The present invention relates to a method for expanding activated adult stem cells or tumor stem cells and/or for maintaining an activated adult stem cell or tumor stem cell culture in vitro in a stem state and/or preventing its senescence and/or differentiation and/or for enriching a cell culture with activated adult stem cells or tumor cells, the method comprising stably providing a target stem cell or target stem cell culture with a molecule capable of chronically inhibiting the interaction of calcineurin (CN) with a substrate containing the PxIxIT motif, where said target stem cell is an adult stem cell or tumor stem cells. The invention also refers to cells obtainable by the method and uses thereof and to a molecule able to chronically inhibit the interaction of calcineurin (CN) with a substrate containing the PxIxIT motifs for use in the expansion of activated adult stem cells and / or in the inhibition of differentiation and / or of senescence of activated adult stem cells.

PRIOR ART

Calcineurin (CN) is a ubiquitously expressed serine (Ser)/threonine (Thr) phosphatase and is composed of a catalytic subunit A (CNA), and a regulatory subunit B (CNB). CN is activated by calmodulin or calpain following an increase in intracellular calcium levels (1). In the calmodulin- mediated activation process, calcium binds to the CNB regulatory sites inducing a conformational change on the CN that allows the interaction of the CN with the calmodulin/calcium complex. This interaction leads to the exposure of the CN catalyst site. When the CN is activated by calpain, the increase in calcium induces the protease activity of calpain that cuts the CN autoinhibitory domain, generating a constitutively active form (2).

CN interacts with substrates that have two main binding sites: the PxIxIT motif, which is considered the primary docking site, and the Lx VP motif (3). Proteins containing either or both of these motifs can be partitioned into regulators and effectors. Regulators are proteins that control CN activity by modulating its subcellular activation and localisation or by recruiting effector molecules. Effectors are transcription factors or kinases. For example, among ubiquitously expressed CN substrates containing the PxIxIT motif, Cabinl, RCAN1/DSCR1, and AKAP79 show regulator functions, while Dynamin 1 GTPase, MKK7 kinase, and NFAT family of transcription factors have effector functions. Clinical and experimental trials have shown the role of CN in controlling cancer progression. Indeed, activation of CN in human colorectal cancer (CRC) is correlated with increased mortality, while inhibiting CN phosphatase function induces tumour regression (4). In line with these results, CN is activated in triple- negative breast cancers and is responsible for tumour growth and metastasis formation (5). A correlation between CN and tumourigenesis has also been observed in other cancers, including melanoma, leukaemia, glioblastoma and cancer of the liver, pancreas, bladder, ovary and prostate (6)(7)(8)(9)(10)(11). Pro-X-Ile-X-Ile-Thr (PxIxIT) sequence peptides (SEQ ID NO:21), derived from the conserved CN-binding motif found in NFAT proteins, compete with NFAT for CN- binding, compromising NFAT binding and dephosphorylation as shown in enzyme assays. In particular, the Val-Ile-Val-Ile-Thr (VIVIT) oligopeptide (SEQ ID NO:l) 16mero possesses a high affinity for CN and effectively inhibits NFAT and its activity, but not NF-kB transcription factor activity as indicated by the activation of the respective genes (12). Thus, the VIVIT peptide is more selective than CsA and FK506, which inhibit the activation of both transcription factors as they block the phosphatase activity of CN. This peptide was identified by random screening of a peptide library. Competing for the main CN binding site, it inhibits the interaction between CN and, not only NFAT, but any substrate containing the PxIxIT motif (Figure 1) (12). It has been shown that the NFAT family of transcription factors is required for differentiation of embryonic stem cells. Indeed, by blocking the activation of NFAT or the phosphatase activity of the CN, the stem cells do not differentiate under conditions that normally induce differentiation (subtraction of growth factors that allow the maintenance of sternness) (Xiang Li et ak, “Calcineurin-NFAT signalling critically regulates early lineage specification in mouse embryonic stem cells and embryos”, Cell Stem Cell , Vol. 8, 46-58, January 2011).

