WO2018020269A1

WO2018020269A1 - Stem cells and cancer

Info

Publication number: WO2018020269A1
Application number: PCT/GB2017/052217
Authority: WO
Inventors: Peter Holland; Thomas DUNWELL; Paul Fairchild; Ignacio MAESO
Original assignee: Oxford University Innovation Limited
Priority date: 2016-07-28
Filing date: 2017-07-28
Publication date: 2018-02-01
Also published as: EP3491124A1; GB201613078D0

Abstract

Provided are methods of producing induced pluripotent stem cells, as well as induced pluripotent stem cell comprising an exogenous nucleic acid, or analogue thereof, encoding an ETCHbox protein, or fragment or variant thereof. The methods involve providing a somatic cell with an ETCHbox protein, or fragment or variant thereof, and further providing the cell with a reprogramming factor. The ETCHbox protein may be selected from the group consisting of: ARGFX; TPRX; LEUTX; PARGFX; and DPRX. Also provided are methods of detecting a teratocarcinoma, or its metastases in a subject, and methods of treating teratocarcinoma, or its metastases, in a subject. These methods make use of the ETCHbox protein ARGFX, or the proteins NANOGNB, or HESX1.

Description

STEM CELLS AND CANCER

FIELD OF THE INVENTION

The present invention relates to methods of producing induced pluripotent stem cells. The invention also relates to the use of exogenous nucleic acids, or analogues thereof, in methods of producing induced pluripotent stem cells. The invention relates to an expression vector, comprising a nucleic acid, or analogue thereof, encoding an ETCHbox protein, fragment, or variant thereof, and a reprogramming factor. The invention further relates to an induced pluripotent stem cell comprising an exogenous nucleic acid, or analogue thereof, encoding an ETCHbox protein, or fragment or variant thereof. The invention also relates to a pharmaceutical composition comprising an induced pluripotent stem cell, and to a kit comprising first and second exogenous nucleic acids, or analogues thereof. Finally, the invention relates to a method of detecting a teratocarcinoma, or its metastases in a subject, and to a method of treating teratocarcinoma, or its metastases, in a subject.

INTRODUCTION

Stem cells are cells with the capacity to develop into cell types of many different lineages. Stem cells can be defined with reference to their source or location, such as mesenchymal stem cells or bone marrow stem cells, or the range of cell types that they are able to give rise to, such as totipotent or pluripotent stem cells.

Pluripotent stem cells are capable of giving rise to any of the three germ layers, endoderm, mesoderm, or ectoderm. While pluripotent stem cells may occur naturally, particularly during development, techniques have also be developed by which somatic cells can be induced to "de-differentiate" and thereby achieve pluripotency.

Such induced pluripotent stem cells (also called iPSCs) are produced using "re-programming factors", typically transcription factors, provided to somatic cells such as adult fibroblasts.

Homeobox genes encode proteins containing a DNA-binding homeodomain. Many different families of homeobox genes, and corresponding proteins, are known. The ETCHbox group of homeobox genes comprises five families, ARGFX, TPRX, DPRX, PARGFX and LEUTX, with PARGFX not present in humans. SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method of producing an induced pluripotent stem cell, the method comprising providing a somatic cell with an ETCHbox protein, or fragment or variant thereof, and providing the cell with a reprogramming factor.

In a second aspect, the invention provides the use of an exogenous nucleic acid, or analogue thereof, encoding an ETCHbox protein, or fragment or variant thereof in a method of producing an induced pluripotent stem cell.

In a third aspect, the invention provides an expression vector, comprising a nucleic acid, or analogue thereof, encoding an ETCHbox protein, or fragment, or variant thereof, and a reprogramming factor.

In a fourth aspect, the invention provides an induced pluripotent stem cell comprising an exogenous nucleic acid, or analogue thereof, encoding an ETCHbox protein, or fragment or variant thereof. An induced pluripotent stem cell, in accordance with this aspect of the invention, may optionally express a reprogramming factor.

In a fifth aspect, the invention provides a pharmaceutical composition comprising an induced pluripotent stem cell according to the fourth aspect.

In a sixth aspect, the invention provides a kit comprising first and second exogenous nucleic acids, or analogues thereof, wherein the first exogenous nucleic acid or analogue encodes an ETCHbox protein, fragment, or variant thereof, and the second exogenous nucleic acid or analogue encodes a reprogramming factor.

In a seventh aspect, the invention provides a method of detecting a teratocarcinoma or its metastases in a subject, the method comprising:

• assaying a sample from the subject to determine the presence of target molecules representative of expression of a protein selected from the group consisting of: ARGFX, NANOGNB and HESX1 ;

• wherein the presence of target molecules representative of expression of a protein selected from the group consisting of: ARGFX, NANOGNB and HESX1 is indicative of teratocarcinoma or its metastases. In an eighth aspect, the invention provides a method of treating teratocarcinoma, or its metastases, in a subject, the method comprising:

• assaying a sample from the subject to determine the presence of target molecules representative of expression of a protein selected from the group consisting of: ARGFX, NANOGNB and HESX1 ; and

• in the event that target molecules representative of expression of a protein selected from the group consisting of: ARGFX, NANOGNB and HESX1 are present, providing the subject with treatment for teratocarcinoma or its metastases.

A method in accordance with this eighth aspect of the invention may, optionally, comprise a further step of assaying a sample from the subject after treatment to determine the presence of target molecules representative of expression of a protein selected from the group consisting of ARGFX, NANOGNB and HESX1. This provides a means to monitor efficacy of the treatment. In the event that such target molecules are present in the sample from the subject after treatment, this indicates that further treatment for the teratocarcinoma or its metastases is necessary. In the event that no such target molecules are present in the sample after treatment, this indicates that the treatment for the teratocarcinoma or its metastases has been effective. Further treatment may be therefore unnecessary, and so treatment may be stopped.

In a ninth aspect, the invention provides an agent that targets ARGFX, and/or NANOGNB, and/or HESX1 for use in the treatment of a teratocarcinoma or its metastases.

In a tenth aspect, the invention provides a method of treating teratocarcinoma or its metastases in a subject, the method comprising the step of providing an agent that targets ARGFX, and/or NANOGNB, and/or HESX1 to a subject in need thereof.

It will be appreciated that the method of treating teratocarcinoma or its metastases according to the eighth aspect of the invention, may involve the use of an agent that targets ARGFX, and/or NANOGNB, and/or HESX1 , according to the ninth aspect of the invention.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows pictures of methylene blue stained cell colonies. A - Fibroblast cells transfected with Yamanaka reprogramming factors (Oct3, c-Myc, Klf4, Sox2), originally plated at a density of 2 x 10³ cells/well in a 96 well plate. B - Fibroblast cells transfected with Yamanaka reprogramming factors, originally plated at a density of 5 x 10³ cells/well in a 96 well plate. C - Fibroblast cells transfected with ARGFX and Yamanaka reprogramming factors, originally plated at a density of 2 x 10³ cells/well in a 96 well plate. D - Fibroblast cells transfected with ARGFX and Yamanaka reprogramming factors, originally plated at a density of 5 x 10³ cells/well in a 96 well plate. E and F - Fibroblast cells transfected with ARGFX but not transfected with Yamanaka factors.

Figure 2 shows correlation between expression of ARGFX RNA (top row) and histological diagnosis of teratocarcinoma (black vertical bars) in testicular cancer samples clustered according to global gene expression (bottom cluster plot).

Figure 3 shows a heat map illustrating gene expression in embryonal, yolk, seminoma and teratoma tissues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the inventors' surprising finding that traditional methods of producing induced pluripotent stem cells can be significantly enhanced by supplementing somatic cells with an ETCHbox protein, such as the exemplary ETCHbox protein ARGFX.

Current methods of making pluripotent stem cells using "traditional" reprogramming factors (such as the "Yamanaka" reprogramming factors) have a low efficiency rate when expression vectors are used for delivery of factors, and this drops to extremely low efficiency when RNA delivery is used (a delivery route which is preferred in the context of the production of induced pluripotent stem cells for therapeutic applications).

Provision of an ETCHbox protein, such as ARGFX, in addition to known reprogramming factors is able to significantly increase the rate of efficiency of induced pluripotent stem cell production. By increasing efficiency in this manner the invention not only enables the generation of higher yields of induced pluripotent stem cells, but also a reduction in the costs associated with induced pluripotent stem cell production.

The finding that ETCHbox proteins can be used in this manner also opens the possibility of using fragments of such proteins, or variants of the proteins or fragments, in the same manner. As discussed further elsewhere in this specification, the ETCHbox protein (such as ARGFX) and/or the reprogramming factor (or factors) can be provided "directly" to the cell as proteins, or can be provided "indirectly" to the cell by means of agents (such as exogenous nucleic acids or analogues thereof) that may be expressed by the cell to yield the ETCHbox protein or reprogramming factor. These various possibilities give rise to a number of the other aspects of the invention, such as the uses of the second aspect, the expression vectors of the third aspect, the induced pluripotent stem cells of the fourth aspect, and the kits of the sixth aspect of the invention. ARGFX represents a particularly suitable ETCHbox protein to be used in these aspects and embodiments of the invention.

The inventors believe that certain embodiments of the invention may address the long-felt need for improved methods of making induced pluripotent stem cells. The inventors also believe that in some embodiments of the invention, an ETCHbox protein (such as ARGFX), or fragment or variant thereof, may be used as a substitute for a reprogramming factor in a method of producing a pluripotent stem cell. Such an embodiment may be particularly beneficial when the substituted reprogramming factor is an oncogene, for example c-Myc, as it could potentially increase the safety of an induced pluripotent stem cell, or of a tissue obtained from an induced pluripotent stem cell. Furthermore, certain embodiments of the invention may significantly speed up the development of novel induced pluripotent stem cell based therapies. The fifth aspect of the invention reflects the pharmaceutical compositions that can be produced in this manner.

Finally, the invention provides novel means to diagnose, treat and monitor teratocarcinoma and its metastases. These are based upon the inventors' finding that ARGFX, which is normally only expressed very briefly in a tightly controlled window during development, is also expressed by teratocarcinomas. Thus the presence of ARGFX, or target molecules indicative of ARGFX expression, in a sample taken from a subject may be used as an indication that the subject has a teratocarcinoma or metastases from such a cancer. If target molecules persist after a primary teratocarcinoma has been removed, this indicates that the original cancer has metastasised, and that further treatment is required.

The following definitions will be helpful in further understanding various aspects and embodiments of the invention.

ETCHbox proteins, and fragments, or variants thereof

The ETCHbox (Eutherian Totipotent Cell Homeobox) genes comprise the phylogenetically- related genes ARGFX, TPRX1, TPRX2, PARGFX, LEUTX, and DPRX. These genes encode corresponding protein families. For the purposes of the present invention, an ETCHbox protein may be a member of the ARGFX family of proteins, a member of the TPRX family of proteins, a member of the LEUTX family of proteins, a member of the PARGFX family of proteins or a member of the DPRX family of proteins. Exemplary ETCHbox proteins may be referred to as ARGFX; TPRX; LEUTX; PARGFX; and DPRX. It will be appreciated that, except for where the context requires otherwise, references to "an ETCHbox protein" in the present specification may be taken as directed to any protein selected from the group consisting of: ARGFX; TPRX; LEUTX; PARGFX; and DPRX.

ARGFX (arginine-fifty homeobox) is any protein encoded by the gene ARGFX. The protein is a DNA-binding protein involved in early embryonic development. One isoform of ARGFX is a 36kDa protein, defined by the amino acid sequence of SEQ ID NO: 1 , with a 315 amino acid sequence length. Another isoform of ARGFX is a 37.5kDa protein, defined by the amino acid sequence of SEQ ID NO: 2, with a 333 amino acid sequence length.

