ES2954867B2 - NUCLEIC ACID AND ITS USE IN THE TREATMENT OF EWING'S SARCOMA - Google Patents

Description

DESCRIPTION

Nucleic acid and its use in the treatment of Ewinq sarcoma

The present invention relates to an isolated nucleotide sequence capable of being specifically expressed in Ewing's sarcoma cells. Said nucleotide sequence is comprised of a regulatory sequence and a sequence encoding a therapeutic protein capable of inactivating the EWSR1-FLI1 oncoprotein or inducing apoptosis of tumor cells or preventing their uncontrolled proliferation, thereby providing a new tool for the treatment of Ewing's sarcoma.

BACKGROUND OF THE INVENTION

Ewing's sarcoma is a highly aggressive tumor that affects children and adolescents. Most tumors are located in long, tubular bones such as the tibia or femur, although 10% of tumors can also develop in soft tissues. Histologically, Ewing's sarcoma is composed of small cells with little cytoplasm and no signs of differentiation, so differential diagnosis with other similar-appearing entities is somewhat complex.

The main molecular characteristic of Ewing sarcoma is the presence of chromosomal translocations specific to this tumor. The chromosomal translocation t(11:22)(q24:q12) is the most common, present in 85% of cases. In this translocation, the EWSR1 gene (Ewing sarcoma breakpoint region 1) is fused to the FLI1 (Friend leukemia integration 1) transcription factor, whose expression gives rise to the EWSR1-FLI fusion protein specific to Ewing sarcoma cells. Although this is the most common, fusions between the EWSR1 gene and other members of the ETS family, such as ERG, ETV1, ETV4, or FEV, can also occur, and are detected in the remaining 15% of Ewing sarcomas. These fusions give rise to chimeric proteins that act as new aberrant transcription factors that govern the expression of genes involved in the regulation of cell proliferation, producing uncontrolled growth that gives rise to tumors.

Currently, conventional treatment consists of three phases: (i) an induction phase, which consists of administering cycles of chemotherapy combining 3-4 drugs; (ii) surgery to remove residual tumors and/or radiotherapy when total removal is not possible; and (iii) a consolidation phase, consisting of a new round of chemotherapy. Despite these aggressive treatments, a significant proportion of patients die from the disease, as a result of refractory tumors that do not respond to treatment or the appearance of late recurrences, the vast majority of which are fatal. All of this indicates that current treatments have reached their limit, and consequently, the development of new, more effective and specific therapies based on the molecular biology of these tumors is essential.

In view of the above, there is a need in the state of the art to provide new alternative treatments to existing ones for Ewing's sarcoma that do not present the disadvantages of chemotherapy and that allow for a response to patients for whom current treatments are ineffective.

DESCRIPTION OF THE INVENTION

The present invention relates to a new therapeutic tool in the treatment of Ewing's sarcoma, which comprises specifically expressing in Ewing's sarcoma cells a protein that is capable of inactivating the EWSR1-FLI1 oncoprotein, or causing the death of said cells or preventing said cells from proliferating and giving rise to a tumor.

The new tool developed by the inventors is based on the presence of polymorphic regions rich in GGAA repeats in Ewing's sarcoma cells. These regions act as expression regulatory sequences and are affinity-bound by the chimeric protein EWSR1-FLI1 and the rest of the fusions characteristic of these tumors, binding to them and acting as transcriptional factors. Thus, the inventors have designed a genetic construct comprising said expression regulatory sequence rich in GGAA sequences to specifically direct the expression of a protein of interest in Ewing's sarcoma cells. In other words, the protein of interest is expressed exclusively in Ewing's sarcoma cells that express EWSR1-FLI1 (or any of the fusions characteristic of these tumors), but not in cells that express the native FLI1 gene or in cells that do not express FLI1.

Thus, if the protein of interest is toxic to the cell, or has the ability to prevent the expression of, or block, the chimeric protein EWSR1-FLI1 (or any of the equivalent fusions), the progression of Ewing's sarcoma would be prevented, thereby promoting treatment of said disease. A schematic of the invention is shown in Figure 1.

The inventors practiced the above-described invention by using a nuclease as the protein of interest and specifically expressing said nuclease together with a guide RNA (gRNA) directed against exon 2 or exon 9 of the FLI1 gene in Ewing's sarcoma cells (see Examples 1 and 2). In this way, genetic inactivation of EWSR1-FLI1 was achieved, and the proliferation of Ewing's sarcoma cells was reduced (see Example 3 and Figure 5).

In summary, this invention is directed to a tool for specifically expressing proteins with therapeutic potential in Ewing's sarcoma cells. The strategy described in this invention can be used to express a nuclease that, in combination with a guide RNA, induces the genetic inactivation of EWSR1-FLI1, to express a protein toxic to Ewing's sarcoma cells, or to express an immunomodulatory protein. Ultimately, based on this invention, it is possible to develop an effective therapeutic approach for the treatment of Ewing's sarcoma. This approach could be applied to all patients, but particularly to those refractory to treatment (approximately 25% of cases) and for whom there are currently no effective therapeutic alternatives.

In view of the above, in one aspect the present invention relates to an isolated nucleotide sequence, hereinafter “nucleotide sequence of the invention”, comprising

(i) a gene expression regulatory sequence wherein said regulatory sequence is specific to Ewing sarcoma cells and comprises n repeats of the sequence GGAA or (its complement) TTCC, where n is an integer equal to or greater than 1; and

(ii) a nucleotide sequence encoding a protein of interest,

where sequence (i) regulates the expression of sequence (ii).

The sequences (i) and (ii) indicated above are operably linked.

In the present invention, a nucleotide sequence is understood to be isolated when said nucleotide sequence is not in the presence of, or associated with, other sequences that accompany it in its natural environment.

In the present invention, "gene expression regulatory sequence" refers to the nucleotide sequences responsible for directing the transcription of a gene (or genes) so that it is expressed. In general, transcription regulatory sequences include, but are not limited to, promoter sequences, transcription initiation and termination sequences, and enhancer or activator sequences (also called enhancers). Therefore, in the context of the present invention, the gene expression regulatory sequence is heterologous to the sequence being regulated (the nucleotide sequence encoding the protein of interest). In the present invention, two sequences are understood to be "heterologous" with respect to each other when both sequences are not found in the same relationship with each other in nature. Examples of this situation include, but are not limited to, when the two sequences originate from different organisms, or when the regulated sequence (the gene) is not the sequence naturally regulated by the regulatory sequence (for example, the promoter).