Adult stem cells (also known as somatic stem cells or tissue stem cells) are rare populations of undifferentiated cells found in the body during most of the postnatal life. They are defined as multipotent in that they are able to self-renew but only give rise to mature cells of the tissue in which they reside. Their progeny replaces cells lost due to tissue turnover or damage, thus ensuring the maintenance of tissue homeostasis. Adult stem cells are usually kept in a quiescent state but, once activated, proliferate to reconstitute damaged tissues. Their division is asymmetrical in that a stem cell, which maintains the tissue niche, and a precursor, which differentiates into the mature cell, are generated from a stem cell. Multiple organs are known to possess a stem niche. Some well-researched examples in mammals include tissues with high regenerative capacity such as blood, skin, intestine, and others to lesser or almost no regenerative capacity, such as the brain. Generally, it is thought that adult stem cells are all the more abundant in a given tissue the greater the latter’s capacity (and need) to regenerate. For example, the adult neural stem cells are numerically small and are located in specific areas of the brain: the hippocampus, the subventricular area and the olfactory bulbs. Considering the easier accessibility of adult stem cells than embryonic stem cells, and considering the extraordinary therapeutic potential in the field of regenerative medicine, there is increasing attention to the methods of isolation and characterisation of these stem cells residing in adult tissues. However, the biggest limitation observed is the difficulty of maintaining adult stem cells in culture and expanding them in an in vitro system to obtain an adequate number for the reinfusion for the shelter of tissue damage.

Within tumours, cancer stem cells (CSCs) mediate primary and acquired resistance to conventional and target therapies. CSCs have similar characteristics to stem cells and have been isolated from many cancers. They are generally characterised by a differentiating potential (recapitulating intratumour heterogeneity), by slow proliferation phenotype, high expression of DNA damage repair mechanisms, anti-apototic proteins, immune system evasion mechanisms (13), and increased activity of the sternness pathways (i.e. Wnt and PI3K/Akt) (14)(15). Isolating CSCs is very difficult because no specific markers are known. There are therefore no standardised protocols, and each laboratory concerned uses non-standardised, secret and expensive protocols. In addition, CSCs may not always be obtained from liquid biopsies or solid tumours. Since CN acting with its phosphatase activity on targets such as NFAT, induces differentiation of embryonic stem cells (Calcineurin-NFAT Signaling Critically Regulates Early Lineage Specification in Mouse Embryonic Stem Cells and Embryos Xiang Li, Lili Zhu, Acong Yang, Jiangwei Lin, Fan Tang, Shibo Jin,l Zhe Wei, Jinsong Li, and Ying Jin Cell Stem Cell 8, 46-58, January 7, 2011), blocking this activity, i.e. the possibility of interacting with substrates containing the PxIxIT motif, could allow CSCs and adult stem cells to be expanded by inhibiting differentiation.

There is therefore still a need to obtain an agent capable of blocking stem cell differentiation. SUMMARY OF THE INVENTION

The present authors found that tumor cells expressing a molecule capable of inhibiting the interaction of calcineurin (CN) with a substrate containing the PxIxIT motif maintained an undifferentiated condition with a migratory phenotype both in vitro and in vivo.

Therefore, an object of the invention is a method for expanding activated adult stem cells or tumor stem cells and/or for maintaining an activated adult stem cell or tumor stem cell culture in vitro in a stem state and/or preventing its senescence and/or differentiation and/or for enriching a cell culture with activated adult stem cells or tumor cells, the method comprising providing, preferably stably providing, a target stem cell or target stem cell culture with a molecule capable of chronically inhibiting the interaction of calcineurin (CN) with a substrate containing the PxIxIT motif, where said target stem cell is an adult stem cell or tumor stem cells.

Preferably, the provision, preferably stably provision, of said molecule to said target stem cell or target stem cell culture allows to obtain activated adult stem cells or tumor stem cells or cultures thereof.

Preferably, the activated adult stem cells or tumor stem cells are cells in active proliferation and/or with migratory capacity.

Preferably, said target cells are adult stem cells of various tissues, preferably neural stem cells, intestinal stem cells, tumor stem cells, preferably of human or mouse origin, more preferably said neural stem cells are obtained from the olfactory bulb and they are preferably human. Preferably, said molecule is a competitor for the binding site of the CN with the nuclear factor of activated T-cells (NFAT).

Preferably, said molecule is expressed, preferably stably expressed, by the target stem cell or target stem cell culture.