The TPRX (tetra-peptide repeat homeobox) gene family comprises various genes. Humans have two genes belonging to this family. These genes are TPRX1 and TPRX2, encoding the proteins known as TPRX1 and TPRX2. Since TPRX1 and TPRX2 genes encode proteins with identical amino acid sequences over part of their length, but of these TPRX1 has higher expression level in normal human embryos, it may generally be preferred to employ TPRX1 as an ETCHbox protein in the various aspects or embodiments of the invention. TPRX1 is a 47kDa protein with a length of 466 amino acids, the sequence of which is set out in SEQ ID NO: 3. TPRX2 is a 33kDa protein with a length of 301 amino acids, the sequence of which is set out in SEQ ID NO: 4.

The DPRX (divergent-paired related homeobox) encodes a 22kDa protein, DPRX, with a length of 191 amino acids. The sequence of the DPRX protein is set out in SEQ ID NO: 5.

The 19kDa LEUTX (leucine twenty homeobox) protein encoded by LEUTX has a sequence length of 198 amino acids. The amino acid sequence of the LEUTX protein is set out in SEQ ID NO: 6.

Any of the ETCHbox proteins referred to above may be utilised in the first to sixth aspects of the invention. However, ARGFX represents a particularly suitable ETCHbox protein to be employed in the production of induced pluripotent stem cells. Accordingly, in a suitable embodiment of the first to sixth aspects of the invention, the ETCHbox protein comprises ARGFX, or a fragment or variant thereof. Suitably the ETCHbox protein may consist of ARGFX, or a fragment or variant thereof. The efficacy of methods of the invention utilising ARGFX as an exemplary ETCHbox protein is illustrated in the Examples later in the specification.

Accordingly, in a suitable embodiment, a method of the invention may employ, or a nucleic acid or analogue as referred to herein may encode, a polypeptide of SEQ ID NO: 1 , or fragment or variant thereof, or a polypeptide of SEQ ID NO: 2, or fragment or variant thereof. Except for where the context requires otherwise, considerations set out here with respect to ARGFX, its fragments and variants (whether of the full length protein or fragments thereof), should be taken as applicable to all embodiments of the first to sixth aspects of the invention.

A fragment of the ARGFX protein (SEQ ID NO: 1) is a polypeptide which consists of a truncation in the amino acid sequence of SEQ ID NO: 1. Such a fragment is less than 315 amino acids in length, but shares 100% sequence identity with the portion of SEQ ID NO: 1 which it corresponds to. A fragment comprising the homeodomain region set out SEQ ID NO: 13 may be especially suitable for use in accordance with such embodiments of the invention.

A fragment of the ARGFX protein (SEQ ID NO: 2) is a polypeptide which consists of a truncation in the amino acid sequence of SEQ ID NO: 2. Such a fragment is less than 333 amino acids in length, but shares 100% sequence identity with the portion of SEQ ID NO: 2 which it corresponds to. A fragment comprising the homeodomain region set out SEQ ID NO:

13 may be especially suitable for use in accordance with such embodiments of the invention.

In a suitable embodiment, a method of the invention may employ, or a nucleic acid or analogue as referred to herein may encode, a polypeptide of SEQ ID NO: 3, or fragment or variant thereof, or SEQ ID NO: 4, or fragment or variant thereof. Except for where the context requires otherwise, considerations set out here with respect to TPRX, its fragments and variants (whether of the full length protein or fragments thereof), should be taken as applicable to all embodiments of the first to sixth aspects of the invention.

A fragment of the TPRX1 protein (SEQ ID NO: 3) is a polypeptide which consists of a truncation in the amino acid sequence of SEQ ID NO:3. Such a fragment is less than 466 amino acids in length, but shares 100% sequence identity with the portion of SEQ ID NO: 3 which it corresponds to. A fragment comprising the homeodomain region set out SEQ ID NO:

14 may be especially suitable for use in an embodiment of the method. A fragment of the TPRX2 protein (SEQ ID NO: 4) is a polypeptide which consists of a truncation in the amino acid sequence of SEQ ID NO:4. Such a fragment is less than 301 amino acids in length, but shares 100% sequence identity with the portion of SEQ ID NO: 4 which it corresponds to. A fragment comprising the homeodomain region set out SEQ ID NO: 15 may be especially suitable for use in an embodiment of the method.

In a suitable embodiment, a method of the invention may employ, or a nucleic acid or analogue as referred to herein may encode, a polypeptide of SEQ ID NO: 5, or fragment or variant thereof. Except for where the context requires otherwise, considerations set out here with respect to DPRX, its fragments and variants (whether of the full length protein or fragments thereof), should be taken as applicable to all embodiments of the first to sixth aspects of the invention.

A fragment of the DPRX protein (SEQ ID NO: 5) is a polypeptide which consists of a truncation in the amino acid sequence of SEQ ID NO: 5. Such a fragment is less than 191 amino acids in length, but shares 100% sequence identity with the portion of SEQ ID NO: 5 which it corresponds to. A fragment comprising the homeodomain region set out SEQ ID NO: 16 may be especially suitable for use in an embodiment of the method.

In a suitable embodiment, a method of the invention may employ, or a nucleic acid or analogue as referred to herein may encode, a polypeptide of SEQ ID NO: 6, or fragment or variant thereof. Except for where the context requires otherwise, considerations set out here with respect to LEUTX, its fragments and variants (whether of the full length protein or fragments thereof), should be taken as applicable to all embodiments of the first to sixth aspects of the invention.

A fragment of the LEUTX protein (SEQ ID NO: 6) is a polypeptide which consists of a truncation in the amino acid sequence of SEQ ID NO: 6. Such a fragment is less than 198 amino acids in length, but shares 100% sequence identity with the portion of SEQ ID NO: 6 which it corresponds to. A fragment comprising the homeodomain region set out SEQ ID NO: 17 may be especially suitable for use in an embodiment of the method.

More than one ETCHbox protein may be used in the production of induced pluripotent stem cells. Accordingly, the first to sixth aspect of the invention may employ two or more ETCHbox proteins, and, optionally, may make use of three or more ETCHbox proteins, or four or more ETCHbox proteins. The ETCHbox proteins ARGFX, TPRX1 , and LEUTX are particularly suitable examples to be employed in the first to sixth aspects of the invention, and so suitably the ETCHbox protein is selected from the group consisting of: ARGFX, TPRX1 and LEUTX, fragments of these proteins, and variants of these proteins or fragments. More suitably the ETCHbox protein is ARGFX (SEQ ID NO: 1).

Suitably the first to sixth aspects of the invention may employ ARGFX in combination with one or more further ETCHbox proteins. Without wishing to be bound by any hypothesis, the inventors believe that the advantages noted when ARGFX is used arise from the ability of this protein to up-regulate expression of other genes, and that other ETCHbox proteins are then able to beneficially down-regulate this increased expression. Thus, suitably the first to sixth aspects of the invention may employ ARGFX, or fragments or variants thereof, in combination with TPRX1 and/or LEUTX, or their fragments or variants. Suitably the first to sixth aspects of the invention may employ ARGFX in combination with TPRX1 (or fragments or variants of either of these proteins).

A suitable fragment of TPRX1 protein consists of up to 465 contiguous amino acids of SEQ ID NO: 3, up to 460 contiguous amino acids of SEQ ID NO: 3, up to 450 contiguous amino acids of SEQ ID NO: 3, up to 440 contiguous amino acids of SEQ ID NO: 3, up to 430 contiguous amino acids of SEQ ID NO:3, up to 420 contiguous amino acids of SEQ ID NO: 3, up to 410 contiguous amino acids of SEQ ID NO: 3, up to 400 contiguous amino acids of SEQ ID NO: 3, up to 390 contiguous amino acids of SEQ ID NO: 3, up to 380 contiguous amino acid of SEQ ID NO: 3, up to 370 contiguous amino acids of SEQ ID NO: 3, up to 360 contiguous amino acids of SEQ ID NO: 3, up to 350 contiguous amino acids of SEQ ID NO: 3, or up to 340 contiguous amino acids of SEQ ID NO: 3.

A suitable fragment of ARGFX or TPRX1 protein consists of up to 330 contiguous amino acids, up to 320 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 3 respectively.

A suitable fragment of ARGFX or TPRX1 protein consists of up to 310 contiguous amino acids of SEQ ID NO: 1 (or SEQ ID NO: 2) or SEQ ID NO:3 respectively.

A suitable fragment of ARGFX, TPRX1 or TPRX2 protein consists of up to 300 contiguous amino acids, up to 290 contiguous amino acids, up to 280 contiguous amino acids, up to 270 contiguous amino acids, up to 260 contiguous amino acids, up to 250 contiguous amino acids, up to 240 contiguous amino acids, up to 230 contiguous amino acids, up to 220 contiguous amino acids, up to 210 contiguous amino acids, or up to 200 contiguous amino acids of SEQ ID NO: 1 (or SEQ ID NO: 2), SEQ ID NO: 3, or SEQ ID NO: 4 respectively.

A suitable fragment of ARGFX, TPRX1 , TPRX2, DPRX or LEUTX protein consists of up to 190 contiguous amino acids, up to 180 contiguous amino acids, up to 170 contiguous amino acids, up to 160 contiguous amino acids, up to 150 contiguous amino acids, up to 140 contiguous amino acids, up to 130 contiguous amino acids, up to 120 contiguous amino acids, up to 1 10 contiguous amino acids, up to 100 contiguous amino acids, up to 90 contiguous amino acids, up to 80 contiguous amino acids, up to 70 contiguous amino acids, up to 60 contiguous amino acids, or up to 50 contiguous amino acids of SEQ ID NO: 1 (or SEQ ID NO: 2), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 respectively.

A variant of an ETCHbox protein is a polypeptide which shares a partial sequence identity with the amino acid sequence of the native an ETCHbox protein, or a fragment of the an ETCHbox protein.

For example, a variant of the ARGFX protein is a polypeptide which shares a partial sequence identity with the amino acid sequence of ARGFX (SEQ ID NO: 1 or SEQ ID NO: 2) or a fragment thereof, whereas a variant of the TPRX1 protein is a polypeptide which shares a partial sequence identity with the amino acid sequence of TPRX1 (SEQ ID NO: 3) or a fragment thereof. A variant of the TPRX2 protein is a polypeptide which shares a partial sequence identity with the amino acid sequence of TPRX2 (SEQ ID NO: 4) or a fragment thereof. A variant of the DPRX protein is a polypeptide which shares a partial sequence identity with the amino acid sequence of DPRX (SEQ ID NO: 5) or a fragment thereof, whilst a variant of the LEUTX protein is a polypeptide which shares a partial sequence identity with the amino acid sequence of LEUTX (SEQ ID NO: 6) or a fragment thereof.

Suitably, a variant of an ETCHbox protein (such as ARGFX) may share at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 78% identity, at least 79% identity, or at least 80% identity with the sequence of the corresponding full length ETCHbox protein, or with a fragment thereof. It will be appreciated that variants may share relatively low identity across their total length, but still remain biologically effective if active areas such the homeobox domain retain sufficient identity to maintain their function.

Indeed, a variant of an ETCHbox protein (such as ARGFX) may share at least 81 % identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91 % identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity with the sequence of the corresponding full length ETCHbox protein, or with a fragment thereof.

The skilled person will appreciate that variants of an amino acid encoding an ETCHbox protein (such as ARGFX), or fragments thereof, may have sequences longer that the amino acid sequences of the relevant ETCHbox protein (or the relevant fragment thereof). In such embodiments, though the sequence identity of a particular region of the variant may be relatively high, the identity shared by the sequence of the variant as a whole may be rather lower.

Somatic Cells

The term "somatic cells" refers to differentiated cells, committed to a particular cell type and/or function. Somatic cells are those cells of the body other than gametes, germ cells, gametocytes, or stem cells. Somatic cells have the ability to be reprogramed to induced pluripotent stem cells through the use of appropriate reprogramming factors.

The first aspect of the invention relates to a method of producing induced pluripotent stem cells from somatic cells. The method may be employed to make induced pluripotent stem cells from a somatic cell obtained from any species. Mammalian somatic cells obtained from humans, non-human primates, cows, horses, dogs, cats, pigs and sheep, are especially suitable in the context of the present invention.

