 72 hours. In some examples, at least 1 , 2, 3, 4, 5, 6, 7 or more days. In some examples, up to 7 days.

[0288] Suitably the method of expanding comprises culturing in the presence of a modified TPO polypeptide or functional fragments thereof of the invention. Suitably therefore, during culturing, the stem cells are contacted with, or exposed to, the modified TPO polypeptides or functional fragments thereof as described herein. Suitably the concentration of the modified TPO polypeptides or functional fragments thereof used may be determined by the culture volume and number of stem cells being cultured. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 0.001 ng / ml to 10000 ng / ml. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 0.01 ng / ml to 1000 ng / ml. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 0.1 ng / ml to 1000 ng / ml. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 1 ng / ml to 100 ng / ml. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 15 ng / ml to 50 ng / ml. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 20 ng / ml to 40 ng / ml.

[0289] In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 2.5x1 O'5nM to 2.5 mM. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 25x10'5nM to 25 nM. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 2.5x1 O'4nM to 2.5 nM. In some examples, the concentration of modified TPO or functional fragments thereof used in the culture may be from about 0.5 nM to 1 nM. In one embodiment, the concentration of modified TPO or functional fragments thereof used in the culture is between 20 ng / m L (0.5 nM) or 40 ng / mL (1 nM).

[0290] Suitably the modified TPO polypeptide or functional fragments thereof may be added to the stem cell culture. Suitably at any time during the culturing of the stem cells. Suitably the modified TPO polypeptide or functional fragments thereof may be added at day 0 of the stem cell culture, and / or at day 1 , 2, 3, 4, 5, 6, 7, of the stem cell culture. In one embodiment, the modified TPO polypeptide or functional fragments thereof may be added to the stem cell culture at day 0 and day 7 of the culture.

[0291] Suitably the stem cells are cultured in the presence of the modified TPO or functional fragments thereof for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 ,15, 20, 24, 30, 32, 46, 58 or 72 hours. In some examples, at least 1 , 2, 3, 4, 5, 6, 7 or more days. For example, 7 days. For example, 72 hours.

[0292] In one embodiment, modified TPO polypeptide or functional fragments thereof may be added to the stem cell culture at day 0 and the stem cells are cultured in its presence for around 7 days, optionally modified TPO polypeptide or functional fragments thereof is further added to the stem cell culture at day 7 and the stem cells are culture in its presence for around 1 hour.

[0293] As described hereinabove, isolated and modified TPO polypeptides or functional fragments thereof disclosed herein may support increased cell growth (expansion or proliferation) compared to cells grown in the presence of wild type TPO or control TPO polypeptides. Modified TPOs or functional fragments thereof described herein may also active downstream pathway signaling in target cells to a greater extent than wild type TPO or a control TPO polypeptide once bound to TpoR. Suitably, these effects may be achieved by the isolated and modified TPO polypeptides or functional fragments thereof disclosed herein.

[0294] Supporting increased cell growth and / or over-activating target cell downstream signaling are particularly advantageous for in vitro (i.e. ex vivo) stem cell expansion protocols such as HSC expansion protocols, as these effects may lead to increased stem cell product.

[0295] Suitably, isolated and modified TPO polypeptides or functional fragments thereof disclosed herein may be directly substituted into existing and known methods of stem cell expansion, such as those described above. The skilled person will be able to make routine parameter changes to standard production protocols to account for the effects of the modified TPO polypeptides or functional fragments thereof described herein, which may result for example in the increased production of stem cells compared to wild type TPO or a control TPO polypeptide.

[0296] A control TPO polypeptide may be a wild type TPO polypeptide which does not contain any modifications, or a modified TPO polypeptide different to the modified TPO polypeptides of the invention, which may contain different modifications to those describe herein.

[0297] As described in the Examples, the modified TPO polypeptides or functional fragments thereof disclosed herein may improve the preservation of HSCs i.e. HSC identity throughout in vitro culturing, or in other words limit HSC exhaustion, without affecting differentiation potential. Modified TPO polypeptides or functional fragments thereof disclosed herein may also expand and / or maintain HSCs without significant loss of their multi-lineage regenerative capacity i.e. differentiation potential. Modified TPO polypeptides or functional fragments thereof disclosed herein can also increase the viability of in vitro cultured, expanded, and / or maintained HSCs. Suitably, these effects may be achieved by exposure of HSCs to isolated and modified TPO polypeptides or functional fragments thereof disclosed herein, suitably during culture of HSCs. Suitably partial-agonist variants, such as modified TPO polypeptides which contain amino acid modifications selected from K35A, K35Q and R119E in SEQ ID NO:1 , or at corresponding positions thereto, may improve the preservation of HSCs.

[0298] These effects are particularly advantageous for ex vivo stem cell expansion protocols, suitably ex vivo HSC expansion protocols, such as those protocols typically employing TPO. In one embodiment, the method of expanding and / or maintaining stem cells described herein is therefore an ex vivo or in vitro method. Cell populations obtained through such cell expansion methods of the invention have a high proportion of cells which are maintained as stem cells (HSCs). Suitably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, up to 100% of the cell population is maintained as stem cells (HSCs). Cell populations obtained through such cell expansion methods of the invention have a low proportion of cells which have differentiated for example a low proportion of MK cells. Suitably therefore the cell populations obtained through such cell expansion methods of the invention have a higher number of stem cells (HSCs) compared to differentiated cell types. Suitably the ratio of stem cells (HSCs) compared to differentiated cell types obtained from the methods of the invention is higher than prior art methods. Suitably the ratio of stem cells (HSCs) compared to differentiated cell types obtained from the methods of the invention may be at least 2: 1 , at least 3: 1 , at least 4: 1 , at least 5: 1 , at least 6: 1 for example. Suitably cell populations obtained through such cell expansion methods of the invention comprise stem cells (HSCs) which are more viable. Suitably cell populations obtained through such cell expansion methods of the invention have stem cells (HSCs) which retain their ability for differentiation. Such stem cells (HSCs) produced from the methods of expansion of the present invention are therefore more clinically useful.

[0299] Suitably, the method of expanding stem cells disclosed herein may be performed using an isolated and modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a functional fragment thereof, and comprising at least one amino acid modification in the site 2 region thereof, at a position selected from: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 or at a corresponding position thereto. Suitably the modifications are at a position selected from R31 , K35, R38, R99, and R119, or at a corresponding position thereto. Suitably the modifications are at a position selected from K35 and R119, or at a corresponding position thereto. Suitably the amino acid modifications are selected from K35A or K35E, or corresponding modifications thereto. In one embodiment, the amino acid modification is R119H, or a corresponding modification thereto.

[0300] Suitably, the method of expanding stem cells disclosed herein may not substantially induce differentiation of the stem cells. That is to say that culturing stem cells such as HSCs using the modified TPO polypeptides or functional fragments thereof as described herein may decouple proliferation from stem cell differentiation. Therefore, the modified TPO polypeptides or functional fragments thereof as described herein may decrease differentiation of the stem cells in comparison to wild type TPO or a control TPO polypeptide. Suitably, the modified TPO polypeptides or functional fragments thereof as described herein may improve preservation of HSCs in comparison to wild type TPO or a control TPO polypeptide. Suitably, the modified TPO polypeptides or functional fragments thereof as described herein reduce exhaustion of HSCs in comparison to wild type TPO or a control TPO polypeptide.

[0301] In particular, modified TPO polypeptides or functional fragments thereof as described herein comprising a modification at position K35 of SEQ ID NO: 1 , or at a corresponding position thereto, may provide improved maintenance of stem cells, preservation of stem cells, reduced exhaustion of stem cells and / or decrease differentiation of the stem cells in comparison to wild type TPO or a control TPO polypeptide. Suitably the modification at position K35 of SEQ ID NO: 1 , or at a corresponding position thereto may be K35A or K35Q. In particular, modified TPO polypeptides or functional fragments thereof as described herein comprising a modification at position R119 of SEQ ID NO: 1, or at a corresponding position thereto, may provide improved maintenance of stem cells, preservation of stem cells, reduce exhaustion of stem cells and / or decrease differentiation of the stem cells in comparison to wild type TPO or a control TPO polypeptide. Suitably the modification at position R119 of SEQ ID NO: 1 , or at a corresponding position thereto may be R119E. Suitably, the modification at position R119 of SEQ ID NO: 1 , or at a corresponding position thereto may be R119H.

[0302] The level of exhaustion of the stem cells may be determined by any suitable means, such as by analyzing cell specific markers. For example, EPCR may be used as a marker specific for non-exhausted and undifferentiated stem cells, such as HSCs Suitably, modified TPO polypeptides or functional fragments thereof as described herein may provide an increased level of EPCR+ stem cells produced from the methods of the invention in comparison to wild type TPO or a control TPO polypeptide.

[0303] Suitably, the increase in EPCR+ stem cells produced by the method of expansion using the modified TPO polypeptides or functional fragments thereof described herein may be about at least 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250% higher in comparison to stem cells cultured using a wild type TPO or control TPO.

[0304] The stem cells cultured and expanded using modified TPO polypeptides or functional fragments thereof as described herein may subsequently be differentiated. For example, by culturing in a differentiation media, or in vivo after being administered as a therapy to a subject. HSCs differentiate sequentially into CFU-GEMMs, BFU-MKs, CFU-MKs, promegakaryoblasts, megakaryoblases, promegakaryocytes, and megakaryocytes, which then become polyploid by endomitosis and undergo a maturation process forming proplatelets, then platelets.

[0305] When the stem cells cultured and expanded using modified TPO polypeptides or functional fragments thereof as described herein are differentiated, the proportion of GEMM, GM and / or BFLI-E cells may be similar to cells cultured using wild type TPO polypeptide. Without being bound by theory, the modified TPO polypeptides as described herein do not negatively impact the differentiation potential of the HSCs. Advantageously, whilst the modified TPO polypeptides or functional fragments thereof of the invention may in an expansion method prevent differentiation of stem cells, this does not reduce the differentiation potential of the stem cells. Suitably, modified TPO polypeptides or functional fragments thereof as described herein may actually increase the total number of each differentiated cell type, when said stem cells are differentiated, for example, increase the total number of GEMM, GM and / or BFLI-E cells produced from stem cells in comparison to stem cells cultured using a wild type TPO or control TPO polypeptide. For example, the amount of each cell type that may be produced from the stem cells may increase by about at least 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% in comparison to cells cultured using a wild type TPO or control TPO polypeptide. Without being bound by theory, this may indicate that the modified TPO polypeptides or functional fragments thereof as described herein may provide an increased number of viable stem cells and / or improve differentiation potential in comparison to stem cells cultured using a wild type TPO or control TPO polypeptides.

[0306] In particular, modified TPO polypeptides or functional fragments thereof as described herein comprising a modification at position K35 of SEQ ID NO: 1 , or at a corresponding position thereto, when used in a method of expanding stem cells may provide increased numbers of viable stem cells and / or stem cells with improved differentiation potential in comparison to stem cells cultured using a wild type TPO or control TPO polypeptide. Suitably, the modification at position K35 of SEQ ID NO: 1 , or at a corresponding position thereto is K35A.

[0307] The properties of the modified TPO polypeptides or functional fragments thereof as described herein detailed above may have a number of advantages. For example, in comparison to a wild type TPO polypeptide. For example, the modified TPO polypeptides or functional fragments thereof as described herein may improve standard in vitro stem cell expansion protocols, especially HSC expansion protocols, for use in gene therapy methods by preserving more HSCs, preventing their differentiation during culturing, but improving their differentiation potential.

[0308] In addition, the modified TPO polypeptides or functional fragments thereof as described herein may provide for improved methods of ex vivo or in vitro platelet production. Without being bound by theory, the Examples provided herein indicate that the modified TPO polypeptides or functional fragments thereof as described herein may increase platelet production by MKs while limiting excessive stem and progenitor cell and MK expansion. Such methods are described below.

[0309] Methods of ex vivo platelet production using modified TPO polypeptides

[0310] The modified TPO polypeptides, or functional fragments thereof, as described herein may also be particularly useful in methods of ex vivo platelet production. As such, also provided herein are methods of ex vivo or in vitro platelet production using the modified TPO polypeptides or functional fragments thereof. Suitably in a further aspect of the invention there is provided the use of a modified thrombopoietin polypeptide, or a functional fragment thereof, according to the first aspect of the invention in a method of ex vivo platelet production.

[0311] According to an aspect of the invention, there is provided a method of ex vivo platelet production, the method comprising:

[0312] (a) Providing one or more stem cells;

[0313] (b) Culturing the one or more stem cells in the presence of a modified thrombopoietin polypeptide, or a functional fragment thereof, according to the first aspect of the invention under suitable conditions for the one or more stem cells to differentiate into platelets; and

[0314] (c) Optionally isolating the platelets.

[0315] The methods include providing a stem cell (such as an HSC) as described above, culturing the stem cell in the presence of a modified TPO polypeptides or functional fragments thereof as described herein. The cells are then differentiated.

[0316] In one embodiment, the stem cells provided in the method of ex vivo platelet production are HSCs. Differentiation of hematopoietic stem cells into MKs involves the generation of a series of progenitors with increasingly restricted differentiation potential. That is, hemopoietic stem cells differentiate sequentially into CFU-GEMMs, BFU-MKs, CFU-MKs, promegakaryoblasts, megakaryoblases, promegakaryocytes, and megakaryocytes, which then become polyploid by endomitosis and undergo a maturation process forming proplatelets, then platelets. Suitably the method comprises a step of culturing HSCs to differentiate them into platelets, suitably via one or more progenitor cells as necessary, in the presence of a modified TPO polypeptide or functional fragments thereof of the invention.

[0317] Methods of producing platelets are described, for example, in US20180237797A and US20180237797A1 which are incorporated herein by reference. The methods described in the field may be carried out as will be known by the skilled person with the replacement of TPO (i.e. wild type TPO) with one or more modified TPO polypeptides or functional fragments thereof as described herein. Alternatively, the methods described may be carried out with addition (i.e. with wild type TPO included) of modified TPO polypeptides or functional fragments thereof as described herein. For general methods of blood cell / progenitor cell differentiation induction culture, see, e.g., Murphy, et al., US Pub. No. 2014 / 0050711 ; Nakamura, et al., US Pub. No. 2014 / 0024118; Lu, et al., Blood, DOI 10.1182 / bllod-2008-05- 157198 (Aug. 19, 2008); Olivier, et al., Stem Cells Transl. Med., 1 :604-14 (2012); Hiroyama, et al., Stem Cells Int'l, DOI: 10.4061 / 2011 / 195780 (2011), Machlus and Italiano, JCB, 201(8):785-96 (2013); Masuda, et al., Cell Research, 23:176-78 (2013); and Reems, et al., Transfus Med Rev, 24(1):33-43 (2010), all of which are incorporated herein in their entirety.

[0318] Cytokines have been found to be the best tool to expand and control the differentiation of uncommitted CD34+ cells (e.g., HSCs or human blood stem cells). Typically, serum-deprived media supplemented with different cytokines are used. Thrombopoietin (TPO) is primarily responsible for the growth and differentiation of MKs. Unlike other lineage-specific cytokines, TPO also plays a vital role in maintaining the hematopoietic stem cell population. TPO synergizes in vitro with multiple cytokines including IL-34, stem cell factor (SCF), IL-6, IL-9, and IL-11 , each of which can increase the number of CFU-MKs and / or MKs. IL-6 and IL-11 particularly have been used in the last stage of culture to induce production of platelets. Suitably the method to produce platelets of the invention may comprise the addition of any one or more of these cytokines to the culture medium, at suitable concentrations and times as is known in the art.

[0319] Other cytokines that have been identified as synergizing with TPO to support the proliferation of immature progenitors, include IL-3, Flt-3 Ligand (FL) and SCF. It has also been shown in vivo that there are two sets of chemokines — stromal-derived factor-1 (SDF-1) and fibroblast growth factor 4 (FGF-4) — that can promote thrombopoiesis in the absence of TPO or c-Mp1. These two chemokines facilitate the migration of MKs toward the bone marrow sinusoidal endothelial, which promotes maturation and release of platelets. Conversely, other growth factors are known to inhibit megakaryocytopoiesis, including TGF-01 , platelet factor 4 (PF4), and IFN-a, such that these factors can be used to arrest differentiation of blood stem cells at different stages (i.e., CFU-GEMM, BFU-MK, CFU-MK, promegakaryoblasts, megakaryoblasts, promegakaryocytes, megakaryocytes), thus immortalizing MKs or MK precursor cell lines. Suitably the method to produce platelets of the invention may comprise the addition of any one or more of these factors to the culture medium, at suitable concentrations and times as is known in the art.