Preferably, said molecule is a peptide comprising or consisting of the sequence SEQ ID NO:l VIVIT, SEQ ID NO: 2 CGGGKMAGP VIVIT GPHEE, SEQ ID NO:3 CGGVIVIT, SEQ ID NO:4 CGGGKMAGPHPVIVITGPHEE or SEQ ID NO:5 C GGGM AGP VI VIT GPHEE . Preferably, the molecule further comprises an amino acid sequence which allows passage through the cell membrane of an eukaryotic cell, preferably said sequence selected from the following group poly-arginine, Arg 11 (RRRRRRRRRRR/SEQ ID NO: 6) ;

Tat / RKKRRQRRR/SEQ ID NO: 7;

Penetratine / RQIKIWF QNRRMKWKK/SEQ BD NO: 8;

Buforin II / TRS SRAGLQFPVGRVHRLLRK/SEQ ID NO: 9; Transportan / GWTLN S AGYLLGKTNLKALAALAKKIL/SEQ ID NO: 10;

MAP (model amphipathic peptide) / KLALKLALKALKAALKLA/SEQ ID NO: 11; K-FGF / AAVALLPAVLLALLAP/SEQ ID NO: 12;

Ku70 / VPMLK/SEQ ID NO: 13

Ku70 / PMLKE/SEQ ID NO: 14; Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP/SEQ ID NO: 15; pVEC / LLIILRRRIRKQAHAHSK/SEQ ID NO: 16;

Pep-1 / KETWWETWWTEWSQPKKKRKV/SEQ ID NO: 17;

SynBl / RGGRLSYSRRRFSTSTGR/SEQ ID NO: 18;

Pep-7 / SDLWEMMMV SLACQY/SEQ ID NO: 19; and HN-I / TSPLNIHNGQKL/SEQ ID NO: 20 Preferably, the provision, preferably stably provision, of the molecule to the target cell comprises:

- the delivery of an expression cassette of the molecule to said target cell where said cassette is preferably comprised in a replicable vector, preferably a viral vector, preferably an adeno-associated virus (AAV), an unintegrated lentivirus, an adenoviral or retroviral vector, or a non-viral vector and / or

- the delivery of nucleic acid encoding the molecule, including mRNA, preferably linked to a nanoparticle, such as a nanovector.

In a preferred embodiment of the invention, the method comprises the steps of: i. culturing neural stem cells in vitro in an appropriate culture medium comprising growth factors, e.g. EGF and / or FGF2, for 5-20 days, preferably 15 days, ii. perform at least one dissociation of the cultured cells and obtain neurospheres, iii. provide the neurospheres with a molecule capable of inhibiting the interaction of calcineurin (CN) with a substrate containing the PxIxIT motif as defined in any one claims 3-5, iv. culture the neurospheres in a culture medium comprising growth factors, for example EGF and / or FGF2, until their natural senescence is reached, preferably for 60-90 days, and optionally v. isolate stem cell colonies from the culture resulting from the step iv.

In a preferred embodiment of the invention, the method comprises the steps of: i. Cultivate in vitro tumor cells, preferably a tumor line, in an appropriate culture medium comprising growth factors, for example EGF and / or FGF2, ii. Perform at least 3 culture passages, iii. provide the cells with a molecule capable of inhibiting the interaction of calcineurin (CN) with a substrate containing the PxIxIT motif as defined in any one of claims 3-5, iv. Perform at least 3 culture passages, v. Bringing culture to a state of complete confluence each time and monitoring the formation of spheres, e.g.7-10 days and optionally vi. isolate stem cell colonies from the culture resulting from the step v.

Preferably, step iii. comprises delivery with a vector expressing the molecule as defined herein and / or comprising a nucleic acid encoding said molecule. Preferably, the vector is a viral vector, preferably a lentivirus.

Preferably, said molecule is a peptide comprising or consisting of the sequence SEQ ID NO:l VIVIT

Another object of the invention is a cell obtainable from the method as defined herein, preferably for medical use, more preferably for use in cell or tissue regeneration.

A further object of the inventions is a cell population comprising at least one cell as defined herein, preferably for medical use, more preferably for use in cell or tissue regeneration. Another object of the invention is the use of the cell or population according as defined herein for in vitro experimentation.

A further object of the invention is a molecule able to chronically inhibit the interaction of calcineurin (CN) with a substrate containing the PxIxIT motifs as defined herein for use in the expansion of activated adult stem cells and / or in the inhibition of differentiation and / or of senescence of activated adult stem cells.

Preferably, the activated adult stem cells are cells in active proliferation and/or with migratory capacity.

Preferably, the activated adult stem cells are obtained by providing, preferably stably providing, said molecule to adult stem cells and/or wherein the activated adult stem cells express, preferably stably express, said molecule.