Suitably, the method may be employed to make induced pluripotent stem cells from a somatic cell obtained from an endangered species.

Somatic cells are generally categorised as mesoderm cells (for example fibroblasts, lymphocytes, adipocytes and myocytes), endoderm cells (for example hepatocytes), and ectoderm cells (for example keratinocytes). Each one of these types of somatic cells may be employed in the methods or uses of the invention to make induced pluripotent stem cells. Suitably, mesoderm cells, especially fibroblasts may be used to make induced pluripotent stem cells. Induced pluripotent stem cell

The term "induced pluripotent stem cell" as used herein refers to a cell derived from a somatic cell which has properties resembling those of an embryonic stem cell. One such property is the ability of the induced pluripotent stem cell to differentiate into a variety of different cell types. Suitably, a pluripotent stem cell has the ability to differentiate into a number of specific cell types, or in fact, into any cell type of the developing embryo. A stem cell with the ability to differentiate into any cell type, including embryonic and extra-embryonic, may be known as a totipotent stem cell.

In the context of the present invention, the term "induced pluripotent stem cell" refers to a stem cell which is at least capable of giving rise to a range of cell types indicative of pluripotency. In fact, it may be capable of giving rise to any embryonic or extra-embryonic cell type. Accordingly, it will be appreciated that for the purposes of the present disclosure, this term may also encompass a totipotent stem cell (since a cell that is totipotent may also meet the requirements of pluripotency). However, in other embodiments, a pluripotent cell may be one that is not totipotent.

An induced pluripotent stem cell may be identified by suitable characteristics, such as specific genetic profiles (for example the expression of OCT4 and NANOG) and/or cell surface markers (such as SSEA1 , SSEA3, or SSEA4). It will be appreciated that the characteristic of an induced pluripotent stem cell may depend upon the species from which the somatic cell was obtained. By way of example, a human or a non-human primate induced pluripotent stem cell may have surface markers such as SSEA3, SSEA4, TRA-1-60 and TRA-1-81 , while a mouse induced pluripotent stem cell may have a surface marker such as SSEA1. Other characteristics of induced pluripotent stem cells will be known to those skilled in the art.

Methods by which the characteristics of a cell can be analysed to determine whether it is an induced pluripotent stem cell will also be known to those skilled in the art. Merely by way of example, methods by which the genetic profile of a cell may be analysed include qPCR, RNAseq or antibody staining. Cell surface markers may be analysed, for example, using fluorescent microscopy or flow cytometry.

Providing

The term "providing" as used herein encompasses any techniques by which a somatic cell may receive an ETCHbox protein (such as ARGFX), or fragment or variant thereof, or a reprogramming factor. As explained further below, such provision may be "direct" which is to say by provision to the cell of the protein, fragment, variant, or reprogramming factor. Alternatively or additionally, the provision may be "indirect", in which the cell is provided with molecular instructions (typically a nucleic acid or analogue thereof) for the cell to synthesize the ETCHbox protein (such as ARGFX), or fragment, or variant thereof, or the reprogramming factor.

For the sake of brevity, the following paragraphs concentrate on embodiments of the invention in the context of the provision of ETCHbox proteins (such as ARGFX), or fragments or variants thereof. However, the skilled person will appreciate that, except for where the context requires otherwise, these same considerations are applicable to the provision of reprogramming factors.

In a suitable embodiment, the ETCHbox protein (such as ARGFX), or fragment or variant thereof is provided to the somatic cell directly. More suitably, the ETCHbox protein (such as ARGFX), fragment or variant thereof is provided to the somatic cell indirectly.

In an embodiment where the ETCHbox protein (such as ARGFX), or fragment or variant thereof is provided to the somatic cell indirectly, it may be provided in the form of a nucleic acid, or an analogue of a nucleic acid, encoding such a protein. Suitably, the nucleic acid or analogue thereof is exogenous, which is to say a nucleic acid or analogue thereof originating from outside the cell, and which does not naturally occur in the cell.

An exogenous nucleic acid or analogue encoding an ETCHbox protein (such as ARGFX), or fragment or variant thereof may be provided to the somatic cell with the use of an expression vector comprising the nucleic acid, or analogue, sequence encoding such a protein. The uses of expression vectors comprising exogenous nucleic acids, or analogues thereof, encoding ETCHbox proteins (such as ARGFX) or reprogramming factors, is discussed further elsewhere in the specification.

Alternatively, the nucleic acid or analogue thereof may be provided as free RNA, DNA or analogue, or as RNA, DNA or analogue packaged with other molecules or chemicals. In a suitable embodiment, the nucleic acid is free RNA or a free analogue thereof.

In an embodiment where the ETCHbox protein (such as ARGFX), fragment or variant thereof is provided to the somatic cell directly, it may be provided in the form of the protein itself. Suitably, the ETCHbox protein (such as ARGFX), fragment or variant thereof is provided to the somatic cell directly in the form of a fusion protein comprising the protein, fragment or variant thereof, and a cell-penetrating domain.

In such an embodiment, the somatic cell may be cultured in the presence of the ETCHbox protein (such as ARGFX), fusion protein, or fragment, or variant thereof. In an embodiment, the somatic cell may be cultured in conditions, such as the presence of an appropriate agent, that facilitate the transport of the ETCHbox protein (such as ARGFX), fusion protein, or fragment or variant thereof into the somatic cell.

The ETCHbox protein (such as ARGFX), or fragment or variant thereof provided to a cell may be exogenous or endogenous. Endogenous nucleic acids, or analogues thereof, encoding ETCHbox protein (such as ARGFX), fragments or variants thereof are described further elsewhere in the specification.

Suitable provision of an "endogenous" ETCHbox protein (such as ARGFX) may make use of a somatic cell's own ETCHbox gene expression. It will be appreciated that although after the 8-cell to morula stage ETCHbox proteins (such as ARGFX) would not normally be expressed, expression can be activated by providing the cell with a transcription factor activator capable of either directly or indirectly activating the expression of ETCHbox protein (such as ARGFX).

In the context of the methods and uses of the invention, the ETCHbox protein (such as ARGFX), fragment or variant thereof, and the reprogramming factor may be provided to the somatic cell at different times and/or simultaneously.

In one embodiment where the ETCHbox protein (such as ARGFX), fragment or variant thereof, and the reprogramming factor (or factors) are provided at different times, such protein may be provided, directly or indirectly, prior to the reprogramming factor or after the reprogramming factor.

In one embodiment, the ETCHbox protein (such as ARGFX), fragment or variant thereof may be provided directly or indirectly 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 24, 48, 72, 96, 120, 144, 168, or more hours prior to the reprogramming factor. More suitably, the ETCHbox protein (such as ARGFX), fragment or variant thereof may be provided 12, 24, or 48 hours prior to the reprogramming factor. Suitably ETCHbox protein (such as ARGFX), fragment or variant thereof may be provided 24 hours prior to the reprogramming factor. In another embodiment, the ETCHbox protein (such as ARGFX), fragment or variant thereof may be provided directly or indirectly 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 24, 48, 72, 96, 120, 144, 168, or more hours after the reprogramming factor. More suitably, the ETCHbox protein (such as ARGFX), fragment or variant thereof may be provided 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or 24 hours after the reprogramming factor. Suitably, the ETCHbox protein (such as ARGFX), fragment or variant thereof may be provided 1 or 2 hours after the reprogramming factor.

Alternatively, the ETCHbox protein (such as ARGFX), fragment or variant thereof may be provided directly or indirectly to the somatic cell simultaneously with the reprogramming factor. By the "simultaneously" it is meant that the ETCHbox protein (such as ARGFX), fragment or variant thereof and the reprogramming factor are provided together, immediately one after the other, or within 0.5 hour from one another.

In an embodiment where more than one ETCHbox protein, fragment or variant thereof, is provided directly or indirectly to a somatic cell, each one of the ETCHbox proteins, fragments or variants thereof may be provided to the cell at a different time and/or simultaneously. By way of example, when more than one ETCHbox proteins (for example ARGFX, and TPRX1 and/or LEUTX), or fragments or variants thereof are provided to the cell, ARGFX may be provided to the cell prior to providing the other ETCHbox protein (such as TPRX1 and/or LEUTX), or fragment or variant thereof.

Exogenous nucleic acids, or analogues thereof, and their uses

The second aspect of the invention provides the use of an exogenous nucleic acid, or analogue thereof, encoding an ETCHbox protein, or fragment or variant thereof in a method of producing an induced pluripotent stem cell. The method may be one as described herein with reference to the first aspect of the invention, and its various embodiments.

The uses of the second aspect of the invention, induced pluripotent stem cells of the fourth aspect of the invention, and kits of the sixth aspect of the invention, all make use of an exogenous nucleic acid, or an analogue thereof, that encodes an ETCHbox protein, or a fragment or variant thereof. For the purposes of the present invention, such an exogenous nucleic acid, or analogue, may be any such nucleic acid or analogue that originates outside a cell in which it is to be used, and encodes the requisite agent. A suitable exogenous nucleic acid may comprise RNA encoding an ETCHbox protein (such as ARGFX), or a fragment or variant thereof.

A suitable exogenous nucleic acid may comprise DNA encoding an ETCHbox protein (such as ARGFX), or a fragment or variant thereof.

An analogue of such a nucleic acid may be one which has increased specificity, affinity and/ or stability as compared to the corresponding nucleic acid. Suitably, the nucleic acid analogue may have increased specificity, affinity and/or stability of at least 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, as compared to the nucleic acid.

In one embodiment a suitable nucleic acid analogue is a morpholino. In another embodiment, a suitable nucleic acid analogue may be selected from the group consisting of: a locked nucleic acid, an unlocked nucleic acid, a glycol nucleic acid, and a peptide nucleic acid.

Exogenous nucleic acids, or analogues, of this sort are suitable for use in contexts where it is desired to indirectly provide an ETCHbox protein (such as ARGFX) or a fragment or variant thereof. Exogenous nucleic acids, or analogues thereof, may also represent suitable agents by which reprogramming factors may be indirectly provided to cells.

Suitable exogenous nucleic acids may be defined with reference to the DNA sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 or SEQ ID NO: 12, which respectively encode two isoforms of ARGFX, TPRX1 , TPRX2, DPRX, and LEUTX.

Suitably, an exogenous nucleic acid may comprise a nucleic acid sharing at least 50% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 12, or a fragment of one of these sequences, at least 55% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment of one of these sequences, at least 60% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment of one of these sequences, at least 65% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 12, or a fragment of one of these sequences, at least 70% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment of one of these sequences, at least 75% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof, at least 80% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 12, or a fragment thereof, at least 85% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof. A suitable exogenous nucleic acid may comprise a nucleic acid sharing at least 90% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof, at least 91 % identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof, at least 92% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof, at least 93% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 12, or a fragment thereof, at least 94% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof, or at least 95% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 12, or a fragment thereof. Suitably, an exogenous nucleic acid may comprise a nucleic acid sharing at least 96% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof, at least 97% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 12, or a fragment thereof, at least 97% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof, at least 98% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12, or a fragment thereof, or at least 99% identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 12, or a fragment of one of these sequences.

A suitable analogue may be based upon any of the nucleic acids defined above.

Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein that is a variant or fragment of ARGFX sharing at least 60% identity with the amino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, at least 65% identity with the amino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, at least 70% identity with the amino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2. The nucleic acid, or analogue thereof, may encode a protein or variant that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity with the amino acid sequence set out in SEQ ID NO: 1 SEQ ID NO:2. Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein sharing at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the amino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2. Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein that is a variant or fragment of TPRX1 sharing at least 60% identity with the amino acid sequence set out in SEQ ID NO: 3, at least 65% identity with the amino acid sequence set out in SEQ ID NO: 3, or at least 70% identity with the amino acid sequence set out in SEQ ID NO: 3. The nucleic acid, or analogue thereof, may encode a protein or variant that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity with the amino acid sequence set out in SEQ ID NO: 3. Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein sharing at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the amino acid sequence set out in SEQ ID NO: 3.

Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein that is a variant or fragment of TPRX2 sharing at least 60% identity with the amino acid sequence set out in SEQ ID NO: 4, at least 65% identity with the amino acid sequence set out in SEQ ID NO: 4, or at least 70% identity with the amino acid sequence set out in SEQ ID NO: 4. The nucleic acid, or analogue thereof, may encode a protein or variant that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity with the amino acid sequence set out in SEQ ID NO: 4. Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein sharing at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the amino acid sequence set out in SEQ ID NO: 4.

Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein that is a variant or fragment of DPRX sharing at least 60% identity with the amino acid sequence set out in SEQ ID NO: 5, at least 65% identity with the amino acid sequence set out in SEQ ID NO: 5, or at least 70% identity with the amino acid sequence set out in SEQ ID NO: 5. The nucleic acid, or analogue thereof, may encode a protein or variant that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity with the amino acid sequence set out in SEQ ID NO: 5. Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein sharing at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the amino acid sequence set out in SEQ ID NO: 5.

Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein that is a variant or fragment of LEUTX sharing at least 60% identity with the amino acid sequence set out in SEQ ID NO: 6, at least 65% identity with the amino acid sequence set out in SEQ ID NO: 6, or at least 70% identity with the amino acid sequence set out in SEQ ID NO: 6. The nucleic acid, or analogue thereof, may encode a protein or variant that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity with the amino acid sequence set out in SEQ ID NO: 6. Suitably an exogenous nucleic acid, or analogue thereof, may encode a protein sharing at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the amino acid sequence set out in SEQ ID NO: 6.

In a suitable embodiment the exogenous nucleic acid, or analogue thereof, further encodes a reprogramming factor.

Alternatively, the exogenous nucleic acid, or analogue thereof, may be for use in combination with a further nucleic acid, or analogue thereof, that encodes a reprogramming factor.

An expression vector

The exogenous nucleic acid, or analogue thereof, encoding the ETCHbox protein (such as ARGFX), or fragment or variant thereof may be provided to the somatic cell with the use of an expression vector comprising a nucleic acid sequence encoding such a protein.

Suitably an expression vector may comprise a nucleic acid of SEQ ID NO: 7, or a fragment thereof, SEQ ID NO: 8, or a fragment thereof, SEQ ID NO: 9, or a fragment thereof, SEQ ID NO: 10, or a fragment thereof, SEQ ID NO: 11 , or a fragment thereof, or SEQ ID NO: 12, or a fragment thereof. Alternatively, the expression vector may comprise an exogenous nucleic acid, or variant thereof, in accordance with any of the embodiments described in the present specification.

In a suitable embodiment, the expression vector may further comprise a nucleic acid encoding a reprogramming factor. Suitably, the expression vector may comprise a nucleic acid encoding one, two, three, four, or more reprogramming factors. Suitable reprogramming factors are discussed elsewhere in this specification. By way of example these may be selected from the group consisting of Oct3, Klf4, Sox2, c-Myc, Lin28 and Nanog. Alternatively, a nucleic acid encoding the ETCHbox protein (such as ARGFX), or fragment or variant thereof, and a nucleic acid encoding the reprogramming factor may be located on separate vectors. Alternatively, the expression vector may encode miRNAs controlling the endogenous expression of these genes. In a suitable embodiment, the expression vector may comprise an Internal Ribosome Entry Site. Alternatively, the polycistronic expression vector may comprise a cleavage site. Merely by way of example, a suitable cleavage site is 2A site (such as F2A, T2A, E2A and P2A).

It will be appreciated that an expression vector may be especially suitable for use in an embodiment of the method of the first aspect, where it is desired for the ETCHbox protein (such as ARGFX), or fragment or variant thereof, and the reprogramming factor to be provided to the somatic cell simultaneously.

To this end, the third aspect of the invention provides an expression vector comprising a nucleic acid encoding an ETCHbox protein (such as ARGFX), or fragment or variant thereof and a reprogramming factor.

In the context of the third aspect, the expression vector may be viral or non-viral. More suitably, the expression vector is viral. By way of example, a suitable viral expression vector may be derived from a virus selected from the group consisting of paramyxovirus, retrovirus, adenovirus, lentivirus, pox virus, alphavirus, and herpes virus. More suitably, the virus is paramyxovirus. An example of a particularly suitable paramyxovirus is Sendai virus. Other viral expression vectors suitable for providing the ARGFX protein, fragment or variant thereof, to somatic cells are known in the art.

Suitable non-viral expression vectors may be selected from the group consisting of inorganic particle expression vectors (such as calcium phosphate, silica, and gold), lipid based particle expression vectors (for example cationic lipids, lipid nano emulsions, and solid lipid nanoparticles) and polymer based particle expression vectors (for example peptides, polyethylenimine, chitosan, and dendimers). Other suitable non-viral expression vectors will be known to those skilled in the art.

Delivery of exogenous nucleic acids, analogues, or expression vectors

Methods of delivering exogenous nucleic acids, or analogues thereof, whether in the form of an expression vector, "free" nucleic acid, or otherwise, to a cell are well known in the art. Merely by way of example, such methods include viral transfection, electroporation and sonoporation. An illustrative technique is described in the Example. A reprogramming factor

The term "reprogramming factor" as used herein, refers to an agent other than an ETCHbox protein (such as ARGFX), fragment, or variant thereof capable of directly or indirectly inducing the conversion of a somatic cell into a pluripotent stem cell. While ARGFX, or a fragment or variant thereof, may itself exhibit activity of a reprogramming factor, it is not encompassed by this term (unless expressly referred to as such) for the purposes of the present disclosure.

In the context of the present invention, a suitable additional reprogramming factor may be selected from the group consisting of: Oct family (for example Oct3, also known as Oct4 or Pou5f1), Sox family (for example Sox2, Sox1 , Sox3, Sox15, or Sox18), Klf family (Klf4, Klf1 , Klf2, or Klf5), Myc family (for example c-Myc, l-Myc, and n-Myc), Lin family (for example Lin 28), Nanog, HHex, Hlx, Essrb, Utf1 , Dppa2, Nr5a2, Sall4, Gata3, Glis1 and Gmnn.

In a suitable embodiment the reprogramming factor is selected from the group consisting of at least one reprogramming factor from the Oct family, at least one reprogramming factor from the Sox family, at least one reprogramming factor from the Klf family, at least one reprogramming factor from the Myc family.

Suitably, the reprogramming factor from the Oct family is Oct3/4.

Suitably, the reprogramming factor from the Sox family is Sox2.

Suitably, the reprogramming factor from the Klf family is Klf4.

Suitably, the reprogramming factor from the Myc family is c-Myc.

Suitable embodiments of the invention may make use of a combination of reprogramming factors.

A suitable embodiment of the invention may make use of a combination of Oct3/4, Sox2, Klf4, and c-Myc as reprogramming factors.

As referred to above, an ETCHbox protein (such as ARGFX), or a fragment or variant thereof, may be used as a replacement for a reprogramming factor. Merely by way of example, an ETCHbox protein may be used as a replacement for a Myc family member that would otherwise be used as a reprogramming factor. A suitable embodiment of the invention may make use of a combination of Oct3/4, Sox2, Klf4, without a Myc family member, as reprogramming factors.

Producing induced pluripotent stem cells

Methods for the production of induced pluripotent stem cells are well known to those skilled in the art. The invention is able to improve the yield of such methods through the provision to the cells to be transformed of an ETCHbox protein (such as ARGFX), or a fragment or variant thereof. Without wishing to be bound by any hypothesis, the inventors believe that the advantages provided by the invention may be conferred on any method for the production of induced pluripotent stem cells known to those skilled in the art.

Increased efficiency of induced pluripotent stem cell production

A notable advantage conferred by the methods and uses of the invention lies in their ability to increase the efficiency of induced pluripotent stem cell production. Accordingly, it will be appreciated that in some embodiments, methods of the invention constitute methods of increasing the efficiency of induced pluripotent stem cell production, and uses of the invention constitute uses of an ETCHbox protein (such as ARGFX) or a fragment of variant thereof, for increasing such efficiency. This is highly desirable, since the efficiency of currently available methods of producing induced pluripotent stem cells is very low.

In the context of the present disclosure, an "increased efficiency of induced pluripotent stem cell production" may be demonstrated by an increase in the proportion of somatic cells reprogrammed to induced pluripotent stem cells. The increase in the proportion of induced pluripotent stem cells may be in relation to comparator methods currently known in the art. These comparator methods do not involve providing somatic cells with an ETCH box protein (such as ARGFX), or fragment or variant thereof.

In suitable embodiments, the methods or uses of the invention may be able to increase efficiency of reprogramming somatic cells to induced pluripotent stem cells by up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, or more as compared to a suitable comparator. Suitably, the methods or uses of the invention may be able to increase efficiency of reprogramming somatic cells to induced pluripotent stem cells by up to 90%, up to 100%, up to 1 10%, up to 120%, up to 130%, up to 140%, up to 150%, or more as compared to a suitable comparator. In suitable embodiments, the methods or uses of the invention may be able to increase efficiency of reprogramming somatic cells to induced pluripotent stem cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more as compared to a suitable comparator. Suitably, the methods or uses of the invention may be able to increase efficiency of reprogramming somatic cells to induced pluripotent stem cells by at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more.

In suitable embodiments, the methods or uses of the invention may be able to increase efficiency of reprogramming somatic cells to induced pluripotent stem cells by up to 10-fold, 9.5-fold, 9-fold, 8.5-fold, 8-fold, 7.5-fold, 7-fold, 6.5-fold, 6-fold, 5.5-fold, 5-fold, 4.5-fold, 4-fold, 3.5-fold, 3-fold, 2.5-fold, 2-fold, 1.75-fold, or 1.5-fold, as compared to a suitable comparator.

In suitable embodiments, the methods or uses of the invention may be able to increase efficiency of reprogramming somatic cells to induced pluripotent stem cells by at least 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7- fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold, as compared to a suitable comparator.

An increase in the proportion of induced pluripotent stem cells may be, for example, observed by an increase in the number of colonies, visualised by methylene blue staining. Other techniques by which it is possible to observe an increase in the proportion of induced pluripotent stem cells will be known to the skilled person.

It will be appreciated that the increased efficiency of induced pluripotent stem cell production that can be achieved in accordance with the invention confers advantages not just in terms of the total yield of stem cells that may be achieved, but also in terms of the ability to reduce the amount of reprogramming factors that must be employed in order to achieve a desired yield. The cost of reprogramming factors represents a major proportion of the total cost associated with current methods for induced pluripotent stem cell production.

Induced pluripotent stem cells of the invention

The fourth aspect of the invention provides an induced pluripotent stem cell, comprising an exogenous nucleic acid, or an analogue thereof, encoding an ETCHbox protein, such as ARGFX, or a fragment or variant thereof. The induced pluripotent stem cell may optionally further comprise an exogenous nucleic acid, or analogue thereof, encoding a reprogramming factor. In such embodiments the ETCHbox protein and reprogramming factor may be encoded by the same nucleic acid, or analogue, or by different nucleic acids, or analogues.

It will be appreciated that induced pluripotent stem cells produced by methods previously known in the art will not incorporate exogenous nucleic acid molecules of the sort herein described, and so the presence of such nucleic acid molecules, or analogues thereof, in induced pluripotent stem cells marks such cells as being in accordance with the present invention.

A kit of the invention

The sixth aspect of the invention provides a kit comprising first and second exogenous nucleic acids, or analogous thereof, wherein the first exogenous nucleic acid or analogue encodes an ETCHbox protein, or fragment or variant thereof, and the second exogenous nucleic acid or analogue encodes a reprogramming factor.

In a suitable embodiment, the first and second exogenous nucleic acids, or analogous thereof, may be located on a single expression vector. Alternatively, the first and second exogenous nucleic acids, or analogous thereof, may be located on separate expression vectors.

As touched upon elsewhere in this specification, the ETCHbox protein (such as ARGFX), or fragment or variant thereof, and the reprogramming factors (or factor) may be provided to a somatic cell at different times and/or simultaneously.