[0320] There are a host of variables that can be exploited to optimize cell culture conditions to maximize platelet production. For example, physical parameters such as oxygen pressure and temperature can be adjusted to maximize MK and platelet production. Elevating O2 conditions increases MK expansion and accelerate MK differentiation, maturation and proplatelet formation. Further, it has been reported that mild hypothermia can be used to favor and accelerate MK differentiation of CD34+ cells such as HSCs. The majority of culture systems employ at least a 2-step culture strategy to generate platelets. For most 2-step strategies, the first step is to amplify blood cell progenitors followed by a second step to differentiate MKs and support platelet biogenesis. For the 3-step strategies, the first step is to amplify the progenitors, the second step is to support MK differentiation, and the third step is to promote platelet biogenesis. Suitably the method of producing platelets of the invention may comprise either the 2-step method, or the 3-step method described herein.

[0321] The first demonstration that functional human platelets could be generated in vitro was reported by Choi et al. using a two-step strategy (Blood, 85:402-13 (1995)). MK formation was first promoted using peripheral blood CD34+ cells cultured with aplastic canine serum. MKs were isolated from the cultures after 11-12 days and replated with fresh media supplemented with human AB plasma without aplastic canine serum. At the peak of proplatelet formation, an average of 40% of the MKs exhibited proplatelets and platelet-sized fragments and the platelet sized fragments aggregated in the presence of agonists (i.e., thrombin and ADP). Subsequent investigators have reported the in vitro production of functional platelets using (TPO) treated human stem cells from different sources using peripheral blood, mononuclear cells, bone marrow, umbilical cord blood, and human embryonic stem cells (see, e.g., Matsunaga, et al., Stem Cells, 24:2877-87 (2006); and Sullenbarger, et al., Exp Hematol., 37:101-10 (2009)).

[0322] Another protocol employs a three step culture system that more closely mimics the in vivo process of MK / platelet development. First HSCs may be cultured and expanded as described herein (for example using the modified TPO polypeptides or a functional fragment thereof of the invention as described herein). Next, the HSCs are transferred to a culture environment to differentiate and expand MKs for another 14 days. This is followed by a 5 day culture period to support the maturation of MKs to produce platelets that exhibit normal morphology and function. In yet another system, umbilical cord blood CD34+ cells may be expanded as described herein (for example using the modified TPO polypeptides as described herein) for three days prior to placement in a 3-dimensional bioreactor continuously perfused with media and free of stromal cells. The advantage of this system is that it permits the continuous collection of platelets, while allowing for an independent control of media and gas flow.

[0323] Suitably therefore the method of ex vivo platelet production described herein may comprise within step (b) a step of (i) culturing the stem cells to differentiate into megakaryocytes in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention and (ii) culturing the megakaryocytes to produce (differentiate into) platelets optionally in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention. Suitably the method may further comprise within step (b) a step of (i) culturing the stem cells in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention, suitably to expand the stem cells, (ii) culturing the stem cells to differentiate into megakaryocytes optionally in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention, and (ii) culturing the megakaryocytes to produce (differentiate into) platelets optionally in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention. Suitably the step of culturing to expand the stem cells comprises the features identified hereinabove for suitable expansion. Suitably the step of culturing the stem cells to differentiate into megakaryocytes is carried out for around 14 days. Sutiably the step of culturing the megakaryocytes to produce (differentiate into) platelets is carried out for around 5 days.

[0324] It may be desired to culture the stem cells on a 2D or 3D cell support structure as reported by Laskey, et al., in US Pub. No. 2010 / 0248361 or a layered material as reported by Peukert, et al., in US Pub. No. 2012 / 0220198.

[0325] In particular, methods of producing platelets described herein may include culturing and differentiating stem cells, preferably HSCs, in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention comprising a modification at positions K35 and / or R119 of SEQ ID NO: 1 , or at a corresponding position thereto. Suitably, the stem cells may be cultured and expanded in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention comprising a modification at positions K35 of SEQ ID NO: 1 , or at a corresponding position thereto. Suitably, the stem cells may be cultured and expanded in the presence of a modified TPO polypeptide comprising a modification of K35A in SEQ ID NO: 1 , or a corresponding modification thereto. Suitably, the stem cells may be cultured and expanded in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention comprising a modification of K35Q in SEQ ID NO: 1 , or a corresponding modification thereto.

[0326] Suitably, stem cells used in methods of producing platelets described herein may be cultured and expanded in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention comprising a modification at position R119 SEQ ID NO: 1 , or at a corresponding position thereto. Suitably, stem cells used in methods of producing platelets described herein may be cultured and expanded in the presence of a modified TPO polypeptide or a functional fragment thereof of the invention comprising a modification of R119E in SEQ ID NO: 1 , or a corresponding modification thereto The generated platelets from the method described herein may be enriched, isolated or purified using any convenient method known in the art, including fluorescence activated cell sorting (FACS), magnetically activated cell sorting (MACS), density gradient centrifugation and the like. In one embodiment, the platelets are isolated by FACs.

[0327] Parameters employed for enriching certain cells from a mixed population include, but are not limited to, physical parameters (e.g., size, shape, density, etc.) and molecule expression (e.g., expression of cell surface proteins or carbohydrates, reporter molecules, e.g., green fluorescent protein, etc.). Because platelets float in media, density gradient centrifugation is particularly cost effective, and can be used safely to separate platelets from any nucleated blood cells likely to be present. Thus, separation by density is a preferred method of isolating the in vitro generated platelets of the invention. Alternatively, an affinity purification method may be utilized to isolate platelets that have cell-surface antibodies that bind to a specific antigen, either naturally, or platelets that have been engineered to do so. The antigen used to immobilize the platelets may be immobilized on a solid phase and used to selectively retain the platelets, while nucleated cells are washed away. The retained platelets may then be eluted by a variety of methods, such as by chaotropic agents, changing the pH, salt concentration, etc. Any of the well-known methods for immobilizing or coupling an antigen to a solid phase may be used. In the instances where the antigen is a protein, the protein may be covalently attached to a solid phase, for example, sepharose beads, by well-known techniques, etc.

[0328] Alternatively, a labeled antigen may be used to specifically label platelets that express an antibody that binds to the antigen and the labeled platelets may then be isolated by cell sorting (e.g., by FACS). In certain cases, methods for antibody purification may be adapted to isolate antibody presenting platelets. Such methods are well known and are described in, for example, Sun, et al., J. Immunol. Methods, 282(1-2):45-52 (2003); Roque et al., J. Chromatogr A., 1160(1-2):44-55 (2007); and Huse, et al., J. Biochem. Biophys. Methods, 51 (3):217-31 (2002). The platelets may also be isolated using magnetic beads or by any other affinity solid phase capture method, protocols for which are known. In some embodiments, antigen-specific antibody presenting platelets may be obtained by flow cytometry using the methods described in Wrammert, Nature, 453: 667-72 (2008), Scheid, Nature, 458: 636-40 (2009), Tiller, J. Immunol. Methods, 329 112-24 (2008); or Scheid, PNAS, 105: 9727-32 (2008), for example, all of which are incorporated by reference for disclosure of those methods. Exemplary antibody-presenting platelet enrichment methods include performing flow cytometry (FACS) of platelets, e.g., through incubating the platelets with labeled antigens and sorting the labeled platelets using a FACSVantage SE cell sorter (Becton-Dickinson, San Jose, Calif. Optical sorting offers an alternative to FACs or MACs, both of which require labelling of the cells (see, e.g., Ashkin, et al., Am. Soc. Of Graviational and Space Biol., 4(2):133-46 (1991)); and MacDonald, et al., Nature, 426:421-24 (2003)).

[0329] Suitably, the isolated platelets can be sterilized by irradiation. Because platelets are enucleated, they remain functional after irradiation. Sutiably therefore the methods of the invention may comprise a further step of sterilizing the platelets, suitably after isolation.

[0330] Platelets

[0331] A primary purpose for generating platelets in vitro or ex vivo is for transfusions in humans. In vitro or ex vivo generated platelets have the advantages that reliance on the current volunteerbased collection system is not necessary; thus the supply of transfusable platelets is not vulnerable to supply chain disruptions; the platelets of the present invention can be chosen to exhibit particular phenotypes such as being, e.g., AB / RhD- or O / RhD-, or with additional, more precise matching for chronically-infused patients; and the risk of contamination by pathogens is greatly reduced. Further, use of in vitro or ex vivo generated platelets has the additional advantage that the platelets transfused are homogenous in age. The in vitro or ex vivo generated platelets of the invention can be used in surgical and chemotherapy settings.

[0332] Suitably, the platelets produced by the methods described herein may be used in the prevention or treatment of disease. Suitable diseases which may be treated with platelets are describe below. Suitably the platelets may be administered by transfusion to a subject in need thereof.

[0333] Suitably, the platelets produced by the methods described herein may be used as carriers or vehicles for the delivery of other agents, such as therapeutic agents. Drug-loaded platelets may have better targeting, superior biocompatibility, and lower immunogenicity than other delivery systems. Drug-loaded platelet therapies include platelet membrane coating, platelet engineering, and biomimetic platelets. Suitably therefore a therapeutic composition comprising platelets produced by the method described herein may further comprise an additional therapeutic agent.

[0334] Peptibodies

[0335] Also provided herein are peptibodies that comprise peptide fragments of the modified TPO polypeptides as described herein. The term “peptibody” refers to a polypeptide comprising one or more bioactive peptides joined together, optionally via linkers, with an Fc region. See U.S. Pat. Nos. 6,660,843, 7,138,370 and 7,511 ,012 for examples of peptibodies. As used herein, the terms “Fc fusion” and “Fc fusion protein” are used interchangeably and refer to a peptide or polypeptide covalently attached to an Fc region. For example, see WO2015150968A2 and W02000024770A2.

[0336] Suitably the use of a peptibody circumvents the issues around immunogenicity of TPO polypeptides for treatment of diseases. Suitably peptibodies are known in the art, specifically TPO based peptibodies are known and described in W02000024770A2 incorporated herein by reference. Suitably the same structure as described in W02000024770A2 may be used to generate peptibodies of the modified TPO polypeptides of this invention, suitably the skilled person would understand that by substituting the peptide fragments therein with modified peptide fragments derived from the modified TPO polypeptides of this invention, a suitable and functional peptibody can be created.

[0337] Therefore in one aspect of the present invention there is provided a peptibody comprising one or more fragments of the modified thrombopoietin polypeptide (TPO) according to the first aspect. Suitably the peptibody may comprise any peptibody structure known in the art.

[0338] In one example, the peptibody may have a structure denoted as (Fc1)-(L1)-P1-(L2)-P2-(L3)- (Fc2).

[0339] Suitably wherein:

[0340] Fc is an Fc region of an immunoglobulin protein; at least one of Fc1 and Fc2 is present;

[0341] L1 , L2 and L3 are optional linkers.

[0342] In one example, the peptibody may have a structure denoted as (Fc1)m-(L1)q-P1-(L2)n-P2- (L3)r-(Fc2)p.

[0343] Suitably wherein:

[0344] Fc is an Fc region of an immunoglobulin protein; at least one of Fc1 and Fc2 is present;

[0345] L1 , L2 and L3 are optional linkers, and m, q, n, r and p are defined hereinbelow.

[0346] Suitably wherein P1 is a peptide fragment between 10 to 20 amino acids in length, derived from the site 1 region of the modified thrombopoietin polypeptide of claim 1 , wherein the site 1 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO:1 , or a corresponding region thereto, which bind to the TPO receptor with high affinity. In some examples, the P1 peptide fragment may also be modified. Suitably the P1 peptide fragment may also comprise one or more amino acid modifications. Suitably the P1 peptide fragment is derived from amino acids within the region of positions 21 to 184 of SEQ ID NO:1 or a corresponding region thereto and comprises at least one amino acid modification at a position therein which binds to the TPO receptor with high affinity. Suitable modifications are described elsewhere herein in relation to the TPO polypeptide. Suitable modifications may increase the affinity of the peptibody to the TPO receptor.

[0347] Suitably wherein P2 is a peptide fragment between 10 to 20 amino acids in length, derived from the site 2 region of the modified TPO polypeptide as described herein. Suitably the P2 peptide fragment is a therefore a modified peptide fragment of TPO. Suitably the P2 peptide fragment is derived from a modified part of the site 2 region of the modified thrombopoietin polypeptide described herein. Suitably the P2 peptide fragment is derived from amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto, and comprises at least one amino acid modification at a position therein which binds to the TPO receptor with low affinity. Suitably the P2 peptide fragment is derived from amino acids 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto, and comprises at least one amino acid modification at a position selected from: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 of SEQ ID NO:1 , or a position corresponding thereto. Suitably said positions, or corresponding positions thereto, make up the site 2 region of the TPO polypeptide.

[0348] Suitably the P2 peptide fragment may comprise any fragment of between 10 to 20 amino acids in length from within the site 2 region of amino acids 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto, having at least one amino acid modification at a position therein which binds to the TPO receptor with low affinity. Suitably the P2 peptide fragment may comprise any fragment of between 10 to 20 amino acids in length from within the site 2 region of amino acids 21 to 184 of SEQ ID NO: 1 or a corresponding region thereto, having at least one amino acid modification at a position therein which binds to the TPO receptor with low affinity. Suitably therefore the P2 peptide fragment may comprise any fragment of between 10 to 20 amino acids in length from within the site 2 region of amino acids 21 to 184 of SEQ ID NO: 1 , or a corresponding region thereto, of the modified TPO polypeptide of the invention. Sutiably therefore the P2 peptide fragment comprises at least one amino acid modification, selected from those described herein in relation to the modified TPO polypeptide of the invention. Suitably the P1 or P2 fragments may comprise amino acids of the site 1 or site 2 regions respectively that are surface-exposed, and may comprise at least one amino acid modification at a position therein which is surface exposed.

[0349] For example, the P2 peptide fragment may consist of amino acids 21 to 41 of SEQ ID NO: 1 or a corresponding region thereto, and comprise at least one amino acid modification at a position selected from: P26, D29, R31 , V32, K35, and R38, or a position corresponding thereto. For example, the P2 peptide fragment may consist of amino acids 90 to 110 of SEQ ID NO: 1 or a corresponding region thereto, and comprise at least one amino acid modification at a position selected from: R99 or a position corresponding thereto. For example, the P2 peptide fragment may consist of amino acids 115 to 135 of SEQ ID NO: 1 or a corresponding region thereto, and comprise at least one amino acid modification at a position selected from: R119, L120, G123, and A124, or a position corresponding thereto. Sutiably the amino acid modifications at these positions may be those modifications described hereinabove in detail.

[0350] L1 , L2 and L3 are optional linkers. When one or more of the linkers are present, it is not critical what each linker chemical structure is, since the linkers serve primarily as spacers. The linker should be chosen so as not to interfere with the biological activity of the final compound and also so that immunogenicity of the final compound is not significantly increased. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, each linker may comprise Yn, wherein Y is a naturally occurring amino acid or a steroisomer thereof and "n" is any one of 1 through 20. Each linker may therefore made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally-occurring amino acids. Suitably the amino acids of each linker may be selected from Gly, Ala, Pro, Asn, Gin, Cys, and Lys. Preferably, the linker is made up of a majority of amino acids that are sterically un-hindered. Specific examples of linkers are:

[0351] (Gly)3Lys(Gly)4(SEQ ID NO: 25);

[0352] (Gly)3AsnGlySer(Gly)2 (SEQ ID NO: 26) (this structure provides a site for glycosylation, when it is produced recombinantly in a mammalian cell system that is capable of glycosylating such sites);

[0353] (Gly)3Cys(Gly)4(SEQ ID NO 27),

[0354] GlyProAsnGly (SEQ ID NO 28), and A Cysteine residue.

[0355] Non-peptide linkers are also possible. For example, alkyl linkers such as -HN- (CH2)S-CO-, wherein s = 2-20 could be used These alkyl linkers may further be substituted by any non- sterically hindering group such as lower alkyl (e g , Cj-Ce), lower acyl, halogen (e g , Cl, Br), CN, NH2, phenyl, etc.

[0356] Another type of non-peptide linker is a polyethylene glycol group, such as -HN-CH2-CH2-(O- CH2-CH2)n-O-CH2-CO- wherein n is such that the overall molecular weight of the linker ranges from approximately 101 to 5000, preferably 101 to 500. In general, it has been discovered that a linker of a length of about 0-14 sub- units (e g , amino acids) is preferred.

[0357] Suitably m, p, q and r are each independently selected from the group consisting of 0 and 1 , wherein at least one of m or p is 1 , and further wherein if m is 0 then q is 0, and if p is 0, then r is 0.