The gene expression regulatory sequence of the present invention is a nucleotide sequence that is specifically recognized by the chimeric proteins EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-ETV4 and/or EWSR1-FEV and therefore will be active only in cells that express these chimeric proteins resulting from the chromosomal translocation characteristic of Ewing's sarcoma. In addition, said gene expression regulatory sequence specific to Ewing's sarcoma cells comprises n repetitions of the sequence GGAA or TTCC, where n is an integer equal to or greater than 1. The presence of these GGAA or TTCC repeats in the regulatory sequence causes them to be specifically recognized by the aforementioned chimeric proteins, and when these act as transcription factors, they trigger the transcription of the gene that is downstream of the regulatory sequence. The number of repetitions (n) of the GGAA or TTCC sequence can vary greatly, from 1 or 2 repetitions to more than 1,000. Thus, in a particular embodiment of the nucleotide sequence of the invention, n is an integer between 2 and 1,000, including both extremes.

Likewise, it should be noted that degenerate GGAA or TTCC sequences are also specifically recognized by the aforementioned chimeric proteins, so the expression regulatory sequence may be characterized by n repetitions of degenerate GGAA or TTCC sequences. Thus, in another particular embodiment of the nucleotide sequence of the invention, alone or in combinations with previous particular embodiments, the GGAA or TTCC sequence is a degenerate GGAA or TTCC sequence, preferably, the degenerate GGAA sequence is the sequence GGGA, GGAG, GAGG, AGGG, GAAA, AGAA, AAGA or AAAG and the degenerate TTCC sequence is the sequence CCCT, CCTC, CTCC, TCCC, CTTT, TCTT, TTCT or TTTC.

In another preferred embodiment, the gene expression regulatory sequence described in (i) comprises, more preferably consists of, SEQ ID NO: 1.

The expression regulatory sequence of the nucleotide sequence of the invention may comprise, in addition to the repetition of the GGAA sequence (or degenerate GGAA sequences) or TTCC (or degenerate TTCC sequences), a promoter, an enhancer, and/or other regulatory sequence(s). Thus, in another particular embodiment of the nucleotide sequence of the invention, the gene expression regulatory sequence further comprises:

(i) a promoter sequence of a gene positively regulated in Ewing sarcoma cells by the chimeric protein EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-ETV4 or EWSR1-FEV, or a fragment of said promoter,

(ii) an expression enhancer sequence, or enhancer, of a gene upregulated in Ewing sarcoma cells by the chimeric protein EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-ETV4 or EWSR1-FEV, or a fragment of said expression enhancer sequence, and/or

(iii) a regulatory sequence, such as a TATA sequence, or a sequence specific to other transcription factors.

The GGAA or TTCC sequence referred to in the present invention may be, but is not limited to:

(i) a sequence of GGAA or TTCC repeats present in the human genome or in any other genome of eukaryotic or prokaryotic cells, with or without relation to the expression of a certain gene, and/or

(ii) a sequence of concatenated GGAA or TTCC repeats not present in nature, but specifically designed to respond to the transcription factors EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-ETV4 or EWSR1-FEV.

In the present invention, the term "promoter" or "promoter sequence" refers to a nucleotide sequence located downstream of a gene and involved in the recognition and binding of RNA polymerase and other proteins responsible for downstream transcription of a gene. In general, the promoter will be suitable for the host cell in which the gene of interest is expressed, which, in this invention, is a nucleotide sequence encoding a protein of interest. In the present invention, a fragment of said promoter is understood to be a "functionally equivalent" fragment of the promoter of a gene upregulated in Ewing's sarcoma cells by the chimeric protein EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-ETV4 or EWSR1-FEV, when said fragment, although not having a nucleotide sequence identical to the promoter sequence, retains the same function as the promoter with the complete nucleotide sequence.

There are a large number of genes positively regulated in Ewing's sarcoma cells by the chimeric proteins resulting from chromosomal translocations in Ewing's sarcoma cells, in particular, by the chimeric proteins EWSR1-FLI1 or EWSR1-ERG, and any of their promoter sequences can be used in the context of the present invention. Examples of genes regulated by the chimeric proteins, in particular, by the EWSR1-FLI1 or EWSR1-ERG proteins, include, but are not limited to, NR0B1, FEZF1, NKX2.2, SOX6, FATE1, PAX7 or EGR2. However, in a particular embodiment of the nucleotide sequence of the invention, the promoter sequence is the promoter of the NR0B1 gene, or a functionally equivalent fragment of said promoter.

The NR0B1 gene (nuclear receptor subfamily 0 group B member 1), also known as AHC; AHX; DSS; GTD; HHG; AHCH; DAX1; DAX-1; NROB1; SRXY2 (NCBI; Gene ID: 190, updated January 25, 2022), has a nucleotide sequence (NCBIReference Sequence: NM_000475.5) that encodes a protein (NCBIReference Sequence: NP_000466.2) comprising a DNA-binding domain and acting as a dominant-negative transcription regulator mediated by the retinoic acid receptor.

Alternatively, or in combination with the promoter sequence, the gene expression regulatory sequence of the invention may comprise an enhancer sequence of a gene upregulated in Ewing's sarcoma cells by the chimeric proteins resulting from chromosomal translocations in Ewing's sarcoma cells, in particular, by the chimeric proteins EWSR1-FLI1 or EWSR1-ERG, and any of its enhancer sequences may be used in the context of the present invention. Examples of such enhancer sequences include, but are not limited to, PRC1, PPP1R1A. Functionally equivalent fragments of such enhancer sequences may also be used in the context of the present invention. The meaning of the term “functionally equivalent fragment” has been defined in previous paragraphs.

Another element of the nucleotide sequence of the invention is a nucleotide sequence that encodes a protein of interest. In the present invention, "protein of interest" is understood to be that protein that is to be specifically expressed in Ewing's sarcoma cells and that, preferably, when present in the cell, exerts some therapeutic effect on Ewing's sarcoma. Thus, therapeutic proteins are considered to be those proteins that, for example, (i) induce apoptosis of the cell in which they are expressed, (ii) prevent the expression of the chimeric protein, (iii) introduce mutations in the chimeric protein that prevent it from being functional, (iv) cut DNA in a sequence-specific manner, such as RNA-induced nucleases and artificial nucleases; (v) induce an immune response directed against Ewing's sarcoma cells; etc. However, as understood by those skilled in the art, it is also possible to use fluorescent proteins (GFP, mCherry and the like) as proteins of interest; luciferase proteins and the like, etc. In a particular embodiment of the nucleotide sequence of the invention, alone or in combination with the particular embodiments above, the protein of interest is a nuclease or a protein toxic to cells.