Therefore, it will be appreciated that whether the first and second exogenous nucleic acids, or analogous thereof are located on a single expression vector or separate expression vectors may depend upon the desired method of providing the ETCHbox protein and reprogramming factors to a somatic cell.

For example, if the ETCHbox protein (such as ARGFX), or fragment or variant thereof, and the reprogramming factors (or factor) are to be provided simultaneously, it may be desirable for the first and second exogenous nucleic acids, or analogous thereof to be located on a single expression vector.

By the same token, if the ETCHbox protein (such as ARGFX), or fragment or variant thereof, and the reprogramming factors (or factor) and the reprogramming factors (or factor) are to be provided at different times, it may be desirable for the first and second exogenous nucleic acids, or analogous thereof to located on separate vectors.

A pharmaceutical composition

The present invention also relates to a pharmaceutical composition comprising an induced pluripotent stem cell, wherein the induced pluripotent cell comprises an exogenous nucleic acid, or an analogue thereof, encoding an ETCHbox protein or a fragment or variant thereof. Suitably, the induced pluripotent stem cell may comprise an expression vector comprising a nucleic acid encoding an ARGFX protein, or fragment or variant thereof, and a nucleic acid encoding a reprogramming factor.

In a suitable embodiment, the pharmaceutical composition may comprise a pharmaceutically acceptable diluent, carrier or excipient. Such compositions may further 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.

The pharmaceutical composition may also include antioxidants and/or preservatives. Antioxidants may include thiol derivatives (e.g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiareticacid. Suitable preservatives may for instance be phenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben, benzalkonium chloride and cetylpyridinium chloride.

The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Assaying

In the context of the present invention the term "assaying" refers to the determination of the presence or absence of target molecules representative of expression of a gene selected from the group consisting of: ARGFX, NANOGNB and HESX1. A suitable method by which such assaying may be carried out is selected depending on the type of target molecule selected.

In one embodiment, gene expression can be detected directly by techniques that allow the assaying of RNA target molecules. Such techniques may include for example RT-PCR (reverse transcription PCR), QPCR (quantitative PCR), SAGE (Serial analysis of gene expression), direct RNA sequencing, Northern blot, RNAse protection, RNA sequencing (RNA-seq), in situ hybridisation, and RNA microarray.

In another embodiment, gene expression can be detected indirectly, by techniques that allow assaying of ARGFX, and/or NANOGNB, and/or HESX1 protein target molecules. Such techniques may include for example ELISA, radioimmunoassay, immunoprecipitation, immunohistochemistry, immunocytochemistry, Western blot and mass spectrometry.

Other methods suitable for assaying gene expression will be known to the person skilled in the art.

Target molecule

In the context of the present disclosure, the term "target molecule" refers to any molecule which is representative of the expression of ARGFX, and/or NANOGNB, and/or HESX1 in a subject. A target molecule may represent the expression of the ARGFX, and/or NANOGNB, and/or HESX1 gene either directly or indirectly.

Merely by way of example, a suitable target molecule which directly represents the expression of ARGFX may comprise an RNA transcript encoding ARGFX. A suitable target molecule which indirectly represents the expression of ARGFX may comprise the ARGFX protein. Alternatively or additionally, a suitable target molecule which indirectly represents the expression of ARGFX may be a protein, or an RNA transcript coding for a protein, the expression of which is controlled by ARGFX.

By the same token, a suitable target molecule which directly represents the expression of NANOGNB may comprise an RNA transcript encoding NANOGNB. A suitable target molecule which indirectly represents the expression of NANOGNB may comprise the NANOGNB protein. Alternatively or additionally, a suitable target molecule which indirectly represents the expression of NANOGNB may be a protein, or an RNA transcript coding for a protein, the expression of which is controlled by NANOGNB. A suitable target molecule which directly represents the expression of HESX1 may comprise an RNA transcript encoding HESX1. A suitable target molecule which indirectly represents the expression of HESX1 may comprise the HESX1 protein. Alternatively or additionally, a suitable target molecule which indirectly represents the expression of HESX1 may be a protein, or an RNA transcript coding for a protein, the expression of which is controlled by HESX1.

A sample

Methods according to the seventh and eight aspect of the invention utilise a sample that provides an indication as to the gene expression in a subject. The sample may be a body fluid sample or a solid sample.

Suitably, the sample may be a fluid sample selected from the group consisting of blood (for example, a whole blood sample, a blood plasma sample, or a serum sample), saliva, seminal fluid, urine, and interstitial fluid.

A solid sample may be a biopsy or smear of a tumour.

A subject

The methods of the seventh, eighth and tenth aspects of the invention respectively relate to methods of detecting or treating a teratocarcinoma or its metastases in a subject. Teratocarcinomas are also referred to as embryonal carcinomas.

The ARGFX and NANOGNB genes are normally expressed only during the 8-cell to morula stage of development, and HESX1 is also only normally expressed during early stages of development. The methods of the seventh and eighth aspects of the invention are based upon the inventors' finding that ARGFX, NANOGNB and HESXIare also expressed by cells of teratocarcinomas, or their metastases. Therefore, in the context of the present disclosure "a subject" is an organism which is past the morula stage (in the case of embodiments relating to ARGFX and/or NANOGNB) or which is past the embryo stage (in the case of embodiments relating to HESX1).

Suitably, the subject may be a mammal. Suitable the subject may be human. More suitably, the subject may be male. The subject in a method of the seventh aspect of the invention may be an individual suspected as having a teratocarcinoma or metastases thereof.

The subject in a method of the eighth aspect of the invention may be an individual identified as having a teratocarcinoma or metastases thereof.

The recognition that, after the morula and early blastocyst stage, strong expression of proteins selected from the group consisting of: ARGFX, NANOGNB and HESX1 is limited to teratocarcinoma cells indicates that targeting cells expressing ARGFX, and/or NANOGNB and/or HESX1 represent a suitable approach for the treatment of teratocarcinoma or its metastases. Suitably, such treatment may make use of agents that target ARGFX, and/or NANOGNB, and/or HESX1.

Accordingly, the ninth and tenth aspects of the invention provide an agent that targets at least one of ARGFX, and/or NANOGNB, and/or HESX1 for use in the treatment of teratocarcinoma or its metastases, and a method of treating teratocarcinoma or its metastases, respectively.

In the context of the ninth and tenth aspect, an agent that targets ARGFX, and/or NANOGNB, and/or HESX1 may target it directly or indirectly. An agent that targets ARGFX, and/or NANOGNB, and/or HESX1 directly may bind to the protein(s) in question. An agent that targets ARGFX, and/or NANOGNB, and/or HESX1 indirectly may bind to the RNA and/or DNA that encodes the relevant protein(s). It will be appreciated that binding of an agent to the RNA that encodes the protein(s) in question, may prevent the expression of the protein(s).

In one embodiment an agent that targets ARGFX, and/or NANOGNB, and/or HESX1 directly (for example by binding to them) may comprise a moiety which binds and/or facilitates binding of the agent to ARGFX, and/or NANOGNB, and/or HESX1. Merely by way of example, such an agent may be selected from the group consisting of an antibody (or antigen-binding fragment thereof), an aptamer, and a T-cell receptor. For example, a suitable agent may comprise an antibody-drug conjugate employing an anti-ARGFX antibody, or a chimeric antigen receptor T- cell with specificity for ARGFX, and/or an anti-NANOGNB antibody, or a chimeric antigen receptor T- cell with specificity for NANOGNB, and/or an anti-HESX1 antibody, or a chimeric antigen receptor T- cell with specificity for HESX1. In a suitable embodiment ARGFX, and/or NANOGNB, and/or HESX1 may serve as a tumour specific antigen against which to raise a cell mediated immune response. Such an embodiment may employ cytotoxic T-cells specific for MHC class I restricted epitopes derived from ARGFX, and/or NANOGNB, and/or HESX1. In one embodiment, an agent that targets ARGFX, and/or NANOGNB, and/or HESX1 indirectly may be a nucleic acid probe. Merely by way of example, a nucleic acid probe may be selected from the group consisting of a DNA probe, a RNA probe, a synthetic nucleic acid analogue probe, a locked nucleic acid (LNA) probe, antisense nucleotide, antisense morpholino nucleotide, siRNA, and a series of oligonucleotide probes with or without sequences permitting amplification through chain reactions. Other agents will be known to those skilled in the art.

In suitable embodiments, the methods of the seventh or eighth aspects of the invention may be of particular use in the detection or treatment of metastases of teratocarcinomas. In such context, the primary teratocarcinoma may have been surgically removed, but the persistence of target molecules representative of expression of proteins selected from the group consisting of ARGFX, NANOGNB and HESX1 is indicative of the presence of metastases of the teratocarcinoma within the subject.

Suitably, the subject may have been diagnosed with embryonal carcinoma, most typically of the testis, and undergone partial or complete bilateral or unilateral orchiectomy. Suitably, the subject may have been diagnosed with testicular tumour through the use of the methods of the seventh aspect of the invention, or other methods know in the art (for example ultrasound). In such embodiments, the presence of target molecules representative of expression of ARGFX, and/or NANOGNB, and/or HESX1 RNA or protein may be indicative of the subgroup of testicular tumour characterised by partial or complete embryonal carcinoma, or by metastasised testicular cancer.

The invention will now be further described, with reference to the Examples.

EXAMPLES

Example 1 : Increased efficiency of reprogramming

Methods 1: Use of the ETCHbox protein ARGFX before Yamanaka reprogramming factors

Human ARGFX coding sequence (SEQ ID NO: 7) was synthesized by GenScript USA. Coding sequences were cloned in-frame with a C-terminal FLAG tag under control of a CMV promoter in GFP-expressing vector pSF-CMV-Ub-daGFP Ascl (Oxford Genetics #OG244). Primary human dermal fibroblasts were obtained from ScienCell (#2320); third passage cells grown in fibroblast medium (ScienCell #2301) were combined with endotoxin-free expression constructs (EMPTY-FLAG or ARGFX-FLAG) in a 2mm gap and electroporated using EPA21 Super Electroporator. Cells were seeded and cultured for 48 hours in fibroblast growth medium.

To purify successfully transfected cells and remove non-transfected cells, FACS sorting was used, as follows:

After 48 hours cells were harvested and separated into a single cell suspension by trypsinisation for FACS sorting. Cells were resuspended in FACS sorting buffer (25mM HEPES pH 7.4, 2mM EDTA, 0.5% BSA in Mg2+/Ca2+ free PBS); GFP positive transfected cells were then enriched using a FACS Aria III Flow Cytometer.

GFP positive FACS sorted cells were seeded back into fibroblast medium in a 96 well plate at either 2,000 or 5,000 cells per well. After 24 hours, the \f?G X-transfected cells were treated with four classical reprogramming factors (Klf4, c-Myc, Oct4 also known as Oct3, Sox2) using two different batches (PH or TJD) of the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher).

After 6 days, the cells were then plated onto a feeder cell layer (mitotically inactivated mouse embryo fibroblasts), and colonies of transformed cells counted from live cell cultures (6 days after plating onto feeder cells) or after methylene blue (7 days after plating onto feeder cells).

Illustrative images of cell cultures are provided in Figure 1. Values of the various fixed colony counts at day 13 are shown in Table 1. These results indicate that providing somatic cells with both the ETCHbox protein ARGFX and reprogramming factors led to a 90% increase in the rate of efficiency of induced pluripotent stem cell production.

Table 1

* Cells in these wells were transfected with ARGFX but not transfected with Yamanaka factors. **Adding ARGFX with the Yamanaka factors improves the efficiency by 90%

Example 2: Diagnosis of testicular tumour subtype, embryonal carcinoma

Methods 2: Correlation of ARGFX, NANOGNB, and HESX1 gene expression and embryonal carcinoma

Raw RNAseq data files for testicular tumour samples were downloaded from The Cancer Genome Atlas (TCGA). The inventors mapped the sequence reads to human genome assembly NCBI GRCh38.p2 using the STAR RNA-seq aligner using the default settings with the addition of -outSAMstrandField intron Motif and -outFilterMultimapNmax 15 to increase the limit for multimapping reads before they would be discarded; this setting improves accuracy of expression analysis from repeated loci.