[0358] In one example, the peptibody may comprise the structure Fc-P1-GPNG-P2 (SEQ ID NO:28). In one example, the peptibody may comprise the structure Fc-P1-GPNG-P2-Fc (SEQ ID NO:28). In one example, the peptibody may comprise the structure P1-GGGGGGGG-P2-Fc (SEQ ID NO: 29). In one example, the peptibody may comprise the structure Fc-GG-P1- GPNG-P2 (“GG” and SEQ ID NO: 28). In one example, the peptibody may comprise the structure Fc-P1-GGGGGGGG-P2 (SEQ ID NO: 29). In one example, the peptibody may comprise the structure Fc-P1-GGGKGGGG-P2 (SEQ ID NO:25). In one example, the peptibody may comprise the structure Fc-P1-GGGCGGGG-P2 (SEQ ID NO:27). In one example, the peptibody may comprise the structure Fc-GGGGG-P1-GGGGGGGG-P2 (SEQ ID NO: 30 and SEQ ID NO: 29).

[0359] Suitably, the peptibodies of the invention may also be covalently or noncovalently associated with a carrier molecule, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see, for example, U.S. Patent 4,289,872 to Denkenwalter et al, issued September 15, 1981 ; 5,229,490 to Tarn, issued July 20, 1993; WO 93 / 21259 by Frechet et al, published 28 October 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide. Other possible carriers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Patent Nos: 4,640,835, 4,496,689, 4,301 ,144, 4,670,417, 4,791 ,192 and 4,179,337. Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)- polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide / ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers.

[0360] Carbohydrate (oligosaccharide) groups may be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligo saccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligo saccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser / Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids not counting proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N- acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.

[0361] Suitably the Fc regions of the peptibody may be selected from the human immunoglobulin lgG-1 heavy chain, see Ellison, J.W. et al, Nucleic Acids Res.10:4071-4079 (1982), or any other Fc sequence known in the art (e.g. other IgG classes including but not limited to lgG-2, lgG-3 and lgG-4, or other immunoglobulins). It is well known that Fc regions of antibodies are made up of monomeric polypeptide segments that may be linked into dimeric or multimeric forms by disulfide bonds or by non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGI, lgG2, lgG3, IgAI, lgGA2) of antibody involved. The term "Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms of Fc molecules. It should be noted that Fc monomers will spontaneously dimerize when the appropriate Cys residues are present unless particular conditions are present that prevent dimerization through disulfide bond formation. Even if the Cys residues that normally form disulfide bonds in the Fc dimer are removed or replaced by other residues, the monomeric chains will generally dimerize through non-covalent interactions. The term "Fc" herein is used to mean any of these forms: the native monomer, the native dimer (disulfide bond linked), modified dimers (disulfide and / or non-covalently linked), and modified monomers (i.e., derivatives). Variants, analogues or derivatives of the Fc region may be constructed by, for example, making various substitutions of residues or sequences or by insertions. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the Fc amino acid sequence. Insertional variants with additional residues at either or both termini can include for example, fusion proteins and proteins including amino acid tags or labels. For example, the Fc molecule may optionally contain an N- terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. In Fc deletion variants, one or more amino acid residues in an Fc polypeptide are removed. Deletions can be effected at one or both termini of the Fc polypeptide, or with removal of one or more residues within the Fc amino acid sequence. Deletion variants, therefore, include all fragments of an Fc polypeptide sequence. In Fc substitution variants, one or more amino acid residues of an Fc polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature, however, the invention embraces substitutions that ore also non-conservative.

[0362] Suitably the Fc regions of the peptibodies may also be derivatized, i.e., bearing modifications other than insertion, deletion, or substitution of amino acid residues. Suitably , the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Derivatives of the invention may be prepared to increase circulating half-life, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs.

[0363] It is also possible to use the salvage receptor binding domain of the intact Fc molecule as the Fc region of the peptibodies, such as described in WO 96 / 32478. Additional members of the class of molecules designated as Fc herein are those that are described in WO 97 / 34631.

[0364] Suitably the Fc regions may be located at the N or C terminus of P1 and / or P2, optionally via a linker . Suitably the Fc regions are fused to the N or C terminus of P and / or P2, optionally via a linker. Suitably Fc1 if present is fused to the N terminus of P1 , optionally via a linker . Suitably Fc2 if present is fused to the C terminus of P2, optionally via a linker . Suitably when the Fc region is fused at the N-terminus of P1 and / or P2 or a linker, such fusion will generally occur at the C-terminus of the Fc chain, and vice versa.

[0365] It is also possible that peptide fragments of the modified TPO polypeptide of the invention can be used as dimers, suitably as tandem dimers, which are also known in the art to have reduced immunogenicity and are suitable for therapeutic uses. Suitable such dimer structures are also described in W02000024770A2 incorporated herein by reference. Suitably the same structure as described in W02000024770A2 may be used to generate tandem dimers of the modified TPO polypeptides of this invention, suitably the skilled person would understand that by substituting the peptide fragments therein with modified peptide fragments derived from the modified TPO polypeptides of this invention, a suitable and functional tandem dimer can be created.

[0366] Therefore in addition to peptibodies, there is provided herein a tandem dimer comprising comprising one or more fragments of the modified thrombopoietin polypeptide (TPO) according to the first aspect. Suitably the tandem dimer may comprise any tandem dimer structure known in the art.

[0367] In one example, the tandem dimer may comprise the structure P1-(L)n-P2. Suitably wherein P1 and P2 are peptide fragments as defined above. Suitably wherein L is a linker as defined above. Suitably wherein ‘n’ is 0 or 1.

[0368] Optionally there may further be provided tandem multimers comprising one or more fragments of the modified thrombopoietin polypeptide (TPO) according to the first aspect, suitably which may comprise more than two linked peptide fragments. For example, a tandem multimer may comprise between 2 to 5 linked peptide fragments. For example: P1-(L)n-P2-(L)n-P3; P1-(L)n- P2-(L)n-P3-(L)n-P4; P1-(L)n-P2-(L)n-P3-(L)n-P4-(L)n-P5.

[0369] The peptibodies or tandem dimers / multimers may be made in a variety of ways. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield, in Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem. Soc. 85:2149 (1963); Davis et al, Biochem. Inti. 10:394-414 (1985); Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Pat. No. 3,941 ,763; Finn et al, The Proteins, 3rd ed., vol. 2, pp. 105-253 (1976); and Erickson et al, The Proteins, 3rd ed., vol. 2, pp. 257-527 (1976).

[0370] Suitably the peptibodies or tandem dimers / multimers may also be made in transformed host cells using recombinant DNA techniques such as those described above in respect of the modified TPO polypeptides.

[0371] There is also provided herein, a nucleic acid encoding a peptibody as described herein, or encoding a tandem dimer / multimer as described herein. There is further provided a vector comprising a nucleic acid encoding a peptibody as described herein, or encoding a tandem dimer / multimer as described herein. There is further provided a host cell comprising said vector or nuclei acids. There is further provided a method of manufacturing a peptibody or tandem dimer / multimer as described herein comprising (a) providing a host cell with a vector or nucleic acid encoding said peptibody or tandem dimer / multimer, and (b) culturing said host cell under suitable conditions to express / produce the peptibody or tandem dimer / multimer and (c) isolating said peptibody or tandem dimer / multimer from the host cell or culture medium. Suitable details regarding nucleic acids, expression vectors, host cells, and methods of culture are described elsewhere herein, and equally apply here.

[0372] Suitably any references herein to peptibodies may be replaced with tandem dimers or tandem multimers. Suitably therefore the tandem dimers or multimers may be comprised in a pharmaceutical composition. Suitably they may be for use in the treatment of a disease, sutiably for use it the treatment of immunological disorders, such as immune thrombocytopenic conditions, haematological disorders or cancers.

[0373] Therapeutic compositions

[0374] The modified TPO polypeptides or functional fragments thereof, host cells for production thereof, stem cells, platelets and peptibodies described herein may be formulated as compositions. Suitably, therapeutic or pharmaceutical compositions.

[0375] As used herein, “pharmaceutical composition” refers to a composition comprising one or more compounds that is formulated for administration to a subject, and the term may be used interchangeably with “therapeutic composition” herein. Pharmaceutical compositions typically comprise one or more active ingredients (e.g. in this case one or more modified TPO polypeptides, or functional fragments thereof, host cells, stem cells, platelets or peptibodies as described herein) and one or more pharmaceutically acceptable materials. The pharmaceutical compositions described herein may therefore comprise one or more modified TPO polypeptides, or functional fragments thereof, host cells, stem cells, platelets or peptibodies as described herein and one or more other components. For example, the pharmaceutical composition may comprise one or more modified TPO polypeptides, or functional fragments thereof, host cells, stem cells, platelets or peptibodies as described herein and a pharmaceutically acceptable excipient, diluent and / or carrier.

[0376] Pharmaceutical compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds. As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound (e.g. one or more modified TPO polypeptides, or functional fragments thereof, host cells, stem cells, platelets or peptibodies as described herein) without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0377] Suitably, the pharmaceutical composition comprises a pharmaceutically acceptable diluent. Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

[0378] Suitably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. Excipients are natural or synthetic substances formulated alongside an active ingredient included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0379] Suitably, the pharmaceutical composition may comprise one or more modified TPO polypeptides, or functional fragments thereof, host cells, stem cells, platelets or peptibodies as described herein and a pharmaceutically acceptable carrier. Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art. Suitably, pharmaceutical compositions are formulated to comprise an acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980). Treatments

[0380] The modified TPO polypeptides or functional fragments thereof, host cells for production thereof, stem cells, platelets and peptibodies or compositions thereof as described herein may be for use as medicaments or therapeutics, suitably in the prevention or treatment of a disease. Methods of treating or preventing a disease in a subject are also provided herein, comprising administering an effective amount of the modified TPO polypeptides, or functional fragments thereof, host cells for production thereof, stem cells, platelets and peptibodies or compositions thereof to a subject in need thereof.

[0381] The subject may be referred to herein as a patient. The terms “subject”, “individual”, and “patient” are used herein interchangeably. As used herein, the term "subject" is intended to include humans and animals. Typically, a subject refers to a human or animal that, within their body, has a cell deficient in homologous recombination. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In some examples, subjects include companion animals, e.g. dogs, cats, rabbits, and rats. In some examples, subjects include livestock, e.g., cows, pigs, sheep, goats, and rabbits. In some examples, subjects include thoroughbred or show animals, e.g. horses, pigs, cows, and rabbits. In one embodiment, the subject is a human subject, which may be an adult or child, male or female.

[0382] Administration may be by any suitable route including, for example, injection, intravenous infusion, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous, intravesicular, transdermally, orally, topically, or other appropriate administration routes. Administration can be "parenteral administration" meaning modes of administration other than enteral and topical administration, usually by injection. Alternatively, modified TPO polypeptides, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein can be administered via a non- parenteral route, such as a topical, epidermal or mucosal route of administration. Local administration may also be carried out, including peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intra cavity infusion, intravesicle administration, and inhalation.

[0383] In particular, modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may administered intraperitoneally. For example, by intraperitoneal injection.

[0384] A suitable dosage of modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be determined by a skilled medical practitioner. Actual dosage levels of modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular antibody employed, the route of administration, the time of administration, the rate of excretion of the antibody, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0385] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, or the method may comprise several divided doses administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation, provided the required interval. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0386] A suitable dose of modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be, for example, in the range of from about 0.01 pg / kg to about 1000 mg / kg body weight of the patient to be treated. For example, a suitable dosage may be from about 0.01 pg / kg to about 100 mg / kg body or from about 0.1 pg / kg to about 10 mg / kg body weight. The amount of modified TPO polypeptides, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein that is administered may be of any appropriate amount such as between 0.01 mg / kg body weight and 2 mg / kg body weight, between 0.04 and 2 mg / kg body weight, between 0.12 mg / kg body weight and 2 mg / kg body weight, between 0.24 mg / kg and 2 mg / kg body weight and most preferably between 1 mg / kg and 2 mg / kg body weight.

[0387] The timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms. For example, an effective dose of the agents or compositions described herein can be administered to an individual at least once daily, at least once every two days, three days, four days, five days, six days, weekly, biweekly, monthly, every six months for an indefinite period of time, or until the individual no longer requires therapy. A person of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions described herein that is administered can be adjusted accordingly.

[0388] Modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be for use in combination with one or more other therapeutic, prophylactic, research or diagnostic agents. By "in combination with," it is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The effect of the two treatments can be partially additive, wholly additive, or greater than additive.

[0389] Also provided herein are methods of prevention or treatment of disease or condition as disclosed herein or the modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein for use in such methods, in a subject, which methods comprises administering the modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein to the subject in a prophylactically or therapeutically effective amount. The modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be co-administered with another agent as described herein. The method of administering modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be carried out by any suitable administration route and at any dosage or as disclosed herein. A “therapeutically effective amount” means a dose or plasma concentration in a subject that provides the desired specific pharmacological effect. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and / or the disease or condition being treated.

[0390] The invention also provides modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein for use in the manufacture of a medicament for the treatment or prevention of a disease or condition as disclosed herein. The invention also provides for a method of treating and / or preventing a disease or condition as disclosed herein by administration of modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets or peptibodies or compositions thereof as described herein in a subject in need thereof. Accordingly, any reference to or embodiments relating to the use of modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets or peptibodies or compositions thereof as described herein for use in treating and / or preventing a disease or condition as disclosed herein can be applied to modified TPO polypeptides or functional fragments thereof, host cells, stem cells, platelets or peptibodies or compositions thereof as described herein for use in the manufacture of a medicament for the treatment or prevention of a disease or condition and methods of treating and / or preventing a disease or condition.

[0391] As used herein, the terms “treat”, “treating” and “treatment” are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom. In other words, terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a condition, symptom or disease.

[0392] In some examples, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other examples, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms), suitably this may be regarded as prophylactic treatment or prevention of a disease. Treatment may also be continued after symptoms have resolved, for example, to delay and / or prevent recurrence, sutiably which may also be regarded as prevention. Treatment may bring about prolonged survival as compared to expected survival if not receiving treatment. Alternatively, or additionally, treatment may provide a patient with an improved standard of life as compared to that which would be expected if not receiving treatment.

[0393] Cell and Gene Therapy

[0394] Suitably the invention provides methods of expanding stem cells, and methods of ex vivo platelet production. Suitably therefore these methods produce stem cells, populations thereof, and platelets. Further aspects of the invention relate to the stem cells, populations thereof, and to the platelets produced by said methods and their therapeutic uses.

[0395] Stem cells and platelets produced by the methods of the invention are capable of being used in a variety of applications, including transplantation, sometimes referred to as cell- based therapies or cell replacement therapies, such as bone marrow transplants, gene therapies, tissue engineering, and in vitro organogenesis. Such applications are useful for the treatment of various disorders, for example, hematological disorders, such as cancers (e.g., leukemia, lymphoma), anemias (e.g., aplastic anemia, sickle cell anemia, Fanconi anemia), lymphocytopenia, neutropenia, thrombocytopenia, etc. Thus, the present invention is also directed to methods of treating a subject comprising administering stem cells produced by methods described herein to the subject, or stem cells produced by the methods herein for use in a method of treating of a subject in need thereof Suitably, the method comprises providing stem cells, such as from the subject or from a donor, culturing the stem cells, such as HSCs as described herein, and introducing or transplanting the cultured stem cells, such as HSCs into the subject. The present invention also provides methods of treating a subject in need of a hematopoietic stem cell-based therapy, comprising providing HSCs from the subject (autologous) or from a donor (allogeneic); culturing the HSCs by the method of expansion as described herein, harvesting the cultured HSCs, and transplanting the cultured HSCs into the subject. Suitably there are also provided HSCs produced by the methods herein for use in a method of treating of a subject in need of a hematopoietic stem cell-based therapy, the method comprising providing HSCs from the subject (autologous) or from a donor (allogeneic); culturing the HSCs by the method of expansion as described herein, harvesting the cultured HSCs, and transplanting the cultured HSCs into the subject.

[0396] Additionally, host cells as described herein, stem cells (such as HSCs, specifically expanded HSCs as described herein), and / or cells of specific lineages produced from expanded stem cells as described herein are suitable for use in ex vivo gene therapy. For example, in ex vivo gene therapy, cells are removed from a subject, and while being cultured in vitro are genetically modified. Generally, a functional replacement gene is introduced into the cells via an appropriate gene delivery vehicle / method (transfection, infection, transduction, homologous recombination, etc.) using an expression system as needed. The genetically modified cells are expanded in culture and returned to the patient. These genetically reimplanted cells will express the transfected genetic material (which may be for example a and modified TPO polypeptide or a functional fragment as described herein) in situ in the subject. Thus, in one example there is provided host cell capable of expressing, in a subject in need thereof, a modified TPO polypeptide having an amino acid sequence according to SEQ ID NO: 1, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, preferably at least 70% identity thereto, or a fragment thereof, and comprising at least one amino acid modification in the site 2 region thereof, or at least one amino acid modification of the surface-exposed amino acids in the site 2 region thereof, at a position selected from: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 or at a corresponding position thereto for use in a method of gene therapy, the method comprising administering an effective amount of the vector or host cell to the subject. Suitably the vector or host cell may comprise a nucleic acid, or an expression construct encoding said modified TPO polypeptides or a functional fragment.