Another object of the invention is a nucleic acid encoding the molecule as defined herein for use in the expansion of activated adult stem cells and / or in the inhibition of differentiation and / or of senescence of activated adult stem cells.

A further object of the invention is a vector expressing the molecule as defined herein or comprising the nucleic acid as defined herein or nanoparticle, such as a nanovector, comprising the molecule as defined herein or comprising the nucleic acid as defined herein for use in the expansion of activated adult stem cells and / or in the inhibition of differentiation and / or of senescence of activated adult stem cells.

Another object of the invention is use of the molecule as defined herein, of the nucleic acid according as defined herein or of the vector as defined herein or the nanoparticle as defined herein to stably inhibit the bond between the CN and one or more of its specific ligands. Another object of the invention is a cell comprising and / or expressing the molecule as defined herein or the nucleic acid as defined herein or the vector as defined herein and / or engineered to comprise and / or express said molecule or said nucleic acid or said vector, preferably for medical use, more preferably in cell or tissue regeneration. Preferably the cell stably express said molecule.

Other objects of the invention are: a cell population comprising at least one cell as defined herein, preferably for medical use, more preferably in cell or tissue regeneration, and the use of the cell or population as defined herein for in vitro experimentation.

DETAILED DESCRIPTION OF THE INVENTION

The molecule according to the invention can be selected from, but is not limited to, nucleic acids, peptides, proteins, antibodies, sugars, synthetic polymers, polynucleotides, fatty acids, small organic molecules or combinations thereof.

The term “the activated adult stem cells” or “activated tumor stem cells” (or cultures thereof) means adult stem cells or tumor stem cells in active proliferation and/or with migratory capacity (or cultures thereof), preferably in active proliferation and with migratory capacity. Such cells may also be defined as non-quiescent or non-quiescent-activated.

Such cells (or cultures thereof) are obtained by respectively providing target adult stem cells or target tumor cells (or cultures thereof) with a molecule capable of chronically inhibiting the interaction of calcineurin (CN) with a substrate containing the PxIxIT motif,

Preferably such molecule is provided to the target cells, e.g. by genetically modifying the cell, e g. by transduction, transformation or transfection with the polynucleotide or with the vector as described herein.

Preferably such molecule is stably provided to the target cells, e.g. by genetically modifying the cell, e.g. by transfection with the polynucleotide or with the vector as described herein. Preferably the target stem cell or target stem cell culture or the activated adult stem cells or activated tumor stem cells transiently or stably express the above molecule. The expression of VIVIT indeed induces the expansion of activated (or non-quiescent-activated) adult stem cells, characterized by a greater migratory capacity compared to cells that do not express VIVT peptide.

For “active proliferation” it intended e.g. a cell state in which stem cells are able to proliferate, proceeding toward the various phases of the cell cycle, rather than being blocked in GO phase. The opposite of ‘active proliferation’ are ‘quiescence’, a cell state in which stem cells do not proliferate but do not die, they simply remain in the GO phase of the cell cycle until they receive specific signals.

The terms “tumor stem cells” or “cancer stem cells” may be used interchangeably. The step of providing target adult stem cells or target tumor cells (or cultures thereof) with a molecule capable of chronically inhibiting the interaction of calcineurin (CN) with a substrate containing the PxIxIT motif may comprise or consists of:

- the delivery of an expression cassette of the molecule to said target cell where said cassette is preferably comprised in a replicable vector, preferably a viral vector, preferably an adeno- associated virus (AAV), an unintegrated lentivirus, an adenoviral or retroviral vector, or a non- viral vector and / or

- the delivery to said target cell of nucleic acid encoding the molecule, including mRNA, preferably linked to a nanoparticle, such as a nanovector and/or

- the delivery to said target cell of a vector expressing the molecule as defined herein and / or comprising a nucleic acid encoding said molecule.

The cells of the invention may also be defined as host cells or genetically modified cell.

Preferably, the stem cells of the invention or the cells of the invention are not human embryonic cells.

The terms “expression vector” or “vector” refer to a nucleic acid that transduces, transforms or infects a host cell, causing the cell to produce nucleic acids and/or proteins other than those that are native to the cell, or causing it to express nucleic acids and/or proteins in a manner not native to the cell.

The term “endogenous” refers to a molecule (e g. A nucleic acid or a polypeptide) or process that occurs naturally, e.g. in a nonrecombinant host cell.