In the context of the present invention, "nuclease" means a hydrolase enzyme (EC 3.1.4) that catalyzes the cleavage of phosphodiester bonds, such as those established in nucleic acids between the pentose group of one nucleotide and the phosphate group of another. Nucleases are also known as phosphodiesterases (PDEs). Nucleases can be classified according to the type of nucleic acid they cleave (if the substrate is RNA, they are called ribonucleases, and if it is DNA, they are known as deoxyribonucleases), or according to the action of the enzyme (exonuclease if it starts at the ends of the nucleic acid chain, or endonuclease if the cleavage occurs at an internal point in the chain). Examples of nucleases that may be used in the context of the present invention include, but are not limited to, engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR/Cas system nucleases with an engineered crRNA/tracrRNA ('single-stranded guide RNA', 'guide sequence', 'guide RNA' or 'gRNA') to guide targeted cleavage. In addition, targeted nucleases based on the Argonaute system (e.g., from T. thermophilus, known as 'TtAgo') are being developed, which may also have potential uses in genome editing and gene therapy.

Thus, in a particular embodiment of the nucleotide sequence of the invention, alone or in combination with the rest of the particular embodiments described above, the nuclease is an RNA-induced nuclease or an artificial nuclease.

RNA-induced nucleases are enzymes that cut DNA guided by a short RNA sequence that recognizes a target sequence. This guide sequence (referred to as guide RNA, gRNA) recruits nuclease containing DNA cutting domains (nuclease domains) to induce a sequence-specific DNA cut. Examples of the RNA-induced nuclease family are CRISPR-Cas proteins, which include, but are not limited to, Cas9, Cpf1 (also known as Cas12a), C2C1 (also known as Cas12b), C2C2 (also known as Cas13a), CasX, CasY, Cas, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Casio, and the like. The RNA-induced nuclease may be a CRISPR-Cas family homolog as described above or the like, but may also be a modified CRISPR-Cas family protein. For example, it may be a modified nuclease in which one (nickase-modified nuclease) or both (dCas9) of the nuclease domains have been inactivated by mutation, or in which an enzyme or the like has been fused to Cas9 such as, but not limited to, the nucleases used in base-editing and prime-editing. Nucleases in which the specificity has been improved by genetic manipulation such as Cas9-HF, HiFi-Cas9, or eCas9 may also be used. Thus, in another particular embodiment of the nucleotide sequence of the invention, alone or in combination with the previous particular embodiments, the nuclease is a Cas family nuclease which, in another even more particular embodiment, said Cas family nuclease is Cas9, Cas12 or Cas13. In the case of the Cas9 nuclease, it may include those derived from Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Geobacillus stearothermophilus and the like. The amino acid sequence of the Cas9 derived from Streptococcus pyogenes in this embodiment is shown in SEQ ID NO: 12 (hereinafter sometimes referred to as SpCas9). In a preferred embodiment, the nucleotide sequence that codes for the protein of interest, as described in the present invention, is SEQ ID NO: 3. In the case of the Cpf1 nuclease, this may include those derived from Acidaminococcus, Lachnospira, Chlamydomonas, Francisella novicida and the like.

In another particular embodiment of the nucleotide sequence of the invention, the nuclease is an artificial nuclease. In the context of the present invention, an artificial nuclease is an artificial restriction enzyme that contains a DNA binding domain specifically designed to recognize a specific target DNA sequence fused to a nuclease domain from, for example, a restriction enzyme. There are several types of artificial nucleases, including, but not limited to, Zinc Finger nucleases (ZFN), Transcription activator-like effector nuclease (TALEN), meganucleases, and the like. Thus, in a particular embodiment of the nucleotide sequence of the invention, alone or in combination with the previous embodiments, the nuclease is a nuclease from the TALEN or ZNF family.

Alternatively to the nuclease, the protein of interest of the nucleotide sequence of the invention may be a protein that is toxic to cells.

In a particular embodiment of the nucleotide sequence of the invention, alone or in combination with the previous particular embodiments, the protein toxic to the cell is the caspase 8 protein, the caspase 9 protein, the thymidine kinase protein, the cytosine deaminase protein, the diphtheria toxin protein or any functionally equivalent variant of said proteins.

Within the context of the present invention, functionally equivalent variants of the caspase 8 protein, the caspase 9 protein, the thymidine kinase protein, the cytosine deaminase protein and the diphtheria toxin protein are also included. A “functionally equivalent variant” of a protein “x” is understood to be that protein that (i) comprises a sequence in which one or more amino acids are substituted by a conserved or non-conserved amino acid residue (preferably a conserved amino acid), or (ii) comprises a sequence comprising an insertion or a deletion of one or more amino acids, and that has the same function as protein “x”, that is, it is toxic to cells. Assays on how to check whether a protein is toxic to cells, and therefore equivalent to the proteins mentioned in the particular embodiment above, are widely known in the state of the art.

As understood by those skilled in the art, the nucleotide sequence of the invention can be incorporated into a vector. Thus, in another aspect, the present invention relates to a vector comprising the nucleotide sequence of the invention, hereinafter "vector of the invention."

A "vector" is capable of transferring gene sequences into target cells. Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and capable of transferring gene sequences into target cells. Thus, the term includes gene cloning and transfer, as well as the integration of vectors or genes.

The vector of the invention is a biologically functional vector or plasmid, such as a cloning vector or an expression vector. Any vector can be used as long as it is replicable and viable in the cells into which it is introduced.