[0397] Suitably the modifications are at one or more positions selected from R31 , K35, R38, R99, and R119 of SEQ ID NO:1 , or corresponding positions thereto. Suitably the modifications are at positions selected from K35 and R119 of SEQ ID NO:1 , or corresponding positions thereto. Suitably the modifications are selected from K35A and K35E of SEQ ID NO:1 , or corresponding modifications thereto. Suitably the modifications are selected from K35A and K35E of SEQ ID NO:1 , or corresponding modifications thereto. Suitably the modifications is R119H of SEQ ID NO:1 , or a corresponding modification thereto. Suitable combinations of modifications are described hereinabove in relation to the TPO polypeptide or a functional fragment.

[0398] Thus, in one example there is provided a stem cell (e.g. HSC or cells produced therefrom), which may have been produced by a method of the invention, as described herein for use in a method of cell therapy, the method comprising administering an effective amount of the stem cell to the subject. Thus, in one example there is provided a host cell as described herein for use in a method of cell therapy, the method comprising administering an effective amount of the host cell to the subject.

[0399] Diseases or conditions that may be treated by gene or cell therapies as described herein include the disease as described below in respect of other methods of treatment described herein. For example diseases or conditions that may be treated by gene or cell therapies as described herein include haematological disorders, immunological disorders and cancer.

[0400] In particular, diseases or conditions that may be treated by gene or cell therapies as described herein include Primary Immune Deficiencies (such as ADA-Deficient Severe Combined Immune Deficiency, X-linked Severe Combined Immune Deficiency, Other Genetic Forms of SCID (Artemis, Rag1 / 2)., Wiskott-Aldrich Syndrome, Chronic Granulomatous Disease, Leukocyte Adhesion Deficiency, Hemophagocytic Lymphohistiocytosis, X-linked Hyper IgM Syndrome, X-linked Lymphoproliferative Disease, X-linked Agammaglobulinemia, and Common Variable Immunodeficiency); Hemoglobinopathies (such as Sickle Cell Diseases and p-thalassemia); Storage and Metabolic Disorders (such as Gaucher Disease and other lipidoses, Mucopolysaccharidoses (l-VII), X-linked Adrenoleukodystrophy, Metachromatic Leukodystrophy, and Osteopetrosis); and Congenital Cytopenias and Stem Cell Defects (such as Fanconi’s Anemia, Schwachman-Diamond Syndrome, and Kostmann’s Syndrome).

[0401] In addition, gene or cell therapies as described may be used to treat cancers such as leukaemia, lymphoma, or myeloma.

[0402] In addition, gene or cell therapies as described may be used to treat aplastic anemia, bone marrow failure syndromes, or Multiple Sclerosis. Bone marrow failure (BMF) refers to the decreased production of one or more major hematopoietic lineages, which leads to diminished or absent hematopoietic precursors in the bone marrow and attendant cytopenias. It can be divided into two categories: acquired and inherited. Inherited bone marrow failure (IBMF) is bone marrow failure that occurs from germline mutations passed down from parents or arising de novo. In addition to symptoms associated with aplastic anemia, such as fatigue, hemorrhage, and recurrent bacterial infections, patients often have extra-marrow features unique to each syndrome. The most common inherited bone marrow failure syndromes (IBMFSs) are Fanconi anemia (FA), dyskeratosis congenita (DC), Shwachman-Diamond syndrome (SDS), congenital amegakaryocytic thrombocytopenia (CAMT), Blackfan-Diamond anemia (BDA), and reticular dysgenesis (RD). Others are less common and share features with the inherited bone marrow failure syndromes listed, for example, short telomerase.

[0403] The methods of cell or gene therapy described herein may be useful in increasing the number stem cells, such as HSCs in a subject, or increasing the number of platelets in a subject. As such, methods of cell or gene therapy described herein may be used in conjunction with treatments such as bone marrow transplantation.

[0404] Other methods of Treatment

[0405] The modified TPO polypeptides or functional fragments thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein are further for use directly in methods of treating a subject in need thereof. The modified TPO polypeptides or functional fragments thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein are further for use directly in methods of treating or preventing immunological disorders, such as immune thrombocytopenic conditions, haematological disorders or cancers.

[0406] Haematological disorders can also be referred to as blood disorders and can affect one or more parts of the blood. They can be both cancerous and non-cancerous. Haematological disorders include erythroid associated disorders such as disorders involving aberrant (increased or deficient) erythroblast proliferation, e.g., an erythroleukemia, and aberrant (increased or deficient) erythroblast differentiation, e.g., an anemia. Erythrocyte-associated disorders include anemias such as, for example, drug (e.g. chemotherapy) induced anemias, hemolytic anemias due to hereditary cell membrane abnormalities, such as hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis; hemolytic anemias due to acquired cell membrane defects, such as paroxysmal nocturnal hemoglobinuria and spur cell anemia; hemolytic anemias caused by antibody reactions, for example to the RBC antigens, or antigens of the ABO system, Lewis system, li system, Rh system, Kidd system, Duffy system, and Kell system; methemoglobinemia; a failure of erythropoiesis, for example, as a result of aplastic anemia, pure red cell aplasia, myelodysplastic syndromes, sideroblastic anemias, and congenital dyserythropoietic anemia; secondary anemia in non-hematolic disorders, for example, as a result of chemotherapy, alcoholism, or liver disease; anemia of chronic disease, such as chronic renal failure; and endocrine deficiency diseases.

[0407] Haematological disorders include disorders involving B-cells which include, but are not limited to precursor B-cell neoplasms, such as lymphoblastic leukemia / lymphoma. Peripheral B-cell neoplasms include, but are not limited to, chronic lymphocytic leukemia / small lymphocytic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and related entities, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma (MALToma), and hairy cell leukemia.

[0408] Haematological disorders include disorders of the bone marrow which include but are not limited to: diseases involving hematopoietic stem cells; committed lymphoid progenitor cells; lymphoid cells including B and T-cells; committed myeloid progenitors, including monocytes, granulocytes, and megakaryocytes; and committed erythroid progenitors. These include but are not limited to the leukemias, including B-lymphoid leukemias, T-lymphoid leukemias, undifferentiated leukemias; erythroleukemia, megakaryoblastic leukemia, monocytic; leukemias are encompassed with and without differentiation; chronic and acute lymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronic and acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic and acute myeloblastic leukemia, chronic and acute myelogenous leukemia, chronic and acute promyelocytic leukemia, chronic and acute myelocytic leukemia, hematologic malignancies of monocyte-macrophage lineage, such as juvenile chronic myelogenous leukemia; secondary AML, antecedent hematological disorder; refractory anemia; aplastic anemia; reactive cutaneous angioendotheliomatosis; fibrosing disorders involving altered expression in dendritic cells, disorders including systemic sclerosis, E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitis localized forms of scleroderma, keloid, and fibrosing colonopathy; angiomatoid malignant fibrous histiocytoma; carcinoma, including primary head and neck squamous cell carcinoma; sarcoma, including kaposi's sarcoma; fibroadanoma and phyllodes tumors, including mammary fibroadenoma; stromal tumors; phyllodes tumors, including histiocytoma; erythroblastosis; neurofibromatosis; diseases of the vascular endothelium; demyelinating, particularly in old lesions; gliosis, vasogenic edema, vascular disease, Alzheimer's and Parkinson's disease; T-cell lymphomas; B-cell lymphomas.

[0409] Hematological disorders include platelet disorders including but not limited to disorders related to reduced platelet number, thrombocytopenia, include idiopathic thrombocytopenic purpura, including acute idiopathic thrombocytopenic purpura, drug-induced thrombocytopenia, HIV- associated thrombocytopenia, and thrombotic microangiopathies: thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome.

[0410] Hematological disorders include thrombosis. Thrombosis can result from platelet dysfunction, e.g. seen in myocardial infarction, angina, hypertension, lipid disorders, diabetes mellitus; myelodysplastic syndromes; myeloproliferative syndromes (including polycythemia vera and thombocythemia); thrombotic thrombocytopenic purpuras; HIV-induced platelet disorders (AIDS-Thrombocytopenia); heparin induced thrombocytopenia; mural cell alterations / interactions leading to platelet aggregation / degranulation, vascular endothelial cell activation / injury, monocyte / macrophage extravasation and smooth muscle cell proliferation; autoimmune disorders such as, but not limited to vasculitis, antiphospholipid syndromes, systemic lupus erythromatosis; inflammatory diseases, such as, but not limited to ilmmune activation; graft Vs host disease; radiation induced hypercoagulation; clotting factor dysregulation either hereditary (autosomal dominant or recessive) such as, but not limited to clotting factor pathways including protein C / S, Anti-thrombin III deficiency, and the Factor V Leiden mutation or acquired such as but not limited to autoimmune, cancer-associated and drug-induced dysregulation of clotting factors. Hematological disorders include red cell disorders including but not limited to, anemias, such as hemolytic anemias, including hereditary spherocytosis, hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphate dehydrogenase deficiency, sickle cell disease, thalassemia syndromes, paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, and hemolytic anemia resulting from trauma to red cells; and anemias of diminished erythropoiesis, including megaloblastic anemias, such as anemias of vitamin B 12 deficiency: pernicious anemia, and anemia of folate deficiency, iron deficiency anemia, anemia of chronic disease, aplastic anemia, pure red cell aplasia, and other forms of marrow failure.

[0411] Hematological disorders include disease of T cells including but not limited to, cell-mediated hypersensitivity, such as delayed type hypersensitivity and T-cell-mediated cytotoxicity, and transplant rejection; autoimmune diseases, such as systemic lupus erythematosus, Sjogren syndrome, systemic sclerosis, inflammatory myopathies, mixed connective tissue disease, and polyarteritis nodosa and other vasculitides; immunologic deficiency syndromes, including but not limited to, primary immunodeficiencies, such as thymic hypoplasia, severe combined immunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory) proliferations of white cells, including but not limited to, leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecific lymphadenitis; neoplastic proliferations of white cells, including but not limited to lymphoid neoplasms, such as precursor T-cell neoplasms, such as acute lymphoblastic leukemia / lymphoma, peripheral T-cell and natural killer cell neoplasms that include peripheral T-cell lymphoma, unspecified, adult T-cell leukemia / lymphoma, mycosis fungoides and Sezary syndrome, and Hodgkin disease.

[0412] Cancers include Adenoid Cystic Carcinoma, Adrenal Gland Cancer, Amyloidosis, Anal Cancer, Ataxia-Telangiectasia, Atypical Mole Syndrome, Basal Cell Carcinoma, Bile Duct Cancer, Birt Hogg Dube Syndrome, Bladder Cancer, Bone Cancer, Brain Tumor, Breast Cancer, Breast Cancer in Men, Carcinoid Tumor, Cervical Cancer, Colorectal Cancer, Ductal Carcinoma, Endometrial Cancer, Esophageal Cancer, Gastric Cancer, Gastrointestinal Stromal Tumor, GISTHER2-Positive Breast Cancer, Islet Cell Tumor, .Juvenile Polyposis Syndrome Kidney Cancer, Laryngeal Cancer, Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic (ALL) Leukemia, Acute Myeloid Leukemia, Adult Leukemia, Childhood Leukemia, Chronic Lymphocytic Leukemia, Chronic Myeloid Liver Cancer, Lobular Carcinoma, Lung Cancer, Lung Cancer - Small Cell, Lymphoma - Hodgkin's Lymphoma, Non-Hodgkin's Malignant Glioma, Melanoma, Meningioma, Multiple Myeloma, Myelodysplastic Syndrome (MDS), Nasopharyngeal Cancer, Neuroendocrine Tumor, Oral Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors, Parathyroid Cancer, Penile Cancer, Peritoneal Cancer, Peutz-Jeghers Syndrome, Pituitary Gland Tumor, Polycythemia Vera, Prostate Cancer, Renal Cell Carcinoma, Retinoblastoma, Salivary Gland Cancer, Sarcoma, Sarcoma - Kaposi Skin Cancer, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymoma, Thyroid Cancer, Uterine (Endometrial) Cancer, Vaginal Cancer, Wilms' Tumor, for example.

[0413] The modified TPO polypeptides or functional fragments thereof, or the peptibodies described herein may be capable of increasing platelet production by MKs while limiting excessive stem and progenitor cell and MK expansion in a subject. As is shown in the examples provided herein the isolated and modified TPOs or a functional fragments described herein may stimulate platelet production while preserving HSCs. These properties may provide advantageous effects in the conditions described above. Suitably therefore, modified TPO polypeptides or functional fragments thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof described herein are for use in the treatment or prevention of diseases which involve a megakaryocyte or platelet deficiency. Such conditions may be the result of a deficiency (temporary or permanent) of active Mpl ligand in vivo. The generic term for platelet deficiency is thrombocytopenia, and hence the modified TPO polypeptides or functional fragments thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof of the present invention are generally available for prophylactically or therapeutically treating thrombocytopenia in patients in need thereof.

[0414] Suitably therefore, modified TPO polypeptides or functional fragments thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof described herein are for use in the treatment or prevention of a disease by increasing megakaryocytes or platelets in a subject in need thereof.

[0415] Suitably therefore, there is provided a method of increasing megakaryocytes or platelets in a subject in need thereof, comprising administering to the subject an effective amount of modified TPO polypeptides or functional fragments thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof described herein.

[0416] Thrombocytopenia (platelet deficiencies) may be present for various reasons, including chemotherapy and other therapy with a variety of drugs, radiation therapy, surgery, accidental blood loss, and other specific disease conditions. Exemplary specific disease conditions that involve thrombocytopenia and may be treated in accordance with this invention are: aplastic anemia; idiopathic or immune thrombocytopenia (ITP), including idiopathic thrombocytopenic purpura associated with breast cancer; HIV associated ITP and HIV-related thrombotic thrombocytopenic purpura; metastatic tumors which result in thrombocytopenia; systemic lupus erythematosus; including neonatal lupus syndrome splenomegaly; Fanconi's syndrome; vitamin B 12 deficiency; folic acid deficiency; May- Hegglin anomaly; Wiskott-Aldrich syndrome; chronic liver disease; myelodysplastic syndrome associated with thrombocytopenia; paroxysmal nocturnal hemoglobinuria; acute profound thrombocytopenia following C7E3 Fab (Abciximab) therapy; alloimmune thrombocytopenia, including maternal alloimmune thrombocytopenia; thrombocytopenia associated with antiphospholipid antibodies and thrombosis; autoimmune thrombocytopenia; drug-induced immune thrombocytopenia, including carboplatin-induced thrombocytopenia, heparin-induced thrombocytopenia; fetal thrombocytopenia; gestational thrombocytopenia; Hughes' syndrome; lupoid thrombocytopenia; accidental and / or massive blood loss; myeloproliferative disorders; thrombocytopenia in patients with malignancies; thrombotic thrombocytopenia purpura, including thrombotic microangiopathy manifesting as thrombotic thrombocytopenic purpura / hemolytic uremic syndrome in cancer patients; autoimmune hemolytic anemia; occult jejunal diverticulum perforation; pure red cell aplasia; autoimmune thrombocytopenia; nephropathia epidemica; rifampicin-associated acute renal failure; Paris-Trousseau thrombocytopenia; neonatal alloimmune thrombocytopenia; paroxysmal nocturnal hemoglobinuria; hematologic changes in stomach cancer; hemolytic uremic syndromes in childhood; hematologic manifestations related to viral infection including hepatitis A virus and CMV-associated thrombocytopenia. Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT). Certain wound healing disorders might also benefit from an increase in platelet numbers. With regard to anticipated platelet deficiencies, e.g., due to future surgery, a compound of the present invention could be administered several days to several hours prior to the need for platelets. With regard to acute situations, e.g., accidental and massive blood loss, a compound of this invention could be administered along with blood or purified platelets. The compounds of this invention may also be useful in stimulating certain cell types other than megakaryocytes if such cells are found to express Mpl receptor.

[0417] In particular, modified TPO polypeptides or functional fragments thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be for use in methods of treating or preventing thrombocytopenia. The method comprising, administering a therapeutically effective amount of modified TPO polypeptides or functional fragments thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a modified TPO polypeptides or functional fragments thereof or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a nucleic acid molecule or vector (encoding and capable of expressing a modified TPO polypeptide as described herein), or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a host cell or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a stem cell or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a peptibody or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a platelets or compositions thereof as described herein to a subject in need thereof.

[0418] Suitably the modified TPO polypeptide or a functional fragments thereof is as defined elsewhere herein.