The terms “polynucleotide” and “nucleic acid” used interchangeably herein refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or functionalised nucleotide bases. The terms “peptide”, “polypeptide” and “protein” are used interchangeably, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatised amino acids, and polypeptides having modified peptide backbones.

As used herein, the terms “operon” and “single transcription unit” are used interchangeably to refer to two or more contiguous coding regions (nucleotide sequences that encode a gene product such as an RNA or a protein) that are coordinately regulated by one or more controlling elements (e.g. a promoter). As used herein, the term “gene product” refers to an RNA encoded by DNA (or vice versa) or protein that is encoded by an RNA or DNA, where a gene will typically comprise one or more nucleotide sequences that encode a protein, and may also include introns and other non-coding nucleotide sequences.

As used herein, the term “heterologous nucleic acid” refers to a nucleic acid wherein at least one of the following statements is true: (a) the nucleic acid is foreign (“exogenous”) to (that is, not naturally found in) a given host cell; (b) the nucleic acid comprises a nucleotide sequence that is naturally found in (that is, is “endogenous”) to a given host cell, but the nucleotide sequence is produced in an unnatural (for example, greater than expected or greater than naturally found) amount in the cell; (c) the nucleic acid comprises a nucleotide sequence the sequence of which differs from an endogenous nucleotide sequence, but the nucleotide sequence encodes the same protein (having the same or substantially the same amino acid sequence) and is produced in an unnatural (for example, greater than expected or greater than naturally found) amount in the cell; or (d) the nucleic acid comprises two or more nucleotide sequences that are not in the same relationship with each other in nature (e.g. nucleic acid is recombinant).

The term “recombinant”, as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps, resulting in the formation of a construct having a structural coding sequence or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non- translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the sequences of interest can also be used in the formation of a recombinant gene or a transcriptional unit. Sequences of non-translated DNA may be present at 5’ or 3’ of the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “DNA regulatory sequences”, below).

Thus, the term “recombinant” polynucleotide or nucleic acid, for example, refers to one which is not naturally occurring, e.g. which is made by the artificial combination of two otherwise separated segments of sequence with human intervention. This artificial combination is often accomplished either by chemical synthesis means or by the artificial manipulation of isolated segments of nucleic acids, e.g. by genetic engineering techniques. Generally, this is done in order to replace a codon with a redundant codon coding for the same or a conservative amino acid, typically by introducing or removing the sequence recognition site at the same time. Alternatively, it is performed to join together nucleic acid segments with desired functions to generate a desired combination of functions. This artificial combination is often accomplished either by chemical synthesis means or by the artificial manipulation of isolated segments of nucleic acids, e.g. by genetic engineering techniques.

The term “transformation” or “genetic modification” refers to a genetic change, permanent or transient, induced in a cell following introduction of a new nucleic acid. Thus, a “genetically modified cell” is a host cell into which a new (e.g. exogenous; heterologous) nucleic acid has been introduced. A genetic change (“modification”) can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. In eukaryotic cells, a permanent genetic change is generally achieved by introducing DNA into the cell’s genome. In prokaryotic cells, a permanent genetic change can be introduced into the chromosome or via extrachromosomal elements, such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell.

The terms “DNA regulatory sequences”, “control elements”, and “regulatory elements”, used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell.

The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship that enables them to function in the intended manner. For instance, a promoter is operatively linked to a nucleotide sequence if the promoter affects the transcription or expression of the nucleotide sequence.

A “host cell,” as used herein, denotes an in vitro eukaryotic cell (e.g. a yeast cell), which eukaryotic cell can be, or has been, used as a recipient for a nucleic acid, and comprises the progeny of the original cell which has been genetically modified with the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g. an expression vector. For example, a suitable eukaryotic host cell is a genetically modified eukaryotic host cell by virtue of introduction into a suitable eukaryotic host cell a heterologous nucleic acid, e g. an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.

As used herein, the term “isolated” is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. The term “polynucleotides” includes DNA, RNA, siRNA and miRNA, whether viral, bacterial, plant or animal (e.g. mammalian), synthetic, single- or double-stranded, comprising natural, non-natural or chemically modified nucleotides. Furthermore, the molecule can also include labelled molecules, such as radioactively or fluorophore labelled molecules.