After first refining several incorrectly annotated homeobox genes (ARGFX, TPRX1, TPRX2) and adding unannotated genes (CPHX1, CPHX2) FPKM (fragments per kilobase per million reads) were calculated using Cufflinks, discarding cases when only a single read mapped from paired-end data. Default settings were used. The FPKM values indicate level of gene expression; these were compared to histological diagnosis data from TCGA.

The same steps were carried out with respect to correlating NANOGNB, and HESX1 gene expression with embryonal carcinoma.

Results

There are application needs for serum biomarkers of tumour subtypes and tissue markers of tumour subtypes. For testicular tumours, the most widely used serum biomarkers are AFP and HCG, but both have poor penetrance and are prone to false positives (Mir M et al. 2016 Urol Clin N Am 43: 119-125). The inventors searched for novel tissue and fluid biological markers of testicular tumours. Global transcriptome, reconstructed from RNAseq data, for 134 human testicular tumours were clustered on the basis of transcriptome similarity. The transcriptome- based clustering correlated with histological diagnosis of the tumours, which divide tumours into seminoma and non-seminoma, and then divide the latter into yolk sac tumours, chorioncarcinoma, teratocarcinoma (also referred to as embryonal carcinoma) and teratoma. Many tumours contain combinations of subtypes.

First, against these categorisations, the inventors compared expression levels of several genes encoding proteins proposed as testicular tumour biomarkers (HGMA1 , HGMA2, SOX2, SOX17, POU5F1/OCT4, and NANOG; Mir et al. cited above). The inventors have found that these have limited ability to distinguish between testicular tumour types. For example, HMGA1 is expressed in all samples, HMGA2 primarily in teratomas, with lower expression in other tissues; these patterns do not match the statements of Mir et al. cited above. NANOG and OCT4 were found to be mainly expressed in testicular tumours except teratomas. SOX17 was found is in all samples, and SOX2 in embryonal carcinoma, yolk sac tumours and teratoma. To their surprise, the inventors discovered that ARGFX demonstrated the most sub-type specific pattern of expression, being expressed almost exclusively in teratocarcinomas. This association has not previously been reported.

Second, the inventors conducted a differential gene expression (DEG) analysis using DESeq2 between the teratocarcinoma-enriched tumours clustered by transcriptome versus all other testicular tumours. From the list of genes upregulated in teratocarcinomas, they:

(i) excluded genes with expression >2 FPKM in adult human tissues to ensure specificity to cancers;

(ii) excluded genes that were up-regulated to <10 FPKM in teratocarcinomas to ensure only robust detectable markers are found;

(iii) excluded genes which were expressed >2 FPKM in teratocarcinomas and non- teratocarcinomas samples present outside of the enriched tumour cluster; and

(iv) excluded genes below the top 8 most differentially expressed genes.

This strategy allowed the inventors to identify three potential teratocarcinoma markers as follows: ARGFX (ranked 1), FOXD3 (ranked 2), DNMT3L (ranked 6).

The identification of expression of ARGFX, NANOGNB, and HESX1 as associated with teratocarcinomas, when these are otherwise only expressed during a very tightly controlled window that occurs in the development process, gives rise to the methods of the seventh and eighth aspects of the invention. These methods allow the detection of teratocarcinomas, or metastases thereof, in a subject by means of a serum test. This is particularly advantageous in the case of metastases, where it may otherwise be very difficult to identify the presence of cancerous cells at sites away from the primary tumour. If the methods of the invention identify the continued presence of expression ARGFX, and/or NANOGNB, and/or HESX1 after a primary teratocarcinoma has been treated, for example by surgical removal, further treatment of the metastases can be initiated, even before these can otherwise be detected. Sequence information

SEQ ID NO: 1 (Amino acid sequence of a first isoform of ARGFX)

MRNRMAPENPQPDPFINRNYSNMKVIPPQDPASPSFTLLSKLECSGTVSAYCSLNLPGST DPPTSASRVAATTAIRRRHKERTSFTHQQYEELEALFSQTMFPDRNLQEKLALRLDLPES TVKVWFRNRRFKLKKQQQQQSAKQRNQILPSKKNVPTSPRTSPSPYAFSPVISDFYSSLP SQPLDPSNWAWNSTFTESSTSDFQMQDTQWERLVASVPALYSDAYDI FQI IELYNLPDEN EISSSSFHCLYQYLSPTKYQVGGQGSSLS I FAGPAVGLSPAQTWPNMTSQAFEAYSLTDS LEFQKTSNMVDLGFL

SEQ ID NO: 2 (Amino acid sequence of a second isoform of ARGFX)

MVTLRGFVTGLPSQISETMRNRMAPENPQPDPFINRNYSNMKVIPPQDPASPSFTLLSKL ECSGTVSAYCSLNLPGSTDPPTSASRVAATTAIRRRHKERTSFTHQQYEELEALFSQTMF PDRNLQEKLALRLDLPESTVKVWFRNRRFKLKKQQQQQSAKQRNQILPSKKNVPTSPRTS PSPYAFSPVISDFYSSLPSQPLDPSNWAWNSTFTESSTSDFQMQDTQWERLVASVPALYS DAYDI FQI IELYNLPDENEISSSSFHCLYQYLSPTKYQVGGQGSSLS I FAGPAVGLSPAQ TWPNMTSQAFEAYSLTDSLEFQKTSNMVDLGFL

SEQ ID NO: 3 (Amino acid sequence of TPRX1 )

MQDPGHLQGPPLALDPPRRQRQERTVYTESQQKVLEFYFQKDQYPNYDQRLNLAEMLSLR EQQLQVWFKNRRAKLARERRLQQQPQRVPGQRGRGARAAPLVPAASASAPQRGPSGILPA AEPTICSLHQAWGGPGCRAQKGIPAALSPGPGPIPAPIPGPAQIPGPLPGSIPGPIPGPA QIPSPIPAPIPGPISGPVQIPGPFRGPIPGPISGPAPIPGPISGPFSGPNPGPIPGPNPG PIPGPISGPIPGPISVPIPGPIPGPISGPISGPNPGPIPGPIPGPISGPNPGPIPGPISG PNPGLIPGPIPGPISGPGPIIGPIPSPAQIPGPGRLQGPGPILSPGRMRSPGSLPGLAPI LGPGSGPGSGSVPAPIPGPGSLPAPAPLWPQSPDASDFLPDTQLFPHFTELLLPLDPLEG SSVSTMTSQYQEGDDSMGKKHSGSQPQEEGGSVNENHSGPRLLLDL

SEQ ID NO: 4 (Amino acid sequence of TPRX2)

MQDPGHLQGPPLALDPPRRQRQERTVYTESQQKVLEFYFQKDQYPNYDQRLNLAEMLSLR EQQLQVWFKNRRAKLARERRLQQQPQRVPGQRGRGARAAPLVPVAAASFPGGPEFPQGRG SWISPQPGPWGVLPAAEPKIYSLPRTWGGPECGTQEGLKAVPAPGPGPIPAPIPGPAQIP GPVPGPAPNLGPMSGPLSVSIPGPIPAPISCPGPIPDPVLGRTLMPGPGSLPTPAPGALW PQSPYASNLSPDTQLYPDFTKLLPLLDRFEESSLSTTTSQYKEEDGFVDKNHSVPRSLLD L SEQ ID NO: 5 (Amino acid sequence of DPRX)

MPGSEDLRKGKDQMHSHRKRTMFTKKQLEDLNILFNENPYPNPSLQKEMASKIDIHPTVL QVWFKNHRAKLKKAKCKHIHQKQETPQPPIPEGGVSTSVGLRNADTLPRLPNAAHPIGLV YTGHRVPSFQLILYPNLKVPANDFIGHRIVHFGCCRDPNIYCLYPILESQVCAPSFHSGS PACSSNQSRER

SEQ ID NO: 6 (Amino acid sequence of LEUTX)

MFEGPRRYRRPRTRFLSKQLTALRELLEKTMHPSLATMGKLASKLQLDLSWKIWFKNQR AKWKRQQRQQMQTRPSLGPANQTTSVKKEETPSAITTANIRPVSPGISDANDHDLREPSG IKNPGGASASARVSSWDSQSYDIEQICLGASNPPWASTLFEIDEFVKIYDLPGEDDTSSL NQYLFPVCLEYDQLQSSV

SEQ ID NO: 7 (DNA sequence encoding a first isoform of ARGFX)

ATGAGGAACAGAATGGCCCCAGAGAATCCCCAGCCAGACCCTTTCATCAATAGGAATTATTC CAACATGAAGGTGATACCACCACAGGATCCAGCTAGTCCCAGTTTCACTCTGTTATCCAAGC TGGAGTGCAGTGGCACGGTCTCGGCTTACTGCAGCCTCAACCTCCCAGGTTCAACTGATCCT CCCACCTCAGCCTCCCGAGTAGCTGCGACTACAGCAATACGGAGAAGGCATAAAGAACGTAC TTCTTTCACCCACCAACAGTATGAGGAGCTAGAAGCTCTGTTTAGCCAGACCATGTTCCCAG ATAGAAATCTTCAGGAGAAACTAGCTTTGAGACTCGACCTACCGGAGTCAACAGTAAAGGTT TGGTTCAGGAACCGGCGATTCAAATTGAAGAAGCAGCAGCAGCAGCAATCAGCAAAGCAACG AAACCAGATCCTTCCATCCAAGAAGAATGTGCCCACCTCCCCCAGAACATCCCCCAGTCCTT ATGCTTTTTCTCCTGTGATTTCAGATTTCTACAGCTCCCTTCCATCTCAGCCCTTAGACCCT TCCAATTGGGCATGGAACTCTACCTTCACTGAGAGTTCTACCAGTGACTTCCAAATGCAAGA TACTCAGTGGGAGAGGCTGGTGGCCTCGGTTCCTGCTTTGTACTCTGATGCCTATGACATAT TCCAAATCATAGAACTGTACAATCTTCCTGATGAGAATGAGATATCCAGCTCTTCTTTCCAC TGTCTGTATCAGTATCTCTCACCCACAAAGTACCAGGTAGGAGGACAGGGTTCCTCTCTCAG CATCTTTGCTGGTCCAGCTGTAGGCCTATCTCCTGCACAAACCTGGCCCAATATGACAAGCC AAGCCTTTGAAGCCTACAGTCTAACAGATAGCCTGGAATTCCAGAAAACCTCCAATATGGTA GACTTGGGATTTCTCTGA

SEQ ID NO: 8 (DNA sequence encoding a second isoform of ARGFX)

ATGGTTACTCTAAGGGGATTCGTGACAGGCCTTCCATCTCAGATTTCAGAAACCATGAGG AACAGAATGGCCCCAGAGAATCCCCAGCCAGACCCTTTCATCAATAGGAATTATTCCAAC ATGAAGGTGATACCACCACAGGATCCAGCTAGTCCCAGTTTCACTCTGTTATCCAAGCTG GAGTGCAGTGGCACGGTCTCGGCTTACTGCAGCCTCAACCTCCCAGGTTCAACTGATCCT C C C AC C T C AG C C T C C C GAG TAG C T G C GAC T AC AG C AAT AC G GAGAAG G C AT AAAGAAC G T AC T T C T T T C AC C C AC C AAC AG TAT GAG GAG C TAGAAG CTCTGTTTAGC CAGAC C AT G T T C C C AGAT AGAAAT C T T C AG GAGAAAC T AG C T T T GAGAC T C GAC C T AC C G GAG T C AAC AG T A AAG GTTTGGTT C AG GAAC C G G C GAT T C AAAT T GAAGAAG C AG C AG C AG C AG C AAT C AG C A AAG C AAC GAAAC C AGAT CCTTCCATC C AAGAAGAAT G T G C C C AC C T C C C C C AGAAC AT C C CCCAGTCCTTATGCTTTTTCTCCTGTGATTTCAGATTTCTACAGCTCCCTTCCATCTCAG CCCTTAGACCCTTCCAATTGGGCATGGAACTCTACCTTCACTGAGAGTTCTACCAGTGAC TTCCAAATGCAAGATACTCAGTGGGAGAGGCTGGTGGCCTCGGTTCCTGCTTTGTACTCT GATGCCTAT GAC AT AT T C C AAAT CAT AGAAC T G T AC AAT CTTCCTGAT GAGAAT GAGAT A TCCAGCTCTTCTTTCCACTGTCTGTATCAGTATCTCTCACCCACAAAGTACCAGGTAGGA GGACAGGGTTCCTCTCTCAGCATCTTTGCTGGTCCAGCTGTAGGCCTATCTCCTGCACAA AC C T G G C C C AAT AT GAC AAG C C AAG C C T T T GAAG C C T AC AG T C T AAC AGAT AG C C T G GAA TTCCAGAAAACCTCCAATATGGTAGACTTGGGATTTCTCTGA