[0419] Thrombocytopenia refers to a decrease in circulating platelet levels which leads to clinical manifestation typically below 50,000 / pL. This condition is commonly associated with defective formation of haemostatic plugs and bleeding, wherein the risk of bleeding is inversely proportional to the platelet count. Currently available treatments for thrombocytopenia and related conditions include, for example, corticosteroids, IVIG, splenectomy, injections of Thrombopoietin (TPO) and TPO mimetics, and whole blood or platelet transfusion, methods which are either palliative and non-specific, or drastic and expensive. Additionally, continuous platelet transfusions can cause immunologic platelet destruction. As such, platelets, modified TPO polypeptides and peptibodies as described herein may be particularly useful in methods of treating thrombocytopenia.

[0420] Suitably, the thrombocytopenia may be caused by or a sequela of an immunological disorder, haematological disorder, cancer or a medical intervention.

[0421] Suitably, the thrombocytopenia may be caused by or a sequelae of surgery or injury.

[0422] Suitably, the thrombocytopenia may be immune thrombocytopenia (ITP). ITP is a condition in which there is bruising or bleeding because there are fewer platelets in the blood than usual (thrombocytopenia) and is usually caused by dysregulation of the immune system

[0423] Immune thrombocytopenia, sometimes referred to as immune thrombocytopenic purpura or idiopathic thrombocytopenia, and more generally as ITP, is characterized by a decrease of peripheral blood platelet counts to less than 100x109per liter, as compared to 150x109to 450x 109per liter in healthy individuals. ITP is an autoimmune disease wherein autoantibodies are formed to glycoproteins. The autoantibodies are believed both to interfere with platelet production and to accelerate platelet destruction. Depressed platelet counts in individuals may be transitory or persistent, and may stem from a variety of causes. ITP with no identified associated causes or disorders is termed primary ITP, while ITP linked to other autoimmune or medical disorders is termed secondary ITP. Whereas ITP in adults typically has an insidious onset with no preceding viral or other illness and it normally follows a chronic course, ITP in children is usually short-lived with at least two-thirds recovering spontaneously within 6 months.

[0424] ITP can manifest itself by platelet-type bleeding (e.g., petechiae; purpura; conjunctival hemorrhage or other types of cutaneous bleeding). ITP can be associated with life-threatening complications such as intracranial bleeding. Even absent serious consequences, ITP patients suffer from a reduced quality of life, similar to other chronic diseases such as diabetes and rheumatoid arthritis. In 2011 the American Society of Hematology (ASH) issued guidelines for the treatment of ITP. Neunert C et al., Blood 117: 4190-4207 (2011). In general five different approaches are available to the treating physician. In general treatment is considered necessary for patients having a platelet count persistently below 30x109 / L. Current treatments for ITP include immunosuppressive agents, such as corticosteroids, intravenous immunoglobulin (IVIg) or anti-RhD immunoglobulin and TPO-R agonists.

[0425] Suitably, the thrombocytopenia may be chronic thrombocytopenia or chronic ITP.

[0426] Given the above, modified TPO polypeptides, peptibodies, host cells (e.g. via gene therapy) and stem cells (e.g. via cell therapy) as described herein may be particularly useful in methods of treating thrombocytopenia. In particular chronic thrombocytopenia or chronic ITP.

[0427] In particular, modified TPO polypeptides or a functional fragment thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be for use in methods of treating or preventing anemia. The method comprising, administering a therapeutically effective amount of modified TPO polypeptides or a functional fragment thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a modified TPO polypeptides or a functional fragment thereof or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a nucleic acid molecule or vector (encoding and capable of expressing a modified TPO polypeptide or a functional fragment as described herein), or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a host cell or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a stem cell or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a peptibody or compositions thereof as described herein to a subject in need thereof. Suitably, the method comprising, administering a therapeutically effective amount of a platelets or compositions thereof as described herein to a subject in need thereof.

[0428] Suitably the modified TPO polypeptide or a functional fragment is as defined elsewhere herein

[0429] Anemia is a condition in which a number of red blood cells (RBCs) is insufficient to meet a body's physiological need. Anemia can be classified from three perspectives: pathogenesis, red blood cell morphology, and clinical presentation. Pathogenic classification refers to the production or the inadequate production of erythrocytes and can be subdivided into two types, hypo-regenerative and regenerative. Hypo-regenerative anemia refers to a pathogenic condition in which the bone marrow production is decreased as a result of impaired function, decreased number of precursor cells, reduced bone marrow infiltration, or a lack of nutrients. Regenerative anemia refers to a pathogenic condition in which bone marrow response appropriately to a low erythrocyte mass by increasing the production of erythrocytes, nemia classified based on red blood cell morphology can be further subdivided into microcytic anemia, normocytic anemia, and macrocytic anemia. Microcytic anemia can be defined as the presence of small, hypochromic red blood cells in a peripheral blood smear and can also be characterized by a low mean corpuscular volume (MCV). Normocytic anemia can be defined as an anemia with a normal MCV range but with decreased hematocrit and / or hemoglobin values. Macrocytic anemia can be defined as the presence of larger than normal red blood cells. Anemia is not necessarily a disease but is instead often a manifestation of an underlying disorder. The etiology behind anemia can be complex and to some degree, difficult to determine. For example, diagnosis of anemia can begin with a general physical checkup and a complete blood count (CBC) work-up. Based on the parameters from the CBC work-up, one or more additional tests are further carried out, sometimes requiring one or more additional hospital or clinical visits by the individual and / or long wait time between each test result. Treatments for anemia include, for example, iron supplements, vitamin supplements (such as folic acid or vitamin B-12), and blood transfusions. Suitably, the anemia may be caused by or a sequelae of an immunological disorder, haematological disorder, cancer or previous medical intervention.

[0430] Suitably, the anemia may be aplastic anemia. Aplastic anemia is a potentially life-threatening, rare and heterogeneous disorder of the blood in which the bone marrow cannot produce sufficiently enough new blood cells due to a marked reduction of immature hematopoietic stem (HSC) and progenitor cells (Scopes et al., 1994, Maciejewski et al., 1994). Accordingly, the main disease manifestations are pancytopenia and marrow hypoplasia which can emerge at any stage of life but are more frequent in young people (age 10-25 years) and the elderly (>60 years) (Marsh et al., 2009). Aplastic anemia can be acquired or inherited. The acquired type is mainly autoimmune-mediated but can also be triggered by environmental factors such as radiation, toxin and virus exposure (Nakao, 1997). The congenital form is rarer, however, mutations in more than 30 genes with functions in DNA repair, ribosome biogenesis and telomere maintenance pathways have been identified to date (Dokal & Vulliamy, 2010). A frequently observed clinical feature of aplastic anemia is short telomere length in peripheral blood leukocytes even in the absence of mutations in the telomere maintenance machinery.

[0431] Suitably, the anemia may be severe aplastic anemia.

[0432] Suitably, the anemia may be severe hypoplastic anemia. Hypoplastic anemia refers to an anemia that results from insufficient differentiation of progenitor cells into erythrocytes. Hypoplastic anemia is usually seen in the first year of life. Patients may have deformed thumbs and other physical problems. They also have an increased risk of leukemia and sarcoma, especially osteosarcoma. Patients with congenital hypoplastic anemia may have a mutation in one of the genes that make proteins found in the cell’s ribosomes. Hypoplastic anemia may also be called Blackfan-Diamond anemia, congenital pure red cell aplasia, DBA, Diamond- Blackfan anemia, erythrogenesis imperfecta, and inherited erythroblastopenia

[0433] Given the above, modified TPO polypeptides or a functional fragment thereof, peptibodies, host cells (e.g. via gene therapy) and stem cells (e.g. via cell therapy) as described herein may be particularly useful in methods of treating anemia. In particular aplastic anemia and hypoplastic anemia.

[0434] In addition to the above, modified TPO polypeptides or a functional fragment thereof, nucleic acid molecules, vectors, host cells, stem cells, platelets and peptibodies or compositions thereof as described herein may be for use in blood transfusions. Suitably, the anemia may be caused by or a sequelae of surgery or injury.

[0435] BRIEF DESCRIPTION OF THE DRAWINGS

[0436] Figure 1. Extracellular structure of the TPO-2xMPL signaling complex. (A) Cryo-EM 3D reconstruction of the TPO-2XMPLECD complex and ribbon model of the top view covered by a transparent surface. Volumes and cartoons are annotated TPO, MPL chain 1 , and MPL chain 2, respectively. The map contour level is set to 9o. (B) Comparison of the extracellular domain architectures amongst MPL, EpoR, GHR, and PRLR. The ligand-receptor complexes are shown side-by-side above a cell membrane cartoon.

[0437] Figure 2. TPO recognition by MPL. (A) Comparison of the cytokine binding mode of the TPO- 2xMPL complex with the EPO-2xEpoR complex. The a-helices A-D are shown as cylinders for TPO and EPO. Loops 1 to 6 (L1 to L6) of MPL are indicated in the panel, where the extremely long L5 is unresolved in the structure. The black arrows indicate the linker between D1 and D2 of MPL. (B) Zoomed-in views of the site 2 (left hand box) and site 1 (right hand box) TPO-MPL interfaces (corresponding to the small boxes in panel A). The sidechains of the interface residues are shown as sticks and labelled. MPL chains are chain 1 , at TPO-TpoR site 1 and chain 2, at TPO-TpoR site 2. (C) Domain diagrams and cartoon representations of TPO and MPL highlighting the loss of function (LOF) mutations. SP: Signal peptide, A-D: TPO a-helices A-D, Pro-rich: proline-rich region, horizontal brackets: disulfide bond in the domain diagrams. LOF single point mutations are indicated with up arrows in the diagrams and annotated in the cartoon models.

[0438] Figure 3. Tuning the TPO-MPL site 2 interaction. (A) Functional determinants of the TPO- MPL site 2 interface, (top) TPO is shown as a ribbon and MPL is shown as transparent surface representation. The five TPO residues selected for the initial mutagenesis screening, including two tunable ones, K35TPOand R119TPO, subjected to further signaling and functional characterization are shown as sticks, (bottom) Zoom-up views of K35TPOand R119TPOinteracting with MPL. The cryo-EM map is shown as gray transparent surface and overlaid on the structure at the counter levels of 10o. (B) UT7-TPO viability assays comparing treatment with increasing concentrations of WT TPO orTPOmoc,supernatantfor 72 hours. Data presented as relative mean cell growth (±SEM) compared to maximal stimulation with WT TPO. (C) Signaling activity was determined by cell barcoded phospho-flow cytometry with either increasing concentrations of WT TPO or TPOmoc / (0-100 ng / mL for 30 mins, left panel) or increasing time (50ng / mL, 0-240 mins, right panel). Data were normalized by generating a z- score of the mean from four independent experiments (±SEM). (D) Schematic outlining the in vitro method used to expand human CD34+cells in 7-day culture followed by TPO stimulation and phosphoflow cytometry to determine signaling response. (E) Intracellular signaling was determined in EPCR+HSCs re-treated with TPO on day 7 for 1 hour and stained for pJAK2 (Y1007 + Y1008), pSTAT5(Y694), pCREB(S133) and pAKT(S473). For all human HSC signaling graphs, a one-way Anova was used to calculate significance of differences. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. (F) Schematic summarizing the changes in signal activation comparing WT TPO and TPOmod. Arrow thickness represents relative level of signaling.

[0439] Figure 4. Quantifying the MPL monomer-dimer equilibrium by single-molecule FRET (smFRET). (A) Schematic outlining the method used to determine MPL monomer-dimer equilibrium Labeling of ALFA-tagged MPL by anti-ALFA nanobodies site-specifically conjugated with Cy3B and ATTO643, respectively. (B) smFRET trajectories highlighting MPL dimers observed in the absence of ligand and upon stimulation with 10 nM WT TPO or TPOmod(scalebar: 2 pm). (C) Quantification of MPL dimerization in the presence of TPOmodEach data point represents the analysis from one cell with the number (N) of cells measured for each condition indicated. (D) smFRET efficiencies in MPL dimers observed in the presence TPO or TPOmod. £ach data pOjnt represents the analysis from one trajectory with the number (N) of trajectories measured for each condition indicated. Data in C and D are presented as box plots and indicate the data distribution of 2ndand 3rdquartile (box), median (line) and 1.5 x interquartile range (whiskers).

[0440] Figure 5. Functional effects of TPOmodon murine hematopoiesis in vivo. (A) Schematic of the experimental workflow for in vivo murine TPO functional analysis. (B) (i) Representative histogram of EPCR+HSCs in the HSPC (LSK CD34- CD135-) compartment and (ii) frequency of EPCR+LT-HSCs in the bone marrow of mice treated with PBS, WT mTPO or mTPOmoc / at day seven (n = 5 from three independent experiments). (C) (i) Representative histogram of CD41+megakaryocyte progenitors (MkP) within the megakaryocyte-erythroid progenitor (Lin- cKit+ScaT CD16 / 32- CD34-) compartment and (ii) frequency of MkPs in the bone marrow of mice treated with WT mTPO or mTPOmoc / (n = 5 from three independent experiments). (D) Enumeration of CD41+BM MKs at day seven post PBS, WT mTPO and TPOmodtreatment (CD41 = cyan labelled; nuclear staining (DAPI) = white). Whole femur sections were analyzed using a fully automated Axio Scan.ZI slide scanner (i) and positive staining was quantified using StrataQuest software (ii) megakaryocyte size was quantified using StrataQuest software. (E) Circulating platelet counts following treatment with PBS, WT mTPO or mTPOmoc / . Each data point represents mean count ± SEM of at least seven mice (range 7-9) for days 0-7 and at least two mice (range 2-4) on days 10-15 from four independent experiments. (F) Schematic summarizing the effects of WT TPO and TPOmoc / on LT-HSC expansion and megakaryopoiesis in vivo.

[0441] Figure 6. Functional effects of TPOmoc / on human HSCs in vitro. (A) Schematic of the experimental workflow for in vitro human TPO functional analysis using human CD34+cells in 7-day expansion cultures. (B) Quantification of total cell expansion after seven days and frequency of primitive progenitors (CD34+CD45RA-) and CD90+EPCR+HSCs in human CD34+cultures treated with WT TPO or TPOmoc / . (C) colony count and frequency from the colony forming unit (CFU) assays generated from 200-cell input following a seven-day expansion of CD34+cells in the presence of TPO variants. (D) Dimensionality reduction of WT and TPOmoc / scRNA sequenced CD34+CD45RA- after 7 days of expansion in vitro. HSC heatmap statistic is mapped based on phenotypic HSC genes (positive: CD34, THY1 , PROCR, HLF, AVP, MLLT3, SPINK2, PROM1 , FLT3, HOXA9, MECOM; negative: FLI1 , GATA1 , HBD, ITGA2B) in each panel, the darker patches are close to 0.0, the lighter patches are between 0.1 -0.3. (E) Venn overlap of HLF+vs HLF differentially expressed genes in each TPO treatment. (F) Venn overlap of differentially expressed genes between WT and TPOmoc / HLF+HSCs. A one-way ANOVA was used to calculate significance of differences where *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. (G). Schematic summarizing the effects of WT TPO and TPOmoc / on ex vivo human HSC expansion.

[0442] Figure 7. Purification of the functional domain of TPO in complex with full-length MPL. (A) Schematic of the recombinant expression constructs for TPO and MPL with purification scheme, and representative final SEC profile and SDS-PAGE gels for structural study. (B) A negative-stain image, (C) a preliminary cryo-EM micrograph, 2D class averages, and 3D reconstruction from initial screening experiments. (D) Representative cryo-EM micrograph and (E) 2D class averages for structural determination.

[0443] Figure 8. Cryo-EM data analysis for the TPO-2xMPL complex. (A) Cryo-EM data processing scheme used to generate the 3D reconstruction of TPO-2XMPLECD. (B) Orientation distribution of the aligned particles in the final 3D reconstruction output from cryoSPARC. (C) Gold- standard Fourier shell correlation (FSC) curves from lowest to highest: no mask to tight and corrected almost overlaid, for the final 3D reconstruction. The FSC mask (outline) for the corrected FSC curve is overlaid onto the 3D map of TPO-2XMPLECD (gray). (D) Local resolution estimates from Phenix, colored on the surface representation of the deepEMhancer sharpened map. 2.8 A across most of the three maps, except the lower tip of the protein in the middle and right hand map which is 6.4 A, respectively. The map contour level is set to 8o. Figure 9. Validation of the cryo-EM model for the TPO-2xMPL complex. (A) Map-model FSC correlation calculated by Phenix. (B, C) Local cryo-EM maps overlaid on (B) the segmented TPO structure or (C) D1 , D2, D3, and D4 of MPL. The map contour levels are indicated in the figure panels.