In the present invention, "expression vector" refers to a gene construct used to introduce and express a specific gene (which in this invention encodes a protein of interest) into a cell of interest (in this invention, an Ewing sarcoma cell). The vector may be a plasmid. A plasmid is an extrachromosomal DNA molecule, separate from the cell's chromosomal DNA and replicating independently of it. Plasmids are generally circular and have two strands of nucleic acid. Alternatively, the plasmid may be designed so that its contents are inserted into genomic DNA by genetic recombination. Many suitable vectors are known to those skilled in the art, and these are commercially available. In general, the nucleotide sequence of the invention is inserted into the vector at an appropriate restriction endonuclease site or sites by standard procedures. These procedures and related subcloning procedures are considered part of the knowledge of those skilled in the art. Examples of vectors that can be used in the context of the present invention include, but are not limited to, viral and non-viral vectors. Illustrative, non-limiting examples of viral vectors that may include the nucleotide sequence or gene construct of the invention include, for example, a lentiviral vector, an adenoviral vector, an adeno-associated vector, a herpes simplex virus, or a retrovirus. Thus, in another particular embodiment of the vector of the invention, the vector is a lentiviral vector, an adenoviral vector, an adeno-associated vector, a herpes simplex virus, or a retrovirus, or a non-viral expression vector. Non-viral vectors that may be cited include bacterial vectors, of which there are numerous examples, including any plasmid (expression or not) described for bacteria (E. coli, Bacillus sp., etc.) or modifications thereof, etc. Additionally, said vectors can be derived from commercial expression vectors, such as those marketed by Promega (e.g., PinPointXa plasmids, etc.), Invitrogen (e.g., Gateway system plasmids, etc.), Amersham (e.g., pGEX plasmids, etc.), Ingenius (e.g., pINK vector, etc.), Sigma (e.g., BICEP vectors, etc.), etc. Alternatively, the vector of the invention can be obtained by conventional methods known to those skilled in the art described.

Other elements that the vector of the invention may comprise are, for example, origins of replication, restriction enzyme recognition sequences and marker genes that allow the identification of cells that have incorporated the vector comprising the nucleotide sequence of the invention.

A "selectable marker," "selectable marker gene," or "reporter gene" includes any gene that confers a phenotype to a cell in which it is expressed to facilitate the identification and/or selection of cells to be transfected or transformed with a vector or nucleic acid construct as described herein. Suitable markers may be selected from markers that confer antibiotic resistance, that introduce a novel metabolic trait, or that allow for visual selection. Examples of selectable marker genes include genes that confer antibiotic resistance (such as nptll which phosphorylates neomycin and kanamycin, or hpt which phosphorylates hygromycin, or genes that confer resistance to, for example, bleomycin, streptomycin, tetracycline, chloramphenicol, ampicillin, gentamicin, geneticin (G418), spectinomycin, or blasticidin), or genes that provide a metabolic trait. The expression of visual marker genes results in the formation of color (e.g. 13-glucuronidase, GUS or 13-galactosidase with their colored substrates, e.g. X-Gal), luminescence (such as the luciferin/lucephalase system) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). This list only represents a small number of possible markers. The skilled person is familiar with such markers. Depending on the organism and the selection procedure, different markers are preferred. If desired, the genetic markers can be removed from the host cell at a later stage.

Once the vector of the invention has been defined, it can be introduced into the host cell. The vector of the invention can be used to transform, transfect, or infect cells that can be transformed, transfected, or infected by said vector. Thus, in another aspect, the present invention relates to a cell comprising the nucleotide sequence of the invention or the vector of the invention, hereinafter "cell of the invention."

Methods for introducing gene constructs or vectors into a cell are widely known to those skilled in the art and their use is standard practice in the laboratory. The term "introduce" in the context of the present invention refers to the incorporation of a nucleotide sequence into a cell by "transfection" or "transformation" or "transduction", where the nucleotide sequence can be incorporated into the genome of the cell (e.g., chromosome, plasmid, mitochondrial DNA), or be an autonomous replicon, or transiently expressed (e.g., transfected mRNA). As used herein, the terms "transformed", "stably transformed" or "transgenic", with reference to a cell, mean that the cell has a non-native (heterologous) nucleotide sequence integrated into its genome, or in an episomal plasmid that is maintained through multiple generations, or transiently expressed.

Examples of methods for introducing vectors into host cells include, but are not limited to, electroporation; nuclear microinjection or direct microinjection into single cells; fusion of bacterial protoplasts with intact cells; use of polycations, e.g., polybrene or polyornithine; membrane fusion with liposomes, lipofectamine-mediated transfection, or lipofection; high-speed bombardment with DNA-coated microprojectiles; incubation with calcium phosphate DNA precipitation; DEAE-dextran-mediated transfection; infection with modified viral nucleic acids; Agrobacterium-mediated DNA transfer, and the like.

Methods for administering the nucleotide sequence of the invention or the vector of the invention to a subject are widely known in the art, such as vesicles, nanoparticles, etc. Thus, in another aspect, the present invention relates to a nanoparticle, an extracellular vesicle or a virus-like particle (VLP) comprising the nucleotide sequence of the invention or the vector of the invention. Hereinafter referred to as "nanoparticle, extracellular vesicle or VLP of the invention".

In general, a “nanoparticle” refers to any particle having a diameter of less than 1,000 nm, e.g., from 20 nm to 500 nm, from 30 nm to 300 nm, from 50 nm to 150 nm. The described nanoparticles may include nanoparticles having a diameter of 60 to 200 nm, or 70 to 150 nm, or 80 to 130 nm.

As understood by those skilled in the art, both the nucleotide sequence and the vector, cell, nanoparticle, extracellular vesicle, or VLP of the invention may be included within a composition. Therefore, in another aspect, the present invention relates to a composition, hereinafter "composition of the invention," comprising the nucleotide sequence of the invention, the vector of the invention, the cell of the invention, and/or a nanoparticle, extracellular vesicle, or VLP of the invention.

The composition of the invention can be used in the treatment of Ewing's sarcoma, so it must be formulated for administration to an individual. Thus, in a particular embodiment, the composition of the invention is a pharmaceutical composition.