SEQ ID NO: 9 (DNA sequence encoding TPRX1)

ATGCAAGACCCTGGTCATCTCCAAGGCCCTCCCCTGGCCCTGGACCCTCCAAGGAGACAGCG G C AG GAG C G C AC G G T C T AC AC T GAAAG C C AG C AGAAAG T G C T AGAAT T T T AC T T T C AGAAG G ACCAGTACCCGAACTACGACCAGCGACTGAATCTGGCGGAGATGCTCAGCCTCAGGGAGCAA CAGCTGCAGGTGTGGTTCAAGAATCGCCGCGCCAAACTAGCTCGGGAGCGGCGGCTCCAGCA GCAGCCCCAGCGCGTCCCTGGGCAGAGAGGCCGAGGAGCCCGCGCTGCGCCCCTAGTCCCTG CAGCCTCTGCCTCCGCACCTCAGCGGGGCCCCTCGGGAATCCTTCCAGCGGCGGAACCCACG ATCTGCAGCCTCCACCAGGCCTGGGGTGGCCCTGGGTGCAGAGCCCAGAAGGGCATCCCAGC TGCCCTGAGTCCAGGCCCTGGCCCGATCCCTGCCCCAATCCCAGGCCCAGCCCAGATCCCAG GCCCACTCCCTGGCTCAATTCCAGGCCCAATTCCAGGCCCAGCTCAGATCCCAAGCCCGATC CCAGCCCCAATCCCAGGCCCAATTTCAGGCCCAGTCCAGATCCCAGGCCCATTCCGTGGCCC AATCCCAGGCCCAATTTCAGGCCCAGCCCCGATCCCAGGCCCAATCTCAGGCCCATTCTCAG GCCCAAACCCAGGCCCGATCCCAGGCCCAAACCCAGGCCCGATCCCAGGCCCAATCTCAGGC CCGATCCCAGGCCCAATCTCAGTCCCGATCCCAGGCCCGATCCCAGGCCCAATCTCAGGCCC AATCTCAGGCCCAAACCCAGGCCCGATCCCAGGCCCAATCCCAGGCCCAATCTCAGGCCCGA ACCCAGGCCCGATCCCAGGCCCAATCTCAGGCCCGAACCCAGGCCTGATCCCAGGCCCAATC CCAGGCCCAATCTCAGGCCCAGGCCCAATTATAGGCCCGATTCCCAGCCCAGCCCAGATCCC AGGCCCAGGCAGACTCCAAGGCCCAGGTCCCATCTTAAGTCCTGGCCGGATGCGAAGCCCTG GCTCACTTCCAGGCCTAGCCCCGATTTTAGGCCCAGGCTCAGGCCCAGGCTCAGGCTCAGTC CCAGCTCCAATCCCAGGCCCAGGATCACTCCCAGCCCCAGCCCCCT TATGGCCTCAGAGCCC CGATGCCTCCGACT TCT TGCCAGACACCCAGT TAT TCCCTCACT TCACAGAGCTGCTCCTAC CCCTAGACCCCT TGGAGGGATCCTCAGTCTCCACCATGACCTCTCAGTACCAAGAAGGGGAT GACTCTATGGGCAAAAAACACTCAGGGTCTCAGCCCCAAGAGGAGGGTGGCTCTGTGAATGA AAAT CAC T CAGGCC C CAGG T TAT T AC TGGAT T TAT AG

SEQ ID NO: 10 (DNA sequence encoding TPRX2)

ATGCAAGACCCTGGTCATCTCCAAGGCCCTCCCCTGGCCCTGGACCCTCCAAGGAGACAGCG G C AG GAG C G CAC G G T C T AC AC T GAAAG C C AG C AGAAAG T G C T AGAAT T T T AC T T T C AGAAG G ACCAGTACCCGAACTACGACCAGCGACTGAATCTGGCGGAGATGCTCAGCCTCAGGGAGCAA CAGCTGCAGGTGTGGT TCAAGAATCGCCGCGCCAAACTAGCTCGGGAGCGGCGGCTCCAGCA GCAGCCCCAGCGCGTCCCTGGGCAGAGAGGCCGAGGAGCCCGCGCTGCGCCCCTAGTCCCTG TAGCCGCTGCCTCCT TCCCTGGGGGTCCTGAGT TCCCGCAGGGCAGGGGT TCCTGGATCTCC CCTCAGCCGGGCCCCTGGGGAGTCCTCCCAGCAGCAGAACCGAAGATCTACAGCCTCCCCCG GACCTGGGGTGGCCCTGAGTGTGGAACACAGGAGGGCCTCAAGGCTGTCCCGGCTCCAGGCC CTGGCCCAATCCCAGCCCCTATCCCAGGCCCAGCCCAGATCCCAGGCCCAGTCCCAGGCCCA GCCCCGAAT T TAGGCCCAATGTCAGGCCCACTGTCAGTCTCGATCCCCGGTCCAATACCAGC CCCCATCTCT TGCCCAGGCCCAATCCCAGACCCAGTCCTAGGCCGAACCCTGATGCCAGGCC CAGGATCACTCCCAACCCCAGCCCCAGGCGCCT TGTGGCCTCAGAGCCCCTATGCCTCCAAC T TGTCGCCAGACACCCAGT TATACCCTGACT TCACCAAGCTGCTCCCGCTCCTAGACCGGT T CGAGGAATCCTCACTCTCCACCACGACGTCTCAGTACAAAGAGGAGGATGGCT TCGTGGACA AAAAT CAC T C AG T C C C C AG G T CAT T AC T G GAT T TAT AG

SEQ ID NO: 11 (DNA sequence encoding DPRX)

AT G C C AG G C T C AGAG GAT C T T C G T AAAG G C AAG GAC C AGAT G CAT T CAC AC AG GAAAC GAAC C AT G T T CAC TAAGAAG C AAC T G GAAGAT C T GAAC AT C T T G T T CAAT GAGAAC C C AT AC C C AA AC C C C AG C C T T C AGAAAGAAAT G G C C T C GAAAAT AGAC AT AC AC C C AAC AG T AC T G C AG G T C TGGT T CAAGAAT CACAGAGCAAAAC T C AAGAAAG C GAAAT G C AAG CAT AT T CAT CAAAAACA AGAAACTCCACAACCGCCAATACCAGAGGGTGGGGTCTCCACCAGTGTCGGCCTGAGAAATG CAGACACACTACCCAGAT TGCCCAACGCTGCTCACCCGATCGGCCTGGTGTACACGGGTCAT CGAGTCCCCTCAT TCCAGCTCATCCTGTACCCCAACCTCAAGGTCCCTGCAAATGACT TCAT TGGCCACAGAATAGTCCAT T T TGGCTGCTGCCGAGATCCTAATATATACTGCCTCTACCCCA T T T TGGAATCCCAAGT T TGCGCTCCAAGCT TCCAT TCTGGCTCTCCTGCCTGT TCATCTAAC C AAAG T C GAGAGAGAT GA SEQ ID NO: 12 (DNA sequence encoding LEUTX)

ATGTTTGAAGGGCCAAGGCGTTATCGTCGGCCACGCACAAGATTTCTCTCCAAACAACTCAC AGCATTGAGAGAATTGCTTGAAAAGACCATGCACCCAAGTTTGGCTACAATGGGGAAACTGG CTTCAAAGCTACAACTTGATCTATCCGTAGTAAAGATCTGGTTCAAGAACCAGCGTGCCAAA TGGAAGAGGCAGCAGCGGCAGCAAATGCAGACACGGCCATCACTAGGGCCAGCAAACCAGAC AACTTCAGTGAAGAAGGAGGAGACTCCCTCAGCCATAACTACTGCAAACATTCGTCCAGTAA GTCCTGGAATCTCTGATGCAAATGACCATGATCTACGTGAGCCTTCTGGTATCAAGAATCCT GGAGGAGCCAGCGCCTCTGCGAGGGTTTCATCCTGGGATTCTCAGTCATATGACATTGAACA GATATGTCTGGGGGCTTCAAATCCTCCTTGGGCCTCCACTCTCTTTGAAATAGATGAATTTG TAAAGATCTATGACTTGCCAGGGGAAGATGACACCAGCAGCCTAAATCAATATCTTTTTCCA GTATGCCTTGAGTATGACCAGCTCCAATCTTCAGTGTAA

SEQ ID NO: 13 (Amino acid sequence of the homeodomain region of ARGFX)

RHKERTSFTHQQYEELEALFSQTMFPDRNLQEKLALRLDLPESTVKVWFRNRRFKLKKQQ

SEQ ID NO: 14 (Amino acid sequence of the homeodomain region of TPRX1 ) and SEQ ID NO: 15 (Amino acid sequence of the homeodomain region of TPRX2)

QRQERTVYTESQQKVLEFYFQKDQYPNYDQRLNLAEMLSLREQQLQVWFKNRRAKLARER

SEQ ID NO: 16 (Amino acid sequence of the homeodomain region of DPRX)

SHRKRTMFTKKQLEDLNILFNENPYPNPSLQKEMASKIDIHPTVLQVWFKNHRAKLKKAK

SEQ ID NO: 17 (Amino acid sequence of the homeodomain region of LEUTX)

YRRPRTRFLSKQLTALRELLEKTMHPSLATMGKLASKLQLDLSWKIWFKNQRAKWKRQQ

Claims

1. A method of producing an induced pluripotent stem cell, the method comprising providing a somatic cell with an ETCHbox protein, or fragment or variant thereof, and providing the cell with a reprogramming factor.

2. The method according to claim 1 , wherein the somatic cell is provided with the ETCHbox protein ARGFX, or a fragment or variant thereof.

3. The method according to claim 1 or claim 2, wherein the somatic cell is provided with the ETCHbox protein TPRX1 , or a fragment or variant thereof.

4. The method according to any preceding claim, wherein the somatic cell is provided with the ETCHbox protein DPRX, or a fragment or variant thereof.

5. The method according to any preceding claim, wherein the somatic cell is provided with the ETCHbox protein LEUTX, or a fragment or variant thereof.

6. The method according to any preceding claim, wherein the somatic cell is provided with both ARGFX, or a fragment or variant thereof, and TPRX1 and/or LEUTX, or a fragment or variant thereof.

7. The method according to claim 6, wherein the somatic cell is provided with both ARGFX, or a fragment or variant thereof, and TPRX1 , or a fragment or variant thereof.

8. The method according to any preceding claim, wherein the ETCHbox protein, or fragment or variant thereof, is provided indirectly to the somatic cell.

9. The method according to claim 8, wherein the ETCHbox protein, or fragment or variant thereof, is provided by an exogenous nucleic acid, or an analogue thereof.

10. The method according to claim 9, wherein the ETCHbox protein, or fragment or variant thereof, is provided by exogenous RNA, or an analogue thereof

1 1. The method according to claim 9, wherein the ETCHbox protein, or fragment or variant thereof, is provided by exogenous DNA, or an analogue thereof

12. The method according to any of claims 9 to 11 , wherein the exogenous nucleic acid, or analogue thereof, is provided as free nucleic acid or analogue.