[0444] Figure 10. Structural characteristics of the TPO-2xMPL complex. (A) Structural features of the extracellular region of MPL with cryo-EM map. A surface representation of MPL chain 1 is colored lighter for CRM 1 and darker for CRM2 and zoomed-in boxes are indicated. The boxed regions are magnified and displayed in ribbon and stick models with black-mesh cryo-EM density overlayed at the contour level of 9o. Boxes are labelled: N-terminal pyroglutamate and surrounding structure; tryptophan mannosylation on the WGSWS motif, disulfide bond bridging D2-D3; domain packing between D2 and D3. (B) Cartoon models showing the structural comparison of the relative CRM geometries amongst the GHR family. Buried surface area between contacting D2 domains is indicated in parentheses.

[0445] Figure 11. TPO recognition by MPL. (A) Magnified views of the site 1 and 2 TPO-MPL interfaces with cryo-EM map contoured at 9o. The views correspond to those shown in Figure 2B. (B) The interaction diagram for the TPO-2xMPL complex at the site 1 (right) and site 2 (left) interfaces created using PDBsum (ebi.ac.uk / pdbsum). Positively charged amino acids; His, Lys, and Arg, negatively charged; Asp, Glu, neutral polar; Ser, Thr, Asn, Gin, Cys, aromatic; Phe, Tyr, Trp, aliphatic; Ala, Vai, Leu, lie, Met, and Pro and Gly.

[0446] Figure 12. Preparation of human TPO variants and assessment of their ability to support UT7- TPO cell growth. (A) Domain diagram of full-length human TPO with sites of mutagenesis indicated with arrows, and introduced mutations tabulated to the right (left panel); expression, preparation and quantification scheme (right panel). TPO variants were used without purification for the cell growth assays. (B) Representative SDS-PAGE (lower panel) and anti- TPO immunoblot (upper panel) analysis showing levels of TPO expression in cell-free supernates. Equal supernate volumes were loaded per lane. (C) Quantification of TPO expression levels by ELISA. (D) UT7-TPO cell proliferation data for TPO variants measured using an XTT assay after 72 hours.

[0447] Figure 13. Dose response signaling assays for TPO K35 variants in UT7-TPO cell cultures. UT7-TPO cells were treated with cell-free supernates containing WT TPO or TPO K35 variants at the indicated concentrations. Phosphorylation of downstream effectors in fixed cells after 30 minutes was measured using barcode-labelled phosphoflow cytometry. Cell growth was measured after 72 hrs using an XTT assay. Data are the mean ± SD for four independent experiments.

[0448] Figure 14. Signaling kinetics assays for TPO K35 variants in UT7-TP0 cell cultures. UT7- TPO cells were treated with cell-free supernates containing 50 ng / mL WT TPO or TPO K35 variants for the indicated times. Phosphorylation of downstream effectors in fixed cells was measured using barcode-labelled phospho-flow cytometry. Cell growth was measured after 72 hrs using an XTT assay after treatment with the indicated TPO K35 variants at the indicated concentrations. Data are the mean ± SD for three independent experiments.

[0449] Figure 15. Dose response signaling assays for TPO R119 variants in UT7-TPO cell cultures. UT7-TPO cells were treated with cell-free supernates containing WT TPO or TPO R119 variants at the indicated concentrations. Phosphorylation of downstream effectors in fixed cells after 30 minutes was measured using barcode-labelled phosphoflow cytometry. Cell growth was measured after 72 hrs using an XTT assay. Data are the mean ± SD for three independent experiments.

[0450] Figure 16. Signaling kinetics assays for TPO R119 variants in UT7-TPO cell cultures. UT7- TPO cells were treated with cell-free supernates containing 50 ng / mL WT TPO or TPO R119 variants for the indicated times. Phosphorylation of downstream effectors in fixed cells was measured using barcode-labelled phosphoflow cytometry. Cell growth was measured after 72 hrs using an XTT assay after treatment with the indicated TPO R119 variants at the indicated concentrations. Data are the mean ± SD for three independent experiments.

[0451] Figure 17. Purification of full-length human TPO variants and assessment of their ability to bind to MPL via the high affinity site 1 using surface plasmon resonance (SPR). (A) Domain diagrams of the C-terminal 8xHis-tagged full-length human and murine thrombopoietin, and schematic of their expression and purification. The mutagenesis sites and the introduced mutations are indicated in the figure panel. (B) Schematic and (C) representative sensorgrams of SPR experiments. (B) High-affinity (site 1) binding of TPO to MPL was examined. MPL extracellular domain (26-491) was captured on the chip surface via a biotinylated Avi-tag and recombinant full-length TPO was injected over the chip as analyte. (C) Representative multicycle kinetics sensorgrams are shown for WT TPO and three TPOmoc / (K35A, K35Q, R119E) note TPO concentrations shown in the inset legend, note that for all panels the order of the lines from top to bottom in the graph are the exact reverse of the order of the lines in the inset legend) binding to MPL and are overlaid with their fit to a 1 :1 interaction model (black lines). Inset shows corresponding steady-state affinity fit. RU, response units. Average KDI ± SD derived from the kinetic fit (1 :1) and steady-state fit (SS) are reported for each TPO variant above the relevant sensorgram plot.

[0452] Figure 18. Cell-surface MPL dimerization detected by single-molecule FRET. Experimental for single-molecule FRET detection by alternating laser excitation. (A) Representative trajectory maps of Cy3B-labeled MPL upon donor excitation (“Donor”), of ATTO643-labeled MPL upon direct acceptor excitation (“Acceptor”) and sensitized acceptor fluorescence upon donor excitation (“FRET”), probed in the presence of 10 nM WT TPO or TPOmoc / . Scalebar: 2 pm. (B) Comparison of the overall MPL density applied in different experiments.

[0453] Figure 19. Workflow of analysis of megakaryocytes in mouse bone marrow following TPO treatment. Quantification analysis was performed on images of bone marrow CD41 + megakaryocytes (MK) from mouse femur sections. Whole section images were taken using the Zeiss AxioScan.ZI from mice treated with PBS, WT TPO or TPOmod. Section data was then analysed using StrataQuest TissueGnostics software by nuclear segmentation using DAPI staining and a CD41 cellular mask applied to regions of interest for measurements. Single MKs were gated on staining intensity and total area (pm). Exported data was analysed using GraphPad Prism 8.0 software.

[0454] Figure 20. Complete blood counts of mice treated with PBS, WT mTPO or mTPOmoc / . Labels along the top of the figure align with the data points in the graphs below. Blood samples were taken via the saphenous vein at day 0 (pre-bleed), and 5, 7, 10 and 15 days post-treatment. Complete blood counts were analyzed using a ProCyte Dx Hematology Analyzer (IDEXX). Each data point represents a single mouse. Data represents 4 independent experiments.

[0455] Figure 21. Dimensionality reduction and pseudotime mapping of scRNAseq. (A) Merged UMAP clustering of all scRNAseq data in labeled areas. (B) Pseudotime projection on UMAP and (C) across clusters for WT and TPOmoc / . (D) Features distinguishing clusters to identify HSCs and downstream progenitors from pseudotime, most of the darker points have an average expression of between 1-2 with the exception of CDK6 at identity 10 which has an average expression of -2.

[0456] Figure 22. Identification of HSCs and differentially expressed genes in scRNAseq. (A) Expression of HSC genes, Megakaryoctye / Erythroid progenitor genes, HLF and Ki67 across UMAP clusters, the darker shading in each panel is at the lowest end of the scales shown, the lighter shading is in the mid-range of the scales shown. (B) Venn overlap of pseudobulk differentially expressed genes between WT and TPOmocte(C) Number of differentially expressed genes between WT and TPOmodsin pseudobulk, HLF+HSCs and HLF+vs HLF' cells.

[0457] EXAMPLES

[0458] Example 1 : Structure of the TPO-MPL complex

[0459] Introduction

[0460] Thrombopoietin (TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. The inventors resolved the 3.4 A resolution cryo-electron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmoc / ) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmoc / decoupled JAK / STAT from ERK / AKT / CREB activation driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utilities of TPOmoc / in hematology research and clinical HSC transplantation.

[0461] Thrombopoietin (THPO or TPO) is a class I cytokine essential for hematopoietic stem cell (HSC) maintenance1 2, and is the primary driver of megakaryocyte differentiation and platelet production3-5. Dysregulation of TPO and the TPO receptor (TpoR or MPL) leads to disease states, with loss-of-function (LOF) mutations leading to thrombocytopenia and bone marrow failure6-8, and gain-of-function (GOF) mutations resulting in the development of myeloproliferative neoplasms (MPNs)9-11. Furthermore, the most common MPN-driver mutations, JAK212 13and calreticulin14 15, cause MPL hyperactivation via interaction with the receptor intracellular and extracellular domains, respectively. Clinically, TPO agonists including the small molecule eltrombopag16and the peptibody romiplostim17have emerged as key therapies for chronic immune thrombocytopenia (ITP) and severe aplastic anemia18 19. However, induction of platelet production comes at the cost of increased HSC proliferation and risk of bone marrow fibrosis20121, highlighting a critical need to decouple these two functions. Additionally, TPO is one of three key cytokines that are essential for the expansion of functional HSCs in vitro2223, and is still used today in the majority of new clinical protocols aimed at increasing HSC expansion for transplantation across a range of malignant and non- malignant hematopoietic disorders24. Unfortunately, issues remain with TPO-induced HSC proliferation, with TPO dose being critical for optimum HSC output25and excessive HSC expansion leading to loss of multi-lineage regenerative capacity26. Thus, balancing TPO-MPL signaling is a critical factor in HSC maintenance and expansion.

[0462] The basic activation mechanism, TPO-driven homodimerization of the monomeric M PL chains, was only recently described using single-molecule techniques27Furthermore, GOF mutations in the receptor, calreticulin, and JAK2 all activate MPL by the shared homodimerization mechanism2728, but with a diverse mode of action, activating distinct downstream signaling pathways which, in turn, drive different physiological outcomes, highlighting the plasticity of this pathway29. The inventors have previously exploited this diversity in activation by using diabodies that bind to and differentially activate MPL to manipulate functional outcomes30, opening up the possibility of developing biased MPL agonists to decouple the functional pleiotropy of MPL.

[0463] Although the TPO-MPL-JAK2 axis remains an attractive target for therapeutic intervention, the structure-guided development of agents that fine tune receptor activation, as seen in other cytokine receptors and G protein-coupled receptors31 32, is hampered by the lack of detailed structural information for the activating TPO-2xMPL complex.

[0464] The inventors describe the 3.4 A cryo-electron microscopy (cryo-EM) structure of TPO bound to two TpoR / MPL extracellular domains (ECDs) in which the cytokine-cytokine receptor interactions are clearly visualized. This provided a blueprint for the design of TPO-based agonists by attenuating the low-affinity interaction site. I ntriguingly, the inventors found several biased agonists which stimulated intracellular signaling pathways in a tunable manner and altered cellular outcomes by uncoupling TPO-stimulated HSC expansion and platelet production.

[0465] Structure of the TPO-MPL complex

[0466] MPL is a “tall” cytokine receptor with the ECD consisting of two cytokine receptor-like modules (CRMs), CRM1 and CRM2, followed by a transmembrane helix and an intracellular domain (ICD)33. The inventors co-expressed and purified the functional domain of TPO and the full- length MPL, consisting of the ECD, TM, and unstructured ICD segments (Figure 7A). After isolating detergent-solubilized MPL from HEK293S GnTL cell membranes by means of a C- terminal His tag, the ligand-receptor complex was purified using an N-terminal FLAG tag on TPO. The complex was further purified by size-exclusion chromatography (SEC) yielding a viable sample for cryo-EM analysis (Figure 7). The 3D reconstruction for the extracellular region of the complex (Figures 1 , 8, 9, and Table 1) revealed both shared and distinct structural features among evolutionarily related class I cytokine receptors. The “tall” ECD dimeric architecture of MPL is twisted in the complex, unique from the “short” homodimeric cytokine receptor family sharing a common ancestor; growth hormone receptor (GHR), prolactin receptor (PRLR), and erythropoietin receptor (EpoR) comprise a single CRM (Figure 1 B)34. The two CRMs of MPL are subdivided into four globular domains, D1 and D2 for MPLCRMI , and D3 and D4 for MPLcRM2 with topological features optimal for TPO-induced dimerization (Figure 10). MPLCRMI is responsible for TPO recognition, and MPLCRM2 is critical to bringing receptor TMs and ICDs into proximity conducive for downstream signal transduction. The TPO-2XMPLCRMI region bears a resemblance to the erythropoietin (EPO)-2XEPORECD complex35and other canonical cytokine-cytokine receptor complexes36’37, where two CRM 1s form a Y-shaped fork to trap TPO (Figure 10B), however, in the case of the TPO-2xMPL complex, there is no direct interaction between D2s. Instead, the cryo-EM map shows the D4s are in close proximity for homodimeric activation2738(Figure 1A).

[0467] Table 1. Data collection and refinement statistics of the cryo-EM analysis.

[0468] *AlphaFold2 accession number. **Phenix local anisotropic sharpening based on half maps.

[0469] The inventors identified several unique structural features in the MPLECD. First, the N-terminal glutamine residue should form pyroglutamate, which, together with a subsequent short a-helix, is folded between D1 and D2 (Figure 10A top-right black box), such that the D2s are twisted away from each other (Figure 10B). The N-terminal a-helix is shorter than, but similar to, the one observed in EpoR, but differs from that in GHR and PRLR. D1 and D2 ‘pinch’ the helix to create a larger D2-D2 distance in the MPL and EpoR complexes. The EPO-2xEpoR complex shows parallel D2s for optimal signaling, but with tenuous direct stem contact (~40 A2interface). An alternative EPO-2xEpoR conformation and the 2x(EMP1-EpoR) structure have also been reported with D2s in a ‘twisted’ arrangement (Figure 10B), where the surrogate peptide agonist EMP1 has much lower efficacy than EPO35,74. These features contrast with the extensive inter-receptor “stem” contacts seen in the GHR and PRLR complexes with interface areas of -470 A2and -670 A2, respectively (Figure 10B). Second, we observed cryo- EM densities corresponding to tryptophan C-mannosylation at the WSXWS motif-like WGSWS sequence within MPLD2 (Figure 10B bottom-left blue box) which has been reported to regulate downstream signaling75, in addition to N-glycans that appear to be crucial for the receptor trafficking and stability76. Note that we observed less-defined mannose densities on MPLD4, and these could not be completely modeled. Third, MPLD2 has an extremely long sequence between a-helices D and E, the so-called DE loop (L5), where the initial -10 amino acids are structured and make a disulfide bond to the CD loop of MPLDS (Figure 10A center purple box). This disulfide linkage between D2 and D3 stabilizes the D2-D3 packing (Figure S4A bottom-right red box) and the relative orientation of CRM1 and CRM2, effectively transmitting the ligand-binding geometry to the membrane-proximal region. The latter ~40 amino acids of L5 are unstructured.

[0470] TPO bridges two MPL chains via the high- and low-affinity interfaces called site 1 and site 2, fostering TPO-specific binding geometry (Figures 2A, 2B, and 11). The interface areas of site 1 and 2 are -750 A2and -620 A2with shape complimentary scores of 0.652 and 0.598, respectively (Figure 2B). For the high-affinity site 1 interface, the a-helix B’ and the adjacent loop of TPO are jointly recognized by D1 and D2 of MPL. The low-affinity site 2 consists of interfaces between (i) MPLDI and TPO helix C with a unique R119TPO:R102MPLstacking39; and (ii) MPLD2 and TPO helix A. The LOF thrombocytopenia mutations R38C / QTPO, R99WTPO, and R119CTPOare all located at the site 2 interface (Figures 2C and 11)33. In a similar manner, the LOF mutant F104SMPLcauses thrombocytopenia due to impaired binding to TPO while homozygous R102PMPLmutations alters receptor trafficking, reducing membrane expression, also leading to thrombocytopenia33. F104MPLand R102MPLshow extensive interactions with TPO at both sites 1 and 2 (Figures 2B, 2C, and 11). LOF mutations also occur at W154MPLand R257MPL, whose sidechains are directed toward the domain core and the mannosylated WGSWS of MPLD2, respectively, stabilizing the protein folding (Figures 2C and 10A).

[0471] Example 2: Cell growth supported by TPO variants (TPOmod) R31TPO, K35TPO, R38TPO, R99TPOand R119TPO

[0472] TPO activates multiple intracellular signaling pathways via JAK2-mediated receptor phosphorylation and STAT activation in hematopoietic stem and progenitor cells (HSPCs) and cells of the megakaryocyte lineage33. In order to probe the molecular determinants of these functional outputs, the inventors exploited the structural information on the low-affinity site 2 interface to design mutants that would alter MPL dimerization to yield differential signaling.