In the context of the present invention, when reference is made to specific DNA sequences, it is intended that RNA molecules identical to said polynucleotides are also comprised in the invention, except that the RNA sequence contains uracil instead of thymine and the skeleton of the RNA molecule contains ribose instead of deoxyribose, RNA sequences complementary to the sequences described therein, functional fragments, mutants and derivatives thereof, proteins encoded by them, functional fragments, mutants and derivatives thereof. Also included in the present invention are nucleic acid or amino acid sequences derived from the nucleotide or amino acid sequences shown in the present invention, e.g. functional fragments, mutants, derivatives, analogues and sequences with a % identity of at least 70, at least 80, at least 85 at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % with the sequences mentioned above. Further molecules capable of inhibiting the interaction of calcineurin (CN) with a substrate containing the PxIxIT motif may be selected by a person skilled in the state of the art using cellular screening methodologies.

Preferably the CSCs were obtained from murine melanoma, e.g. from B16 cells and/or from murine triple negative breast cancer e.g. 4T1 cells.

The cells are preferably murine neural stem cells obtained from the subventricular area of the brain and/or intestinal stem cells extracted from the mouse intestine

In the context of the present invention, the term “complete confluence” denotes the state in which the cells are at such a density that there is no space between them and can be assessed by means of a microscope. The term denotes, for example, a confluence of more than 80 per cent.

Any method known to the person skilled in the state of the art to provide the molecule of the invention to the cells can be used in the present invention. In a preferred embodiment of the invention, the cells described herein are characterised by the fact that exogenous nucleic acid has been introduced by the use of a viral vector, for example in the form of a viral expression construct, more preferably a lentiviral or retroviral vector. Alternatively, the cells described herein are characterised by the fact that exogenous nucleic acid is or comprises a construct of non-viral expression.

Preferably, in the vector as described above, the polynucleotide (or exogenous nucleic acid) is under the control of a promoter capable of expressing said polynucleotide efficiently.

The polynucleotide sequence in the vector is operatively linked to an appropriate expression control sequence (promoter) to direct the synthesis of the mRNA. Examples of promoters include the immediate promoter of early cytomegalovirus (CMV) genes, thymidine kinase HSV, early and late SV40, LTRs from retrovirus, preferably derived from murine leukemia viruses (MLV). The vectors may also contain one or more selectable gene markers.

As used herein, the term “genetically modified cell” refers to a host cell that has been transduced, transformed or transfected with the polynucleotide or with the vector as described above.

The introduction of the polynucleotide or vector previously described in the host cell may be carried out using methods known to the person skilled in the state of the art, such as calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, lipofection, microinjection, viral infection, thermal shock, cell fusion, and so forth.

It should be noted that, as used herein and in the appended claims, the singular indefinite articles “a” and “an” and the singular or plural definite article “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of such proteins, and so forth.

The present invention will now be illustrated by means of non-limiting examples, with reference to the following figures.

Figure 1. Molecular model of the CN with the two anchoring sites of the substrates indicated. In particular, the site indicated in yellow is the binding site of the substrates that have the PxIxIT motif. The figure also lists the known substrates of the CN.

Figure 2. Vector used to translate the VIVIT peptide into murine and human cells.

Figure 3. B16-VIVIT or 4T1-VIVIT cells and the respective control cells were cultured in suspension plates in Neurocult commercial culture medium (stem cell technologies). One million cells have been plated in a 10 cm diameter petri dish for two weeks. The selected B16-VIVIT and 4T1 -VIVIT cells were able to form spheres like stem cells while the control cells were not. Figure 4. Expression of stem cell genes in B16 and 4T1 cells following constitutive expression of the VIVIT peptide and subsequent culture as compared to control cells (B16-BFP and 4T1- BFP). Figure 5. Molecular analysis of 4T1-VIYIT and 4Tl-control by RNAseq. 4T1-VIVIT and 4T1- control cells were cultured in suspension plates in serum-free culture medium. One million cells were plated in 10cm diameter petri dishes for two weeks. The RNA was then extracted and RNAseq analysis was performed. (A) Dot plot showing GSEA results of 4T1-VIVIT vs 4T1- control for Gene Ontology Biological Process category. The top 10 pathways ordered by normalized enrichment score (NES) are shown. Dot colour denotes NES and dot size denotes - Iogl0(adjusted p-value). Notably, 4T1-VIVIT significantly upregulates genes involved in the epithelium-mesenchymal transition indicating an enrichment of stem cells in the culture. B) Overlap between the input gene list and the Stem Cell types present in StemChecker. In left panel, the overlap significance is plotted as -loglO(p-value); in right panel, table with the results is shown. C) Overlap between the input gene list and the Transcription Factors. In left panel, the overlap significance is plotted as -loglO(p-value); in right panel, the table displays the significance of enrichment of genes included as transcription factor targets among the input genes found in StemChecker. Significance (p-value) is calculated by the hypergeometric test and the adjusted p-value is calculated by Bonferroni correction.