13. The method according to any of claims 9 to 11 , wherein the exogenous nucleic acid, or analogue thereof, is provided in an expression vector.

14. The method according to claims 1 to 2, wherein the ETCHbox protein, or fragment or variant thereof is provided prior to the additional reprogramming factor.

15. The method according to any preceding claim, wherein the ETCHbox protein, or fragment or variant thereof is exogenous.

16. The method according to any of claims 1 to 7, wherein the ETCHbox protein, or fragment or variant thereof, is provided indirectly to the somatic cell.

17. The method according to any preceding claim, wherein the somatic cell is from a mammal.

18 The method according to claim 17, wherein the mammal is selected from the group consisting of human, non-human primate, cow, horse, sheep, dog, cat or pig.

19. The method according to any of claims 1 to 16, wherein the somatic cell is from an endangered species.

20. The method according to any preceding claim, wherein the somatic cell is selected from the group consisting of mesoderm cell, endoderm cell, and ectoderm cell.

21. The method according to any preceding claim, wherein the reprograming factor is selected from the group consisting of Oct family, Sox family, Klf family, Myc family, Lin family, Nanog, Hhex, Hlx, Essrb, Utf1 , Dppa2, Nr5a2, Sall4, Gata3, Glis1 , and Gmnn.

22. The method according to claim 21 , the reprogramming factor is selected from the group consisting of at least one reprogramming factor from the Oct family, least one reprogramming factor from the Sox family, at least one reprogramming factor from the Klf family, and at least one reprogramming factor from the Myc family.

23. The method according to claim 21 or claim 22, wherein the Oct family reprograming factor is Oct3.

24. The method according to any of claims 21 to 23, wherein the Sox family reprograming factor is Sox2.

25. The method according to any of claims 21 to 24, wherein the Klf family reprograming factor is Klf4.

26. The method according to any of claims 21 to 25, wherein the Myc family reprograming factor is c-Myc.

27. The method according to any preceding claim, wherein the ETCHbox protein, or fragment or variant thereof is provided in an amount sufficient to increase the proportion of induced pluripotent cells obtained from somatic cells by at least 1.5-fold as compared to the proportion of induced pluripotent cells obtained from somatic cells in the absence of the ETCHbox protein, fragment, or variant thereof.

28. A use of an exogenous nucleic acid, or analogue thereof, encoding an ETCHbox protein, or fragment or variant thereof in a method of producing an induced pluripotent stem cell.

29. The use according to claim 28, wherein the method is one according to any of claims 1 to 26.

30. The use according to claim 28 or 29, wherein the exogenous nucleic acid, or analogue thereof, encodes ARGFX

31. The use according to any of claims 28 to 30, wherein the exogenous nucleic acid, or analogue thereof, comprises SEQ ID NO:2.

32. The use according to any of claims 28 to 31 , wherein the exogenous nucleic acid further encodes a reprogramming factor.

33. The use according to any of claims 28 to 32, wherein the exogenous nucleic acid, or analogue thereof, is for use in combination with a further nucleic acid, or analogue thereof, that encodes a reprogramming factor.

34. The use according to claim 32 or 33, wherein the exogenous nucleic acid, or analogue thereof, encodes a reprogramming factor is selected form the group consisting of Oct family, Sox family, Klf family, Myc family, Lin family, Nanog, Essrb, Utf1 , Dppa2, Nr5a2, Sall4, Gata3, Glis1 , and Gmnn.

35. The use according to any of claims 28 to 34, wherein the exogenous nucleic acid is RNA, or an analogue thereof.

36. An expression vector comprising a nucleic acid encoding an ETCHbox protein, fragment, or variant thereof, and a reprogramming factor.

37. An expression vector according to claim 36, comprising a nucleic acid encoding ARGFX, or a fragment or variant thereof.

38. An expression vector according to claim 36 or 37, comprising a nucleic acid encoding TPRX1 , or a fragment or variant thereof.

39. An expression vector according to any of claims 36 to 38, comprising a nucleic acid encoding DPRX, or a fragment or variant thereof.

40. An expression vector according to any of claims 36 to 39, comprising a nucleic acid encoding LEUTX, or a fragment or variant thereof.

41. An expression vector according to claim 36, comprising a nucleic acid encoding both ARGFX, or a fragment or variant thereof, and TPRX1 and/or LEUTX, or a fragment or variant thereof.

42. An expression vector according to any of claims 36 to 41 , wherein the reprogramming factor is selected form the group consisting of Oct family, Sox family, Klf family, Myc family, Lin family, Nanog, Essrb, Utf1 , Dppa2, Nr5a2, Sall4, Gata3, Glis1 , and Gmnn.

43. An expression vector according to any of claims 36 to 42, wherein the expression vector encodes at least one reprogramming factor from the Oct family, at least one reprogramming factor from the Sox family, at least one reprogramming factor from the Klf family, and at least one reprogramming factor from the Myc family.

44. An expression vector according to claim 42 or claim 43, wherein the Oct family reprograming factor is Oct3.

45. An expression vector according to any of claims 42 to 44, wherein the Sox family reprograming factor is Sox2.

46. An expression vector according to any of claims 42 to 45, wherein the Klf family reprograming factor is Klf4.

47. An expression vector according to any of claims 42 to 46, wherein the Myc family reprograming factor is c-Myc.

48. An induced pluripotent stem cell comprising an exogenous nucleic acid, or analogue thereof, encoding an ETCHbox protein, or fragment or variant thereof.

49. An induced pluripotent stem cell according to claim 48, wherein the exogenous nucleic acid, or analogue thereof, encodes ARGFX, or a fragment or variant thereof.

50. An induced pluripotent stem cell according to claim 48 or claim 49, wherein the exogenous nucleic acid, or analogue thereof, encodes TPRX1 , or a fragment or variant thereof.

51. An induced pluripotent stem cell according to any of claims 48 to 50, wherein the exogenous nucleic acid, or analogue thereof, encodes DPRX, or a fragment or variant thereof.

52. An induced pluripotent stem cell according to any of claims 48 to 51 , wherein the exogenous nucleic acid, or analogue thereof, encodes LEUTX, or a fragment or variant thereof.

53. An induced pluripotent stem cell according to claim 48, wherein the exogenous nucleic acid, or analogue thereof, encodes both ARGFX, or a fragment or variant thereof, and TPRX1 and/or LEUTX, or a fragment or variant thereof.

54. An induced pluripotent stem cell according to claim 48, wherein the exogenous nucleic acid, or analogue thereof, encodes both ARGFX, or a fragment or variant thereof, and TPRX1 , or a fragment or variant thereof.

55. An induced pluripotent stem cell according to any of claims 48 to 54, wherein the exogenous nucleic acid, or analogue thereof, further encodes a reprogramming factor.

56. An induced pluripotent stem cell according to any of claims 48 to 54, wherein the exogenous nucleic acid, or analogue thereof, comprising a further exogenous nucleic acid, or analogue thereof, encoding a reprogramming factor.

57. An induced pluripotent stem cell according to claim 55 or claim 56, wherein the reprogramming factor is selected form the group consisting of Oct family, Sox family, Klf family, Myc family, Lin family, Nanog, Essrb, Utf1 , Dppa2, Nr5a2, Sall4, Gata3, Glis1 and Gmnn.

58. An induced pluripotent stem cell according to any of claims 48 to 57, wherein the exogenous nucleic acid is RNA, or an analogue thereof.

59. A pharmaceutical composition comprising an induced pluripotent stem cell according to any of claims 48 to 58.

60. A kit comprising first and a second exogenous nucleic acids, or analogues thereof, wherein the first exogenous nucleic acid or analogue encodes an ETCHbox protein, fragment, or variant thereof, and the second exogenous nucleic acid or analogue encodes a reprogramming factor.

61. A kit according to claim 60, wherein the first exogenous nucleic acid, or analogue thereof, encodes ARGFX, or a fragment or variant thereof.

62. A kit according to claim 60 or claim 61 , wherein the first exogenous nucleic acid, or analogue thereof, encodes TPRX1 , or a fragment or variant thereof.

63. A kit according to any of claims 60 to 62, wherein the first exogenous nucleic acid, or analogue thereof, encodes DPRX, or a fragment or variant thereof.

64. A kit according to any of claims 60 to 63, wherein the first exogenous nucleic acid, or analogue thereof, encodes LEUTX, or a fragment or variant thereof.

65. A kit according to claim 60, wherein the first exogenous nucleic acid, or analogue thereof, encodes both ARGFX, or a fragment or variant thereof, and TPRX1 and/or LEUTX, or a fragment or variant thereof.

66. A kit according to claim 60, wherein the first exogenous nucleic acid, or analogue thereof, encodes both ARGFX, or a fragment or variant thereof, and TPRX1 , or a fragment or variant thereof.

67. A method of detecting a teratocarcinoma or its metastases in a subject, the method comprising:

• wherein the presence of target molecules representative of expression of a protein selected from the group consisting of: ARGFX, NANOGNB and HESX1 is indicative of teratocarcinoma or its metastases.

68. A method of treating teratocarcinoma, or its metastases, in a subject, the method comprising:

69. A method according to claim 68, further comprising assaying a sample from the subject after treatment to determine the presence of target molecules representative of expression of a protein selected from the group consisting of: ARGFX, NANOGNB and HESX1.

70. A method according to any of claims 67 to 69, wherein expression of a gene selected from the group consisting of: ARGFX, NANOGNB, and HESX1 is detected directly by techniques that assay for RNA target molecules.

71. A method according to claim 70, wherein the technique is selected from the group consisting of: RT-PCR (reverse transcription PCR), qPCR (quantitative PCR), SAGE (Serial analysis of gene expression), direct RNA sequencing, Northern blot, RNAse protection, RNA sequencing (RNA-seq), in situ hybridisation, and RNA microarray.

72. A method according to any of claims 67 to 69, wherein expression of a gene selected from the group consisting of: ARGFX, NANOGNB, and HESX1 is detected directly by techniques that assay for the corresponding ARGFX, and/or NANOGNB, and/or HESX1 protein target molecules.

73. A method according to claim 72, wherein the technique is selected from the group consisting of: ELISA, radioimmunoassay, immunoprecipitation, immunohistochemistry, immunocytochemistry, Western blot and mass spectrometry.

74. A method according to any of claims 67 to 73, wherein the sample is a body fluid sample

75. A method according to claims 74, wherein the body fluid sample is selected from the group consisting of: blood (for example, a whole blood sample, a blood plasma sample, or a serum sample), saliva, seminal fluid, urine, and interstitial fluid.

76. A method according to any of claims 67 to 73, wherein the sample is a solid sample

77. A method according to claim 76, wherein the solid sample, is a biopsy or smear of a tumour.

78. An agent that targets ARGFX, and/or NANOGNB, and/or HESX1 for use in the treatment of a teratocarcinoma or its metastases.

79. The agent that targets ARGFX, and/or NANOGNB, and/or HESX1 for use according to claim 78, wherein the agent comprises a moiety which binds and/or facilitates the binding of the agent to ARGFX, and/or NANOGNB, and/or HESX1.

80. The agent that targets ARGFX, and/or NANOGNB, and/or HESX1 for use according to claims 78 to 79, wherein the agent is selected from the group consisting of an antibody, an antigen-binding fragment of an antibody, an aptamer, and a chimeric antigen receptor T- cell.

81. A method of treating teratocarcinoma or its metastases in a subject, the method comprising the step of providing an agent that targets ARGFX, and/or NANOGNB, and/or HESX1 to a subject in need thereof.

82. The method of claim 81 , wherein the agent comprises a moiety which binds and/or facilitates the binding of the agent to ARGFX, and/or NANOGNB, and/or HESX1.

83. The method of claims 81 to 82, wherein the agent is selected from the group consisting of an antibody, an antigen-binding fragment of an antibody, an aptamer, and a chimeric antigen receptor T- cell.