[0473] The inventors selected five basic residues on TPO surface that mediate interactions with MPL, namely residues R31 , K35, R38, R99, and R119 (Figures 3A top and 12A left). These encompass a range of chemical properties that could affect the interface in different ways40, including 1) Ala (small hydrophobic), 2) Gin (medium polar), 3) His (aromatic with positive, maintained charge), and 4) Glu (negative, opposite charge). The inventors investigated the following TPOmodvariants: R31TPOvariants R31A, R31 E, R31 H, R31Q; K35TPOvariants K35A, K35E, K35H, K35M, K35Q; R38TPOvariants R38A, R38E, R38H, R38Q; R99TPOvariants R99A, R99E, R99H, R99Q, R99W; and R119TPOvariants R119A, R119C, R119E, R119H, R119Q. In particular, the inventors hypothesized that TPO-R119E and TPO-R119H would be worth quering as R119TPOforms cationic TT-TT stacking with the counterpart arginine residue at MPL. Two disease mutants, TPO-R99W and TPO-R119C33, are also included, and TPO-K35M was tested to include a hydrophobic residue with similar size to the wild-type.

[0474] The inventors then screened sidechain substitutions at each site R31 , K35, R38, R99, and R119 for their ability to support cell growth using cell culture supernatant containing TPO variants (Figures 12A-12C). These included R31TPOvariants R31A, R31 E, R31 H, R31Q; K35TPOvariants K35A, K35E, K35H, K35M, K35Q; R38TPOvariants R38A, R38E, R38H, R38Q; R99TPOvariants R99A, R99E, R99H, R99Q, R99W; and R119TPOvariants R119A, R119C, R119E, R119H, R119Q. K35TPOand R119TPOvariants showed the most “tunable” nature, including WT-like, neutral antagonists, partial-agonists, and super-agonists (Figures 3A and 3B). In cell proliferation assays, TPO-K35A and TPO-K35Q were partial agonists, with TPO- K35Q being less potent compared to TPO-K35A. TPO-R119H appeared to be a more potent agonist than WT TPO, while TPO-R119E was a partial agonist (Figure 3B).

[0475] Example 3: Molecular signaling supported by TPOmodvariants K35TPOand R119TPO

[0476] The inventors next determined how TPOmoc / variants, namely K35TPOvariants K35A, K35E, K35H, K35M, K35Q and R119TPOvariants R119A, R119C, R119E, R119H, R119Q, altered signaling using barcoded phospho-flow cytometry in UT7-TPO cells. Signaling was determined by dose (0-100 ng / mL for 30 min) and kinetics (50 ng / mL for 0-240 min). All tested TPOmodvariants Were able to activate signaling (Figures 3C, 13-16), although maximal stimulation was generally lower than WT TPO, in particular activation of JAK2, STAT3 and STAT5. Of the TPOmoc / variants, JAK / STAT activation was highest in TPO-K35A, which also exhibited comparable levels of ERK / AKT / CREB activation to WT TPO. Although maximal activation of AKT was comparable to WT TPO, TPO-K35A was not able to maintain activation of AKT for as long as WT TPO. These data are consistent with the finding that levels of phospho-mTOR (S2448), which is phosphorylated by AKT, were lower after treatment with TPO-K35A. Interestingly, TPO-K35Q was less effective than WT TPO at activating all pathways, especially JAK / STAT, although it was able to stimulate CREB activation to a level comparable to TPO-K35A. TPO-R119E shows a similar “fingerprint” to TPO-K35Q, exhibiting low levels of JAK / STAT and ERK / AKT activation, while stimulating CREB activation at higher concentrations. Consistent with the proliferation assays, TPO-K35E exhibited neutral antagonism by failing to activate any of the signaling pathways investigated (Figures 13 and 14), whereas TPO-R119H activates JAK / STAT more potently than WT TPO, especially at earlier time points (5-30 min; Figures 15 and 16).

[0477] In comparison to other synthetic TPO agonists, TPOmoc / are unique in that they are predicted to maintain the normal binding geometry of the dimeric receptor, which has been shown to be a key determinant of signaling outcome41, whilst showing differential efficacies in MPL-JAK2 dimer formation compared to the endogenous complex31141. The synchronized changes in JAK / STAT activation and cell growth in UT7-TPO cells confirmed the ability of TPOmoc / variants to differentially regulate cytokine-induced proliferation via the JAK / STAT signaling pathway. Interestingly, however, attenuation of JAK2 and STAT3 / 5 phosphorylation was not necessarily coupled to changes in the phosphorylation of other downstream effectors, such as AKT and CREB, and differed among TPOmoc / variants.

[0478] Example 4: Signaling in primary hematopoietic stem and progenitor cells (HSPCs) of TPO"100' variants K35A, K35Q and R119E

[0479] To enable more precise probing of TPO pharmacology, the inventors purified the WT TPO and selected TPOmod, the partial agonists TPO-K35A / Q and TPO-R119E (Figure 17A). K35TPON^-H forms a hydrogen bond with E160MPLbackbone carbonyl, whilst R119TPOmakes an arginine-arginine stack with R102MPLand a possible salt bridge with E99MPL(Figures 3A bottom and 11). Surface plasmon resonance (SPR) demonstrated that all the recombinant TPOmoc / retained the wild-type site 1 affinity to MPL-ECD (Figures 17B, 17C, and Table 2), confirming that the proteins were properly folded.

[0480] These purified TPOmoc / variants, namely K35A, K35Q and R119E, were then used to determine whether the signaling differences were also observed in human primary HSPCs. Human CD34+cells were expanded with SCF, FLT3L and TPO for seven days prior to stimulation with WT TPO or TPOmoc / for one hour and changes in signaling activity were determined by phospho-flow cytometry (Figure 3D). The inventors observed similar trends in the activation of JAK2 and STAT5, with reduced activation in the presence of TPOmoc / , especially with TPO- R119E, compared with WT TPO (Figure 3E). However, TPOmoc,-mediated activation of both AKT and CREB was comparable to WT TPO, mirroring the differential signaling observed in UT7-TPO cell line. Changes in signaling activity are summarized in a schematic (Figure 3F). Table 2: Kinetic and steady-state parameters, site 1 binding constants and quality control data from two independent surface plasmon resonance (SPR) experiments measuring the binding of TPO as analyte to biotinylated MPL extracellular domain as ligand. KD values were derived by fitting the data to a 1:1 interaction model (kinetic fit) and also to an equilibrium binding model (steady-state fit).

[0481] Example 5: MPL dimerization induced by TPOmo< / variants K35A, K35Q and R119E The inventors next probed by live cell single molecule Forster resonance energy transfer (smFRET) how the recombinant TPOmoc / variants, namely K35A, K35Q and R119E, altered ligand-induced MPL dimerization. To this end, N-terminally ALFA-tagged MPL and C- terminally mEGFP-tagged JAK2 with tyrosine kinase deletion (JAK2ATK) were transiently expressed in HeLa cells and the cell-surface MPL was selectively labelled with ALFA-tag nanobodies conjugated to Cy3B and ATTO643, respectively (Figure 4A). Cells were then stimulated with PBS, 10 nM WT TPO, or 10 nM TPOmoc / , and MPL dimerization reported via smFRET was quantified by time lapse total internal fluorescence (TIRF) microscopy with alternated dual-color laser excitation (Figure 18A). Very low levels of unstimulated MPL dimerization and strong dimerization by WT TPO were confirmed by smFRET (Figures 4B and 4C). The mean FRET efficiency of 32% observed for WT TPO-induced MPL dimers was well in line with the ~7.2 nm distance between the MPL N-termini observed in the structure (Figure 4D). All three TPOmoc / variants K35A, K35Q and R119E induced significantly lower levels of MPL dimerization than WT TPO (Figures 4C and 18B), in line with an attenuated site 2 interaction affecting the monomer-dimer equilibrium. Although differences among the three different TPOmoc / variants were minor, lower dimerization by TPO-R119E as compared to TPO- K35A was statistically significant. Since the geometry of cytokine receptors dimers has been shown to influence signaling41, the inventors used FRET to gauge effects of the TPOmoc / variants versus WT TPO. No significant differences in the FRET efficiencies were observed among the TPOmoc / variants (Figure 4D), confirming that the MPL dimer geometries were not affected by the TPO mutations, and the signaling effects are due second MPL chain efficiency. Consistently, more frequent association and dissociation of MPL dimers was observed for -|-pQmodas comparec| t0WT TPO.

[0482] As the high-affinity site 1 interaction of TPO with MPL is merely altered, the changes in signaling and cell growth are not a result of an overall reduction in TPO binding to target cells. Likewise, the geometry of MPL dimers is not affected by TPOmoc / as confirmed by the unaltered FRET efficiencies. Thus, differences in the maximum number and / or lifetime of the MPL dimers formed by TPOmoc / compared to WT TPO likely confers selective signaling in TPO responsive cells.

[0483] Example 6: Stimulation of platelet production while preserving HSCs in mice by TPOmo< / variants K35A, K35Q and R119E

[0484] As TPO is essential for both HSC maintenance and megakaryocyte differentiation1-5, the inventors next determined whether TPOmoc / variants K35A, K35Q and R119E altered hematopoiesis in vivo. TPO and MPL sequences, including K35TPOand R119TPO, are highly conserved in human and mouse, allowing the inventors to generate equivalent TPOmoc / mutations in murine TPO variants (mTPOmoc / ) K35A, K35Q and R119E, and to purify the recombinant proteins (Figure 17A). To characterize the biological effect and function of mTPOmoc / in vivo, mice were treated with PBS or 5 ig recombinant mTPO (WT or mTPOmoc / ) daily for five days, taking complete blood counts at days 5, 7, 10 and 14 (Figure 5A). For bone marrow (BM) analysis, mice were euthanized at day 7 (Figure 5A), as this was the time point at which maximal hematological impact was observed.

[0485] WT mTPO induced a significant expansion of EPCR+long-term (LT)-HSCs and increased numbers of BM CD41+megakaryocyte progenitors (MKPs) compared to PBS at day 7 (Figures 5B and 5C). In comparison, while tested mTPOmoc / variants did increase the number of EPCR+LT-HSCs compared with PBS, the expansion was reduced compared to WT mTPO (Figure 5B) and the inventors observed no significant change in the number of MKPs with mTPOmoc / variants compared to PBS (Figure 5C). To determine whether the differences in MKPs was also observed in mature MKs, day 7 BM was sectioned and the number and size of CD41+MKs quantified (Figures 5D and 19). Only WT mTPO treatment significantly increased the number of BM MKs compared to PBS, while the overall size of BM MKs was not altered by mTPO treatment. WT mTPO and mTPOmoc / variants K35A, K35Q and R119E increased circulating platelet counts to a similar extent compared to PBS, peaking at day 7, and returning to normal levels by day 15 (Figure 5E). All other circulating blood lineages were unchanged (Figure 20). Taken together, these data demonstrate that K35A, K35Q and R119E modifications of TPO uncouple platelet production in vivo from HSC and progenitor expansion (Figure 5F).

[0486] The clinical use of TPO receptor agonists (TPO-RAs) for chronic immune thrombocytopenia18 19and severe aplastic anemia is now well-established. However, the currently available TPO-RAs can act as a double-edged sword, partially due to their impact on various levels of the hematopoietic hierarchy. One example is the development of morphologically-abnormal MKs in response to TPO-RA treatment, including MKs with hyperlobulated nuclei and MK clustering, both of which are hallmarks of MK development in essential thrombocythemia49. Therefore, the ability to fine-tune TPO signaling to specifically increase platelet count in ITP patients without impacting progenitor expansion or normal MK differentiation would be clinically attractive. In vivo mouse platelet response to the treatment of mTPOmoc / variants K35A, K35Q and R119E, was similar to WT mTPO, however, HSC, MkP and MK expansion in the bone marrow was lower for mTPOmoc / than WT mTPO. These results indicated that -|-pQmodvariantscould increase platelet production by MKs while limiting excessive stem and progenitor cell and MK expansion, thus providing a route to precision targeting of MPL. Example 7: Preservation of human hematopoietic stem cells (HSCs) during in vitro expansion by TPOmo< / variants K35A, K35Q and R119E

[0487] State-of-the-art human ex vivo hematopoietic stem cells (HSC) expansion for transplantation currently relies on supplementation with TPO42or TPO mimetics43. Without the addition of supplementary molecules, human HSCs reach near-exhaustion over seven days of culture due to rapid stimulation and expansion44. Encouraged by the effect of TPOmoc / on murine HSCs in vivo, the inventors tested the ability of TPOmoc / variants K35A, K35Q and R119E to maintain human HSCs ex vivo (Figure 6A). In the inventors’ culture conditions, WT TPO led to a greater expansion and differentiation of all cells, of which only a small proportion was EPCR+HSCs, suggesting HSC exhaustion (Figure 6B). However, substituting WT TPO with the TPOmoc / variants under investigation significantly diminished total cell count by day seven while the proportion of EPCR+HSCs was significantly increased, particularly with TPO-K35A (Figure 6B). This suggested improved preservation of the HSCs in vitro at the expense of cell proliferation, implying a uncoupling of proliferation / differentiation from stem cell maintenance by the tested TPOmoc / variants.

[0488] To assess the functional impact of TPOmoc / variants, colony formation assays were performed following seven days of CD34+cell expansion. After 14 days of differentiation, total colony count, representing the proportion of colony forming units in the initiating population, was significantly higher in cultures from cells expanded with TPO-K35A (Figure 6C). However, the frequency of colony types was not different between the groups suggesting that expansion with TPOmoc / variants did not skew cell differentiation potential.

[0489] The inventors further studied the transcriptome of primitive cells (CD34+CD45RA-) treated with TPOmoc / variants K35A, K35Q and R119E at single cell resolution (Figure 21). The inventors identified similar profiles of differentiation trajectory (Figures 21 B and 21C) and cellular subtypes (Figures 21 D and 22A), which led to few differentially expressed genes in pseudobulk total cells between TPOmoc / and WT controls (Figures 22A-22C). Focusing on HSCs characterized by HLF expression45(Figures 6D and 22A), the inventors noted a strong overlap between the transcriptomes of cells treated with WT and TPOmoc / variants in differentially expressed genes in / 7Z_F-positive versus / 7Z_F-negative HSCs (Figures 6E and 22C). The / 7Z_F-positive HSC populations demonstrated few differentially expressed genes in WT versus TPOmoc / variants, showing minimal overlap between TPO-K35Q and TPO-R119E (Figure 6F), and a large proportion were downregulated ribosomal genes in line with lower activation of HSCs. These data suggest that, while WT TPO drives greater total expansion of HSCs and progenitor cells, TPOmoc / maintains primitive HSCs without leading to excessive proliferation and differentiation of progenitor cells (Figure 6G).

[0490] TPO is also critical for both murine and human HSC maintenance and expansion ex vivo50, and there is increasing evidence that TPO dose may play a major role in stem cell production in vitro25. Over seven days of in vitro culture in the presence of WT TPO, HSCs exhaust to near complete depletion without additional cytokine treatment25. Similar to the inventors’ observations in mice, the overall level of proliferation in human HSPC cultures was significantly lower with TPO-K35A / Q and TPO-R119E treatment and, in turn, preserved a higher frequency of EPCR+HSCs. Examination of the transcriptome of TPOmoc,-expanded HSCs demonstrated high levels of similarity to WT TPO-expanded HSCs suggesting these cells are not compromised and potential for the application of TPOmoc / in tuning functional outcomes in regenerative medicine. The ability to tune TPO signaling activity by altering how the cytokine interacts with its receptor raises the exciting possibility of improving standard in vitro HSC expansion protocols for allogeneic transplantation. Indeed, the need to stimulate HSCs for gene therapy can itself reduce the stem cell pool pre-transplantation and there are major efforts underway to preserve more HSCs during gene therapy protocols51. Bone marrow transplantation was the first regenerative medicine approach to enter the clinic and protocols for expansion of HSCs (the long-term source of reconstitution) have largely remained unchanged52. Newer protocols aimed at expanding HSCs from umbilical cord blood have shown promise5354, but still rely on WT TPO supplementation and are thus balancing proliferation with loss of long-term reconstitution potential in basal conditions. The ability to direct TPO-MPL signaling to support new expansion protocols offers much promise for the future of HSC transplantation.

[0491] Example 9: Methods used in Examples 1-8

[0492] Data and code availability

[0493] The cryo-EM map has been deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-29644 and the model coordinate has been deposited in the Protein Data Bank (PDB) under accession number 8G04. The scRNAseq 10x genomics data is available under PRJEB61988 via EBI RNA repository.

[0494] Mammalian cell lines and culture conditions

[0495] For recombinant production of the TPO-2xMPL complex, suspension HEK293S GnTI- cells (ATCC CRL-3022) were grown in Freestyle 293 Expression Medium (Thermo Fisher) supplemented with 1% (v / v) FBS (Sigma-Aldrich) and maintained at 37°C with 5% C02 and gentle agitation.

[0496] Insect cell lines and culture conditions

[0497] For BacMam expression, baculovirus was produced in Spodoptera frugiperda (Sf9) ovarian cells (ATCC CRL-1711) maintained in Sf-900 III medium (Thermo Fisher) with 10% (v / v) FBS (Sigma-Aldrich) and GlutaMAX (Thermo Fisher). Insect cells were grown at 27°C with ambient CO2 and gentle agitation.