As used herein, the term “pharmaceutical composition” refers to a formulation adapted to administer a predetermined dose of the compound of the invention to a cell, group of cells, organ, tissue, or subject. Furthermore, the elements comprising the pharmaceutical composition, i.e., the nucleotide sequence, vector, cell, nanoparticle, extracellular vesicle, and/or VLP of the invention, may be in a therapeutically effective amount. The term “therapeutically effective amount,” as used herein, is understood to mean an amount capable of providing a therapeutic effect, and which can be determined by the person skilled in the art by commonly used means. The amount of the compound of the invention that can be included in the pharmaceutical compositions according to the invention will vary depending on the subject and the particular mode of administration. The appropriate dosage of the active ingredient(s) within the pharmaceutical composition will depend on the type of disease to be treated, the severity and course of the disease, whether the composition is being administered for preventative or therapeutic reasons, prior therapy, the patient's medical history and response to the composition, and the physician's judgment. The amount of the compound of the invention is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an appropriate dosage level will generally be about 1x10 to 1x10 viral particles or nanoparticles, extracellular vesicles, or VLPs of the invention. The compounds may be administered on a regimen of 1 to several times per day or for two, three, four, five, six, or seven days, preferably once every other day. The pharmaceutical composition may be administered for at least 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, or more days, preferably for 14 days. The pharmaceutical composition may preferably be administered once every two days for 14 days.

The composition or pharmaceutical composition of the invention may also contain one or more carriers and/or excipients. Thus, in another particular embodiment, the composition of the invention also comprises a carrier and/or excipient, which, in another more particular embodiment, is a pharmaceutically acceptable carrier or excipient.

Carriers and excipients used in the preparation of compositions are widely known in the art, and their use is routine practice for those skilled in the art. In a particular embodiment, the carrier or excipient is pharmaceutically acceptable. The term "pharmaceutically acceptable excipient" refers to a therapeutically inactive substance used to incorporate the active ingredient and is acceptable to the patient from a pharmacological/toxicological standpoint and to the pharmaceutical chemist who produces it from a physical/chemical standpoint with respect to composition, formulation, stability, patient acceptance, and bioavailability. The excipient or carrier also includes any substance that serves to improve the administration and effectiveness of the active ingredient within the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include one or more of water, saline solution, phosphate buffer solution, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may also comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which improve the shelf life or effectiveness of the compositions that form part of the pharmaceutical compositions. Examples of suitable carriers are well known in the literature. Examples of carriers, without limitation, include a number of saccharides such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; a number of starches such as corn starch, wheat starch, rice starch, and potato starch; a number of celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; and a number of fillers such as gelatin and polyvinylpyrrolidone. In some cases, a disintegrant such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added.

The composition of the invention may further comprise compounds useful in the treatment of cancer, in particular, in the treatment of Ewing's sarcoma, to complement and/or increase the efficacy of the treatment. Therefore, in another particular embodiment, alone or in combination with the rest of the particular embodiments, the composition of the invention further comprises a chemotherapeutic agent. Examples of chemotherapeutic agents useful in the treatment of Ewing's sarcoma include, but are not limited to, vincristine, doxorubicin, cyclophosphamide, ifosfamide, etoposide, irinotecan, temozolamide, actinomycin D, gencitabine, docetaxel or topotecan.

The pharmaceutical compositions of the invention may be administered by any suitable route, such as oral, nasal, ocular, topical, intradermal, intracranial, intravenous/parenteral, or intratumoral administration. In another particular embodiment of the composition of the invention, alone or in combination with the preceding particular embodiments, the composition is formulated for oral, parenteral, or intratumoral administration.

"Oral" administration refers to the pharmaceutical composition being ingested into the body after swallowing. "Intravenous" or "parenteral" administration refers to the administration of the pharmaceutical composition by injection into the bloodstream. "Intratumoral" administration refers to the administration of the pharmaceutical composition by injection directly into or near the tumor.

In another aspect, the present invention relates to a kit comprising the nucleotide sequence of the invention, the vector of the invention, the cell of the invention, the nanoparticle, the extracellular vesicle or the VLP of the invention, and/or the composition of the invention.

As explained at the beginning of this description, the invention relates to a tool for specifically expressing proteins with therapeutic potential in Ewing's sarcoma cells. Thus, in another aspect, the present invention relates to the nucleotide sequence of the invention, the vector of the invention, the cell of the invention, the nanoparticle, the extracellular vesicle or the VLP of the invention, the composition of the invention, and/or the kit of the invention for use as a medicament.

Thus, in another aspect, the present invention relates to the nucleotide sequence of the invention, the vector of the invention, the cell of the invention, the nanoparticle, the extracellular vesicle or the VLP of the invention, the composition of the invention, and/or the kit of the invention for use in the treatment of Ewing's sarcoma.

In the present invention, "therapy" or "treatment" refers to the administration to an individual of a compound, or combination of compounds, with the goal of inhibiting a disease or pathological condition, i.e., halting its development; alleviating a disease or pathological condition, i.e., causing the regression of the disease or pathological condition; and/or stabilizing a disease or pathological condition in an individual. In the present invention, the disease or pathological condition is Ewing's sarcoma.

In the present invention, the term "individual" is equivalent to the term "subject," so both terms may be used interchangeably throughout this description. The term "individual" refers to any animal belonging to any species. However, in a particular embodiment, the subject is a mammal, preferably a primate, more preferably a human of any race, sex, or age. The individual object of the present invention suffers from Ewing's sarcoma. The disease Ewing's sarcoma has been described at the beginning of this description.

DESCRIPTION OF THE FIGURES

Figure 1 is a graphic representation of the invention where the isolated nucleotide sequence object of this invention is represented, which comprises a regulatory sequence formed by n GGAA repeats and a nucleotide sequence that codes for a protein of interest that can be a nuclease (e.g. Cas9), a protein toxic to cells (T Protein, e.g. Casp9) or an immunomodulatory protein (Protein I that induces an immune response). This isolated nucleotide sequence can be transferred to the target cells (Ewing's sarcoma cells) by different types of viral and non-viral vectors. In a particular embodiment of the invention, the protein of interest is a Cas9 nuclease that is specifically expressed in Ewing's sarcoma cells thanks to the specificity conferred by the regulatory sequence, which allows the Cas9 protein of interest to be expressed only in Ewing's sarcoma cells that express the EWSR1-FLI1 chimeric protein (or some other of the equivalent chimeric proteins). In the presence of a guide RNA directed against EWSR1-FLI1, the EWSR1-FLI1 DNA is cut and inactivated, which ultimately causes the tumor cells to stop proliferating. In normal cells, there is no expression of Cas9 and therefore no cuts are made on the DNA.