Figure 6. 4T1 cells, expressing or not the VTVTT peptide, were grown as spheres and then adoptively transferred in vivo in the indicated amounts. Tumor growth curves over time (days after transfer) are shown. Notably, tumor derived from 4T1 cells that express VIVIT shows a higher growth rate.

Figure 7. Vascularisation of B16-VIVIT or control tumours (B16-BFP). (top panels) At day 6 after in vivo tumour cell transfer, mice were treated with fluorescent dextran (injected IV). The implanted tumours were then analysed under a two-photon microscope to highlight the vessels. Note that the number of vessels is much higher in control tumours. The lower panels instead show sections of tumours implanted on day six and analysed in immunohistochemistry for expression of CD31, a marker of endothelial cells.

Figure 8. Flow cytometry analysis of BFP protein expression in neurospheres SVZ-VIVIT (red dots) SVZ-BFP (green dots) compared with uninfected SVZ (blue dots), 8 days after infection with 100 MOI lentivirus.

Figure 9. Images of neurosphere cultures derived from SVZ-VIVIT and SVZ-BFP at 5 and 29 days of culture in conditioned medium with EGF and FGF2.

Figure 10. Growth curve obtained by counting neural cells of SVZ-VIVIT and SVZ-BFP cultures every 8 days during 60 days of in vitro maintenance in medium conditioned with EGF and FGF2. B) Migration assay of SVZ-VIVIT and SVZ-BFP adult neural stem cells after 5 days of culture in transwell. Figure 11. A) Expansion of adult NSCs expressing the VIVIT peptide in vivo. Histological sections of mouse brain after 30 days (left panel) or 90 days (right panel) of VIVIT or BFP intracranial injection in the adult stem cells niches of the subventricular zone NSCs are stained with anti-Nestin antibody (NSC marker). Images show the expansion of NSCs in brain of mice who received VIVIT peptide as compared to control mice (BFP treatment) and their migration toward the corpus callosum (CC) (arrows). The expansion of NSCs in distal sections from the site of injection (right panels) observed in VIVIT-treated mice indicates an increase in their ability to migrate along the brain as compared to NSCs of BFP -treated mice.

B) Expansion of intestinal adult stem cells (identify as cells that express LGR5 protein, also referred as LGR5 positive cells) expressing the VIVIT peptide in vivo. For these experiments we used mouse model in which the expression of VIVIT is induced by tamoxifen treatment selectively in cells that express LGR5 protein. Images shows intestinal section of LGR5-CRE- VIVIT mice that were treated with tamoxifen once a week for 3 weeks to obtain the expression of VIVIT peptide. Untreated mice were used as control. (Right panel). Images show the induction of VIVIT expression (red) in tamoxifen treated mice only in LGR5 positive cells (green). (Left panel) Images shows only the LGR5 staining. Notably, an increase in the number of intestinal stem cells (LGR5 positive, green) in mice that express VIVIT peptide as compared to control mice has been observed.

EXAMPLE 1 Materials and Methods

Infection of tumour cell lines with lentiviral vector expressing VIVIT

The lentiviral vector containing the VIVIT peptide or just the GFP protein (control vector) is described in Monica Soldi et ak, “Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering”, Molecular Therapy: Methods & Clinical Development Vol. 19, December 2020.

Two different tumour cell lines were transduced with the lentivirus expressing VIVIT or with the control lentivirus (BFP): murine melanoma tumour cells (B16), murine mammary tumour cells (4T1). The cells were infected with 100 MOI of virus for 18 hours. After infection, the cells were left to rest for 72 hours, adding fresh culture medium each day. Subsequently, the infection was checked by assessing the expression levels of the reporter protein BFP in the flow cytometer. All of the lines used for the experiments showed a BFP positivity of more than 97%.

Culture condition All tumour cell lines transduced with the lentivirus expressing the VIVIT or the control lentivirus were plated at the physiological confluence (2 million cells in T 175 cm ² flask), in commercial medium NeuroCult ™ (Neural Stem Cell Culture Media) added with 1% glutamine and 1% penicillin / streptomycin and placed in an incubator at 37 ° C with 5% C02. After 48 hours the cells reached maximum physiological confluence and were divided 1:10 for two consecutive times. At this point the cells were used for analyses.