[0498] Preparation of the ligand-receptor complex for structural study

[0499] The coding sequences of MPL (UniProt ID: P40238, residues 1-635) with C-terminal Protein C and octahistidine tags, and N-terminal FLAG-tagged TPO (UniProt ID: P40225, residues 22-184: TPO163) were each cloned into the BacMam vector pVLAD55. We used endogenous signal peptide for MPL and influenza hemagglutinin (HA) signal peptide for TPO (Figure 7A). P0 viruses were prepared in Sf9 by co-transfecting the expression vector and BestBac Linearized Baculovirus DNA (Expression Systems, CA, USA) using a standard method, and the virus was amplified from P0 to P2. The TPO-MPL complex was produced by co-expression in HEK293S GnTL cells using BacMam baculovirus transduction. 3% (v / v) and 7% (v / v) of P2 viruses encoding MPL and TPO, respectively, were added to the cells at a density of 2-3 x 106cells / mL, and the culture flasks were shaken at 37°C for 24 hrs under 5% CO2. After centrifugation, cells were washed with phosphate-buffered saline (PBS, pH 7.2, Gibco) supplemented with 5 mM EDTA-Na (pH 8.0) and 1 :1000 protease inhibitor cocktail (PIC, Sigma Aldrich, MO, USA), weighed and stored at -20°C. The cell pellet was thawed and lysed with a Dounce homogenizer in a lysis buffer composed of 20 mM Tris-HCI pH 8.0, 5 mM EDTA-Na (pH 8.0), and 1 : 1000 PIC. The lysate was centrifuged at 48,000 g for 30 mins, and the membrane pellet was resuspended and nutated for 2 hrs in a solubilization buffer consisting of HBS (10 mM HEPES-Na pH 7.2, 150 mM NaCI), 1 % (w / v) Lauryl Maltose Neopentyl Glycol (LMNG, Anatrace; OH, USA), 10% (v / v) glycerol, and complete PIC (Roche, Basel, Switzerland). After centrifugation at 48,000 g for 1 hr, 8 mL High Affinity Ni-Charged Resin (GenScript, NJ, USA) was added to the supernatant, and the mixture was incubated at 4 °C for 2 hrs on a rotator. The resin was then collected in a column, washed with HBS containing 0.1% (w / v) LMNG, 0.01 mM TCEP (pH 7.0), and 20 mM imidazole (pH 8.0). The bound protein was eluted in HBS containing 0.1% (w / v) LMNG, 0.01 mM TCEP (pH 7.0), and 250 mM imidazole (pH 8.0) and further purified over an in-house anti-FLAG M1 affinity Sepharose column as follows. The eluate was supplemented with 2 mM CaCh before loading over the anti-FLAG M1 affinity column. The column was then washed with wash buffer 1 (HBS with 0.1% (w / v) LMNG, 0.01 mM TCEP (pH 7.0), and 2 mM CaCI2), wash buffer 2 (HBS with 90 mM HEPES-Na pH 8.3, 150 mM KCI, 5 mM ATP, 0.1% (w / v) LMNG, 0.01 mM TCEP (pH 7.0), and 2 mM CaCI2), wash buffer 1 , and finally wash buffer 3 (HBS with 0.01 % (w / v) LMNG, 0.01 mM TCEP (pH 7.0), and 2 mM CaCI2). The complex was eluted in HBS supplemented with 0.001% (w / v) LMNG, 0.01 mM TCEP (pH 7.0), 0.2 mg / mL FLAG peptide and 5 mM EDTA- Na (pH 8.0), and further purified by size-exclusion chromatography (SEC) using a Superose 6 10 / 300 column (Cytiva) equilibrated with HBS containing 0.001% (w / v) LMNG and 0.01 mM TCEP (pH 7.0). The SEC-purified sample was concentrated to ~0.2 mg / ml and gently crosslinked with 1 mM bis(sulfosuccinimidyl)suberate (BS3) (Thermo Fisher Scientific, MA, USA) overnight at 4°C, quenched by adding 20 mM Tris-HCI pH 8 and incubation for 30 mins, and re-purified by passing over the Superose 6 10 / 300 equilibrated in HBS supplemented with 0.001 % (w / v) LMNG. The peak fraction containing the crosslinked TPO-2xMPL complex was kept for negative-stain EM analysis, and the fractions were concentrated to -3 mg / mL for cryo- EM (Figure 7A).

[0500] EM sample preparation and data acquisition

[0501] The crosslinked TPO-2xMPL complex was first imaged with negative-stain EM to confirm the sample quality. The peak SEC fraction was diluted 2-fold and applied onto the glow- discharged carbon grid (Ted Pella, Carbon Type-B Support Films, 200 copper mesh). The excess solution was blotted to a filter paper, and the specimen was stained with 1% (w / v) uranyl acetate (Electron Microscopy Sciences). The grid was loaded into a Tecnai F20 operated at 200 kV and equipped with a Gatan K2 camera, and images were acquired with Gatan DigitalMicrograph software. For cryo-EM, 0.1 volumes of 0.5% (w / v) digitonin in HBS was added to the concentrated TPO-2xMPL complex in solution, and 3 pL sample was applied onto glow-discharged 200 mesh gold grids (Quantifoil R1.2 / 1.3). Excess sample was blotted to a filter paper for 3 sec before plunge-freezing using a Leica EM GP (Leica Microsystems) at 8°C and 90% humidity. The cryo-EM movies were collected on a Titan Krios at the CryoEM facility, the HHMI Janelia Research Campus. The microscope was equipped with a Gatan K3 camera and BioQuantum imaging filter and operated at 300 kV in correlated double sampling mode at a nominal magnification of 81 ,000x, corresponding to the super-resolution pixel size of 0.5039 A and a calibrated magnification of 46,382x. Each movie was recorded for a total of 6.125 sec with 0.1225 sec exposure per frame at an exposure rate of 9.5 electrons / pixel / second at the sample, that yielded an electron count of -7.5 electrons / pixel / second at the camera. The datasets were collected using SerialEM software with the defocus range between -0.8 and -2.0 pm and beam-image shift set to collect 9 movies from 9 holes per stage shift with autofocusing. Cryo-EM data processing

[0502] A total of 10,494 movies were subjected to beam-induced motion correction using MotionCor256with binning to a pixel size of 1.078 A. Dose-weighted motion-corrected micrographs were imported into cryoSPARC57, and the patch contrast transfer function (CTF) values were estimated with a default setting. The template-based particle picking was performed using the 2D templates generated from the preliminary 3D map obtained from ab-initio 3D reconstruction followed by non-uniform refinement of selected particles during the screening experiments (Figure 7C). 9,537,683 “particles” were extracted with a box size of 300 pixels and binning to 128 pixels, and two rounds of 2D classifications were performed to remove buffer features and contaminants yielding -3,358,637 protein particles. Heterogeneous refinements were performed with custom 3D templates to identify a particle set for a more complete extracellular signaling complex (Figure 8A). After further 2D classification, 901 ,465 selected particles were extracted with a box size of 300 pixels without binning, and ...

Claims

CLAIMS1. An isolated and modified thrombopoietin (TPO) polypeptide having an amino acid sequence according to SEQ ID NO: 1 or at least 70% identity thereto, or a functional fragment thereof, and comprising at least one amino acid modification of the amino acids in the site 2 region thereof, wherein the site 2 region consists of amino acids within the region of positions 21 to 184 of SEQ ID NO: 1 , or a corresponding region thereto, which bind to the TPO receptor with low affinity.

2. The isolated and modified thrombopoietin polypeptide of claim 1 , wherein the amino acids in the site 2 region thereof bind to the TPO receptor with an affinity in the micromolar range3. The isolated and modified thrombopoietin polypeptide of claim 1 or 2, comprising at least one amino acid modification at a position in the site 2 region thereof selected from: P26, D29, R31 , V32, K35, R38, R99, R119, L120, G123, and A124 of SEQ ID NO:1 , or at a position corresponding thereto.

4. The isolated and modified thrombopoietin polypeptide of claim 1 or 2, comprising at least one amino acid modification at a position in the site 2 region thereof selected from: R31 , K35, R38, R99, and R119 of SEQ ID NO:1 , preferably K35 and R119 of SEQ ID NO: 1 , or at a position corresponding thereto.

5. The isolated and modified thrombopoietin polypeptide of any one of claims 1 to 4, wherein the at least one amino acid modification at position R31 of SEQ ID NO:1 , or at a position corresponding thereto, is selected from: R31A, R31 E, R31 H, and R31Q,: wherein the at least one amino acid modification at position K35, or at a position corresponding thereto, is selected from: K35A, K35E, K35H, K35M, and K35Q; wherein the at least one amino acid modification at position R38, or at a position corresponding thereto, is selected from: R38A, R38E, R38H, and R38Q; wherein the at least one amino acid modification at position R99, or at a position corresponding thereto, is selected from: R99A, R99E, R99H, R99Q, and R99W; and / or wherein the at least one amino acid modification at position R119, or at a position corresponding thereto, is selected from: R119A, R119C, R119E, R119H, and R119Q.

6. The isolated and modified thrombopoietin polypeptide of any preceding claim, wherein the at least one amino acid modification in the site 2 region thereof is selected from K35A, K35Q, R119E, and R119H of SEQ ID NO:1 , or a corresponding modification thereto.

7. The isolated and modified thrombopoietin polypeptide, or fragment thereof, of any of preceding claim, comprising or consisting of the amino acid sequence shown in any of SEQ ID NOs: 2 to 24.

8. An isolated nucleic acid molecule encoding the modified thrombopoietin polypeptide of any preceding claim.

9. An expression construct comprising the isolated nucleic acid molecule of claim 8, operably linked to a promoter.

10. A vector comprising the nucleic acid molecule of claim 8, or the expression construct of claim 9.

11. A host cell transformed with the nucleic molecule of claim 8, the expression construct of claim 9, the vector of claim 10.

12. A method of manufacturing the modified thrombopoietin polypeptide according to any one of claims 1 to 7, the method comprising:(a) Providing the host cell of claim 11;(b) Culturing the host cell under suitable conditions for the cell to produce the modified thrombopoietin polypeptide;(c) Isolating the modified thrombopoietin polypeptide from the host cell or from the cell culture thereof.

13. A method of expanding and / or maintaining stem cells, the method comprising:(a) Providing one or more stem cells;(b) Culturing the one or more stem cells in the presence of a modified thrombopoietin polypeptide of any one of claims 1 to 7, under suitable conditions for the one or more stem cells to proliferate.

14. The method of claim 13, wherein the stem cells are hematopoietic stem cells.

15. The method of claim 13 or 14, wherein the modified thrombopoietin polypeptide comprises at least one amino acid modification selected from: K35A, K35Q, R119E, and R119H, preferably R199H, of SEQ ID NO:1, or a corresponding modification thereto.

16. The method of any one of claims 13 to 15, wherein the method does not substantially induce differentiation of the stem cells.

17. A Stem cell or a population of stem cells produced by the method of any one of claims 13 to 16 or a therapeutic composition thereof.

18. The stem cell, the population of stem cells, or the therapeutic composition thereof of claim 17, for use in the prevention or treatment of a disease.

19. The vector of claim 10, the host cell of claim 11, or the stem cell, the population of stem cells or the therapeutic composition thereof of claim 17, for use in a method of gene therapy or cell therapy, preferably for the treatment of an immunological disorder, a haematological disorder or cancer, more preferably for the treatment of aplastic anemia, bone marrow failure syndromes, Multiple Sclerosis, leukaemia, lymphoma, or myeloma, optionally wherein the cell therapy is autologous or allogeneic stem cell transplantation.

20. A method of gene therapy or cell therapy for preventing or treating a disease in a subject, the method comprising administering an effective amount of vector of claim 10, the host cell of claim 11 , or the stem cell the population of stem cells, or the therapeutic composition thereof of claim 17 to the subject in need thereof, preferably for the treatment of an immunological disorder, a haematological disorder or cancer, more preferably for the treatment of aplastic anemia, bone marrow failure syndromes, MultipleSclerosis, leukaemia, lymphoma, or myeloma optionally wherein the cell therapy is autologous or allogeneic stem cell transplantation.

21. A method of ex vivo platelet production, the method comprising:(a) Providing one or more stem cells;(b) Culturing the one or more stem cells in the presence of a modified thrombopoietin polypeptide of any one of claims 1 to 7 under suitable conditions for the one or more stem cells to differentiate into platelets; and(c) Optionally isolating the platelets.

22. The method of claim 21 wherein the modified thrombopoietin polypeptide comprises at least one amino acid modification selected from: K35A, K35Q, R119E and R119H of SEQ ID NO:1, or a corresponding modification thereto.

23. The method of claim 21 or 22 wherein step (b) comprise (i) culturing the stem cells to differentiate into megakaryocytes and (ii) culturing the megakaryocytes to differentiate into platelets.

24. Platelets produced by the method of any one of claims 21 to 23, or a therapeutic composition thereof.

25. The platelets or the therapeutic composition thereof of claim 24, for use in the prevention or treatment of a disease.

26. The platelets or the therapeutic composition thereof of claim 24,, for use in the prevention or treatment of an immunological disorder, a haematological disorder or cancer, preferably wherein the immunological disorder, haematological disorder or cancer comprise thrombocytopenia and / or anemia.

27. The platelets, or the therapeutic composition thereof for use according to claim 25 or 26, wherein the platelets are for use in preventing or treating thrombocytopenia or anemia in an immunological disorder, haematological disorder or cancer.

28. The platelets, or the therapeutic composition thereof for use according to claim 26 or 27, wherein: the thrombocytopenia comprises immune thrombocytopenia, chronic thrombocytopenia, or chronic immune thrombocytopenia; or the anemia comprises aplastic anemia, severe aplastic anemia, hypoplastic anemia, or severe hypoplastic anemia.

29. A method of treating or preventing a disease in a subject, the method comprising: administering an effective amount of the platelets of claim 24 to the subject in need thereof.

30. A method of treating or preventing an immunological disorder, haematological disorder or cancer in a subject, the method comprising administering an effective amount of the platelets of claim 24 to the subject in need thereof, preferably wherein the immunological disorder, haematological disorder or cancer comprise thrombocytopenia and / or anemia.

31. The method of claim 29 or 30, wherein the platelets treat or prevent thrombocytopenia or anemia in an immunological disorder, haematological disorder or cancer.

32. The method according to claim 30 or 31 , wherein: the thrombocytopenia comprises immune thrombocytopenia, chronic thrombocytopenia, or chronic immune thrombocytopenia; or the anemia comprises aplastic anemia, severe aplastic anemia, hypoplastic anemia, or severe hypoplastic anemia.

33. A peptibody comprising one or more fragments of the isolated and modified thrombopoietin polypeptide according to any of claims 1-7.

34. A peptibody or pharmaceutical composition thereof according to claim 33, for use in the prevention or treatment of a disease.

35. A peptibody or pharmaceutical composition thereof according to claim 33, for use in the prevention or treatment of an immunological disorder, haematological disorder, or cancer preferably wherein the immunological disorder, haematological disorder or cancer comprise thrombocytopenia and / or anemia.

36. The peptibody or pharmaceutical composition thereof for use according to claim 34 or35, in the prevention or treatment of thrombocytopenia or severe aplastic anemia in an immunological disorder, haematological disorder or cancer.

37. The peptibody or pharmaceutical composition thereof for use according to claim 35 or36, wherein the thrombocytopenia is immune thrombocytopenia, chronic thrombocytopenia, or chronic immune thrombocytopenia; or the anemia is aplastic anemia, severe aplastic anemia, hypoplastic anemia, or severe hypoplastic anemia.

38. A method of treating or preventing a disease in a subject, the method comprising: administering an effective amount of the peptibody or pharmaceutical composition thereof of claim 33 to the subject in need thereof.

39. A method of treating or preventing an immunological disorder, haematological disorder or cancer in a subject, the method comprising administering an effective amount of the peptibody or pharmaceutical composition thereof of claim 33, to the subject in need thereof, preferably wherein the immunological disorder, haematological disorder or cancer comprise thrombocytopenia and / or anemia.

40. The method of claim 38 or 39, wherein the peptibody or pharmaceutical composition thereof prevents or treats thrombocytopenia or severe aplastic anemia in an immunological disorder, haematological disorder or cancer.41 . The method of claim 39 or 40, wherein the thrombocytopenia comprises immune thrombocytopenia, chronic thrombocytopenia, or chronic immune thrombocytopenia; or the anemia comprises aplastic anemia, severe aplastic anemia, hypoplastic anemia, or severe hypoplastic anemia.

42. A pharmaceutical composition comprising the isolated and modified thrombopoietin protein of any one of claims 1 to 7, or the peptibody of claim 33.

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