Figure 2 depicts the experiments performed to demonstrate the ability of the GGAA repeat-rich regulatory sequence to regulate the expression of the protein of interest, in this case Cas9. Ewing sarcoma cells A673 (A) and MHH-ES1 (B) were infected with lentiviruses containing the promGGAA-Cas9 sequence and the promGGAA-less-Cas9 sequence (without GGAA repeats) and Cas9 levels were analyzed by western blot. EWSR1-FLI1 protein expression and tubulin expression were used as loading controls. The A673 cell line, which was genetically modified to constitutively express Cas9, was used as a positive control for Cas9 expression. The Cas9 protein could only be detected in cells infected with promGGAA-Cas9 but not with promGGAA-less-Cas9, demonstrating that the promGGAA regulatory sequence is capable of regulating Cas9 expression in Ewing sarcoma cells.

Figure 3 shows experiments performed to demonstrate that the regulatory sequence is not active in another type of sarcoma (fibrosarcoma) other than Ewing sarcoma despite expressing native FLI1. HT1080 fibrosarcoma cells were infected with lentiviruses containing the promGGAA-Cas9 sequence and the promGGAA-less-Cas9 sequence (without GGAA repeats), and Cas9 levels were analyzed by western blot. FLI1 protein expression and tubulin expression were used as loading controls. The A673 cell line, which was genetically modified to constitutively express Cas9, was used as a positive control for Cas9 expression. The Cas9 protein could not be detected in HT1080 cells infected with either promGGAA-Cas9 or promGGAA-less-Cas9, demonstrating that the promGGAA regulatory sequence is not active in cells such as HT-1080, even though they express high levels of the native FLI1 protein.

Figure 4 shows the effect of Cas9 expression and a guide RNA directed against FLI1 exon 2 in A673 cells. A673 Ewing sarcoma cells were co-infected with the promGGAA-Cas9 and promGGAA-less-Cas9 lentiviral vectors and lentiviral vectors expressing a guide RNA (sgRNAs) designed to produce cuts in the DNA encoding FLI1 exon 2. When A673 cells were co-infected with the promGGAA-Cas9 lentiviral vectors and sgRNA exon 2, a gene editing percentage of around 50% was observed, which remained stable for approximately 40 days of culture. However, when cells were co-infected with the promGGAA-less-Cas9 and sgRNA exon 2 lentiviral vectors, the level of gene editing detected was much lower than that observed previously and in any case remained stable throughout the experiment at levels close to the detection limit of the technique.

Figure 5 shows the therapeutic potential of targeted Cas9 expression in Ewing sarcoma cells. A673 Ewing sarcoma cells were co-infected with the promGGAA-Cas9 and promGGAA-less-Cas9 lentiviral vectors and guide RNAs targeting FLI1 exon 2 and FLI1 exon 9, and cell doubling times (measured in hours) were quantified. (A) Results obtained in cells infected with the promGGAA-less-Cas9 lentiviral vector or the promGGAA-Cas9 lentiviral vector and guide RNA targeting FLI1 exon 2 are shown. In this case, no differences in the doubling time of A673 cells were detected. In (B) the results obtained in cells infected with the promGGAA-less-Cas9 lentiviral vector or the promGGAA-Cas9 lentiviral vector and the guide RNA directed against FLI1 exon 9 are shown. In this case, it was observed that the doubling time in cells infected with the FLI1 exon 9 gRNA and the promGGAA-Cas9 lentiviral vector was doubled compared to the doubling time observed in cells infected with the FLI1 exon 9 gRNA and the promGGAA-less-Cas9 lentiviral vector.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

I - MATERIAL AND METHODS

Cell culture

Two Ewing sarcoma lines, A673 and MHH-ES1, which express the chimeric protein EWSR1-FLI1 were used, with the difference that A673 cells express the type I fusion protein and MHH-ES1 cells express the type II fusion protein. A fibrosarcoma line, HT1080, which does not express the chimeric protein EWSR1-FLI1 but does express native FLI1, was also used. A673 and HT1080 cells were cultured in DMEM with 10% fetal bovine serum (FBS) supplemented with penicillin and streptomycin. MHH-ES1 cells were cultured in RPMI with 10% FBS supplemented with penicillin and streptomycin. For selection media after infection with lentiviral vectors, Blasticidin (5 pg/mL) was added for selection of cells infected with the Cas9 promoter-linked lentiviral vector and Hygromycin B Gold (350 pg/mL) was added to select for cells infected with the RNA guide lentiviral vector.

Preparation of lentiviral vectors with the GGAA promoter linked to Cas9To generate lentiviral vectors with the GGAA promoter (promGGAA-Cas9, SEQ ID NO: 1) linked to Cas9, the services of a specialized company (VectorBuilder, Chicago, USA) were used. The GGAA repeat sequence present in the promoter of the NR0B1 gene of Ewing sarcoma A4573 cells was cloned in front of the coding sequence for the hCas9 protein (SEQ ID NO: 3). In addition, another lentiviral vector was manufactured in which the GGAA sequences were eliminated from SEQ ID NO: 1 (promGGAA-less-Cas9, negative control) (SEQ ID NO: 2). The rest of the elements of the lentiviral vector were those considered standard for third-generation lentiviral vectors.

Lentivirus infection

The cells were co-infected with lentiviruses containing the different constructs of the aforementioned promoters, and in parallel with lentiviruses containing expression vectors for the guides (sgRNA) directed against exon 2 of FLI1 (not present in the EWSR1-FLI1 chimeric protein, used as an editing control that does not affect EWSR1-FLI1) and against exon 9 of FLI1 (present in the EWSR1-FLI1 chimeric protein and therefore its editing affects the functionality of the EWSR1-FLI1 protein). For the generation of these lentiviral expression vectors of the guide RNAs, the services of a specialized company (VectorBuilder, Chicago, USA) were used. In these vectors, the expression of the guide RNA is under the control of a U6 promoter, being the rest of the elements of the lentiviral vector those considered standard for third generation vectors. An MOI of 5 was used for both promoter and sgRNA lentiviruses. At the indicated times, cell aliquots were collected for DNA and protein extraction.

Protein expression analysis

Cas9 expression in cells infected with the promGGAA-Cas9 and promGGAA-less-Cas9 vectors was analyzed by western blot. Cells were lysed in standard RIPA buffer, and protein concentration was quantified using the BOA method (Pierce). A total of 20 pg of protein extract was loaded onto a PAGE-SDS gel. Proteins were transferred to a PVDF membrane using the semi-dry transfer technique. Cell extracts from cells expressing Cas9 under the constitutive CMV promoter were used as a positive control for Cas9 (described in Cervera ST et al. 2021 doi:10.3390/cancers13153783). Membranes were incubated overnight with anti-Cas9 (Abcam #ab191468, 1:1,000 dilution), anti-FLI1 (Abcam #ab133485, 1:250 dilution) (recognizing EWSR1-FLI1 and native FLI1), and anti-tubulin (Abcam #185067, 1:10,000 dilution) antibodies as loading controls. Membranes were subsequently incubated with an HRP-linked secondary antibody and revealed using IMMOBILON chemiluminescent reagent (MERCK).

Gene editing analysis

To determine the levels of gene editing, cellular DNA was extracted at different times. This DNA was used as a template to amplify by PCR the genomic region of interest that included the junction regions of the guide RNAs of exon 2 and exon 9 of FLI1 and adjacent regions. The primers used in the PCR were the following: Forward Exon 9 FL1: 5’-TCTCTGGGCTGAGGTGTTCT-3’ (SEQ ID NO: 8); Reverse Exon 9 FLI1: 5’- ATTCATGTTGGGCTTGCTTT- 3’ (SEQ ID NO: 9); Forward Exon 2 FLI1: 5’- TGTCACTTGCTTGGGTGAAG-3’ (SEQ ID NO: 10); Reverse Exon 2 FLI1: 5’- GTTGACCCTCACTGGCTGAT-3’ (SEQ ID NO: 11). Each of the generated amplicons was sequenced using Sanger sequencing. The percentage of gene editing (indel generation) was determined by analyzing Sanger electrophoregrams using the SYNTHEGO application (Synthego Corporation, California, USA).

Cell proliferation analysis

To determine the effect of gene editing on cell proliferation, an experiment was performed to quantify cell doubling time. To do this, cells infected with the promGGAA-Cas9 and promGGAA-less-Cas9 lentiviral vectors and the FLI1 exon 2 and exon 9 guide RNAs were maintained in continuous growth for 45–50 days in 100-mm dishes. The number of cell doublings and the average doubling time were determined in each trypsinization cycle. Cells were seeded at a set density in 100-mm dishes, and when they reached 80–85% confluence, they were trypsinized, and the number of viable cells was counted using Trypan Blue solution and an automatic cell counter (NanoEntek). The number of doublings and the doubling time (t) were calculated using the following formulas:

n0 of duplications = log2 (n0 of cells (initial time) / n0 of cells (final time))

t duplication = t (between each cycle) / n0 of cell duplications (between each cycle) At the end of each cycle, an aliquot of the cells was stored for later extraction of DNA, RNA and proteins.

II - RESULTS

Example 1. Expression of a protein of therapeutic interest in a specific manner in Ewing sarcoma cells

The ability of the promGGAA sequence to specifically induce the expression of a protein of therapeutic interest in Ewing sarcoma cells was then analyzed. A673 and MHH-ES1 Ewing sarcoma cells were infected with the promGGAA-Cas9 and promGGAA-less-Cas9 lentiviral vectors, and Cas9 protein expression was determined by western blot. Figure 2 shows the results obtained in these experiments. Cas9 expression was clearly detected in both Ewing sarcoma cells when they were infected with the promGGAA-Cas9 vector, but not when the same cells were infected with the promGGAA-less-Cas9 vector. Both cells were infected with similar efficiency since the expression of the GFP protein present in the lentiviral vectors was similar in all cases, so the differences in expression in the Cas9 protein can only be attributed to the presence of GGAA repeats in the promoter.

Example 2. Specificity analysis of the expression of a protein of interest in cells other than those of Ewing sarcoma.

To demonstrate that the expression of a protein of interest under the regulation of promGGAA is exclusive to Ewing sarcoma cells, an experiment was performed in a cell line that does not express EWSR1-FLI1. HT1080 cells (derived from a fibrosarcoma) were chosen because these cells do not express EWSR1-FLI1, but do express native FLI1, and are therefore an ideal model to evaluate the specificity of promGGAA as a regulatory sequence. These cells were infected with the lentiviral vectors promGGAA-Cas9 and promGGAA-less-Cas9, and Cas9 protein expression was determined by western blot. Figure 3 shows the results obtained in these experiments. Cas9 expression could not be detected in cells infected with either the promGGAA-Cas9 or the promGGAA-less-Cas9 vector, demonstrating that the GGAA promoter is only capable of inducing the expression of a gene of interest in the case of Ewing sarcoma cells expressing the oncogenic protein EWSR1-FLI1. It is worth noting that HT-1080 cells express high levels of native FLI1 as demonstrated by western blot, despite which there was no induction of Cas9 expression. Both cells were infected with a similar efficiency as demonstrated by the expression of the GFP protein present in the lentiviral vectors, so the absence of Cas9 expression cannot be attributed to a deficient infection.

Example 3. Therapeutic potential of Cas9-specific expression in Ewing sarcoma cells.

To evaluate the therapeutic potential of Cas9-specific expression in Ewing sarcoma cells, A673 Ewing sarcoma cells were co-infected with the lentiviral vectors promGGAA-Cas9 and promGGAA-less-Cas9 and lentiviral vectors expressing a guide RNA (sgRNAs) designed to produce cuts in the DNA coding for FLI1 exon 2. We chose this guide RNA in FLI1 exon 2 for these experiments because gene editing in this exon does not affect EWSR1-FLI1 expression since FLI1 exon 2 is not included in the EWSR1-FLI1 fusion. In this way, it is possible to study the editing efficiency without compromising cell viability. The sequences corresponding to the gRNA and sgRNA of exon 2 are shown in SEQ ID NO: 4 and SEQ ID NO: 6, respectively. The results obtained in these experiments are shown in Figure 4. When A673 cells were coinfected with the promGGAA-Cas9 and sgRNA exon 2 lentiviral vectors, a gene editing percentage of around 50% was observed, which remained stable for approximately 40 days of culture. However, when the cells were coinfected with the promGGAA-less-Cas9 and gRNA exon 2 lentiviral vectors, the level of gene editing detected was much lower than previously observed and, in any case, remained stable throughout the experiment at levels close to the detection limit of the technique.

Next, and to demonstrate the therapeutic potential of Cas9-specific expression in Ewing sarcoma cells, the effect of gene editing on cell proliferation was analyzed. To this end, Ewing sarcoma cells were co-infected with the lentiviral vectors promGGAA-Cas9 and promGGAA-less-Cas9 and RNA guides directed against FLI1 exon 2 and FLI1 exon 9. The sequences corresponding to the gRNA and sgRNA of exon 9 are shown in SEQ ID NO: 5 and SEQ ID NO: 7, respectively. In this case, and unlike the guide directed to FLI1 exon 2, the guide directed to exon 9 causes gene inactivation of EWSR1-FLI1 since mutations occur in FLI1 exon 9, which is part of the EWSR1-FLI1 fusion and also encodes the DNA-binding domain. The results of these experiments are shown in Figure 5. When A673 cells were co-infected with FLI1 exon 2 gRNA and either the promGGAA-less-Cas9 lentiviral vector or the promGGAA-Cas9 lentiviral vector, no differences in A673 cell doubling time were detected. This result is consistent with the fact that gene editing in FLI1 exon 2 does not affect Ewing sarcoma cell proliferation, since although Cas9 expression is induced in A673 cells infected with the promGGAA-Cas9 lentiviral vector (as shown in Figure 2), and editing is observed in FLI1 exon 2 (as shown in Figure 4), this does not translate into a decrease in cell doubling rate. However, when A673 cells were infected with FLI1 exon 9 gRNA and either the promGGAA-less-Cas9 lentiviral vector or the promGGAA-Cas9 lentiviral vector, significant differences in doubling times were observed. Specifically, the doubling time in cells infected with FLI1 exon 9 gRNA and the promGGAA-Cas9 lentiviral vector was doubled compared to the doubling time observed in cells infected with FLI1 exon 9 gRNA and the promGGAA-less-Cas9 lentiviral vector. This means that there is no Cas9 expression in cells infected with FLI1 exon 9 gRNA and the promGGAA-less-Cas9 lentiviral vector (Figure 2) and therefore no significant editing. However, in cells infected with the FLI1 exon 9 gRNA and the promGGAA-Cas9 lentiviral vector, there is a high expression of Cas9 and consequently cuts can occur in FLI1 exon 9 that affect the integrity of the EWSR1-FLI1 fusion gene, consequently leading to a slowdown in cell proliferation.

Claims (21)

1. An isolated nucleotide sequence comprising (i) a gene expression regulatory sequence, wherein said regulatory sequence is specific to Ewing sarcoma cells and comprises n repeats of the sequence GGAA or TTCC, where n is an integer equal to or greater than 1; and (ii) a nucleotide sequence encoding a protein of interest, wherein sequence (i) regulates the expression of sequence (ii), for use in the treatment of Ewing's sarcoma. 2. Nucleotide sequence for use according to claim 1, wherein the gene expression regulatory sequence further comprises (i) a promoter sequence of a gene positively regulated in Ewing sarcoma cells by the chimeric protein EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-ETV4 or EWSR1-FEV, or a fragment of said promoter, (ii) an expression enhancer sequence of a gene upregulated in Ewing sarcoma cells by the chimeric protein EWSR1-FLI1, EWSR1-ERG, EWSR1-ETV1, EWSR1-ETV4 or EWSR1-FEV, or a fragment of such an expression enhancer sequence, or (iii) a TATA regulatory sequence. 3. Nucleotide sequence for use according to claim 2, wherein the promoter sequence is the promoter of the NR0B1 gene or a functionally equivalent fragment of said promoter. 4. Nucleotide sequence for use according to any one of claims 1 to 3, wherein the sequence GGAA or TTCC is a degenerate GGAA or TTCC sequence, preferably, the degenerate GGAA sequence is the sequence GGGA, GGAG, GAGG, AGGG, GAAA, AGAA, AAGA or AAAG and the degenerate TTCC sequence is the sequence CCCT, CCTC, CTCC, TCCC, CTTT, TCTT, TTCT or TTTC. 5. Nucleotide sequence for use according to any one of claims 1 to 4, wherein n is an integer from 2 to 1,000. 6. Nucleotide sequence for use according to any one of claims 1 to 5, wherein the protein of interest is a nuclease. 7. Nucleotide sequence for use according to claim 6, wherein the nuclease is a nuclease of the TALEN, ZNF or Cas family. 8. Nucleotide sequence for use according to claim 7, wherein the Cas family nuclease is Cas9, Cas12 or Cas13. 9. Nucleotide sequence for use according to any one of claims 1 to 6, wherein the protein of interest is the caspase 8 protein or the caspase 9 protein or any functionally equivalent variant of said protein. 10. Nucleotide sequence for use according to any one of claims 1 to 6, wherein the protein of interest is the thymidine kinase protein or any functionally equivalent variant of said protein. 11. Nucleotide sequence for use according to any one of claims 1 to 6, wherein the protein of interest is the cytosine deaminase protein, or any functionally equivalent variant of said protein. 12. Nucleotide sequence for use according to any one of claims 1 to 6, wherein the protein of interest is the diphtheria toxin protein, or any functionally equivalent variant of said proteins. 13. A vector comprising the nucleotide sequence according to any one of claims 1 to 12 for use in the treatment of Ewing's sarcoma. 14. A vector for use according to claim 13, wherein the vector is a lentiviral vector, an adenoviral vector, an adeno-associated vector, a retroviral vector, a herpes simplex vector or a non-viral expression vector. 15. A cell comprising the vector according to any of claims 13 or 14, for use in the treatment of Ewing's sarcoma. 16. A nanoparticle, an extracellular vesicle or a virus-like particle comprising a nucleotide sequence according to any one of claims 1 to 12, for use in the treatment of Ewing's sarcoma. 17. A composition comprising the nucleotide sequence according to any one of claims 1 to 12, the vector according to claim 13 or 14, the cell according to claim 15, and/or the nanoparticle, extracellular vesicle or virus-like particle according to claim 16, for use in the treatment of Ewing's sarcoma. 18. Composition for use according to claim 17, further comprising a pharmaceutically acceptable carrier or excipient. 19. Composition for use according to claim 17 or 18, further comprising a chemotherapeutic agent. 20. Composition for use according to any one of claims 17 to 19, wherein the composition is formulated for oral, parenteral or intratumoral administration. 21. A kit comprising the nucleotide sequence according to any one of claims 1 to 12, the vector according to claim 13 or 14, the cell according to claim 15, the nanoparticle, extracellular vesicle or virus-like particle according to claim 16, or the composition according to any one of claims 17 to 20, for use in the treatment of Ewing's sarcoma.