Real-time PCR

Expression of sternness genes was performed by real-time PCR in both in vitro grown cell samples and tumours surgically removed from animals. Samples were homogenised in the TRIzol reagent, then RNA was extracted using Quick-RNA MiniPrep or microPrep kits (Zymo, cat R2050 and R1051 research respectively). Complementary single-stranded DNA (cDNA) was synthesised using high-capacity cDNA reverse transcription kits (catalogue number 4368814, Applied Biosystems). NanoDrop (Thermo Scientific) was used to titrate the mRNA. Amplification of the cDNA of Oct4, Sox2 and NanoG was performed using sybr green Master Mix.

Inoculation of tumours

Mice were inoculated with 2.5 x 10 ⁵ B16 or 4T1 by subcutaneous injection into the flank at day 0. tumours were measured every 2 days for length, width and thickness using calipers.

Histological analysis of explanted tumours

Tumours explanted 6 days after inoculation were incorporated into OCT (Bio-optics) freezing medium. The sections (5 pm) were cut on a cryostat, adhered to the Superfrost Plus slide (Thermo Scientific), fixed with acetone and blocked with PBS containing 0.3% of Triton X-100 (Sigma-Aldrich) and 10% FBS. The sections were then stained with purified anti-mouse CD31 antibody (1 :200) in blocking buffer, 1 hour at room temperature. Sections were washed with TBS buffer and incubated with the Rat-on-Mouse HRP - Polymer (Biocare Medical) kit. Dako EnVision System - HRP was used as a chromogen and counterstaining with Mayer’s Hematoxylin (Bio-optics) After dehydration, the coloured slides were mounted with Eukitt and images were acquired with a dotSlide Vsl20 (Olympus). Image analysis was performed using FIJI - ImageJ (Schindelin et al., 2012, 2015). ROI colour deconvolution was used to separate the DAB signal, and particle analysis was used to automatically recognise, count, and measure all CD31 -positive points within the total tumour area.

Analysis of vascularisation using a two-photon microscope The direct optical microscope (BX51; Olympus) was used for intravitally imaging the explanted tumours. The infrared laser source (Mai Tai HP + DeepSee, Spectra Physics, USA; with 120 fs pulses at full width at half maximum and 80 MHz repetition frequency) is coupled to the head of FV300 scanning (Olympus, Japan). All measurements were acquired using a 20 x, 0.95 - NA, 2 - mm - WD lens (XLUMPlan FI; Olympus, Japan). TPE allows limited photodamage of samples, simultaneous excitation of multiple fluorescent probes and deep penetration into thick tissues such as tumours. The fluorescence signal is directed to an unscanned unit and divided into three channels (blue, green and red channel) by two dichroic beam dividers. For the analysis of the tumour implants, the entire microscope was surrounded by a custom-made thermostatic cabinet in which the temperature was maintained at 37°C (air thermostatation from “The Cube”; Life Imaging Services, Basel, CH) and the physiological conditions were guaranteed during the entire analysis by flowing a buffer solution at 37°C saturated with a mixture of 95% 02-5% C02. Volocity (Perkin-Elmer Inc.) was used to analyse recorded footage. The extracted traces were then analysed for the measurement of interaction time using a specially designed MatLab code (MathWorks Inc.).

Results

Generation of tumour lines expressing the VIVIT peptide

The authors generated stable tumour lines expressing the VIVIT peptide and tumour control lines (empty vector) to study the effects of inhibiting CN interaction with molecules containing the PxIxIT motif. We selected two types of cancer cell lines: 4T1, a breast tumour model, and B16, a melanoma model. We infected these cell lines with a lentiviral vector expressing a single transcript encoding both the VIVIT peptide and the blue fluorescent protein (BFP) bound by the self-cleaving 2A peptide sequence. After translation, the peptide 2A undergoes self-cleavage leading to expression of the two separate peptides (BFP and VIVIT). (Figure 2).

Selection of CSCs

To select CSCs, tumor cells transduced with the VIVIT peptide and control cells (tumor-BFP) were cultured for approximately two weeks, undergoing at least two 1:10 dilution steps after reaching complete confluence. At the end of the two weeks all the BFP cells continued to grow in adhesion or aggregated in random structures, while the cells expressing the VIVIT peptide grew into a sphere, a typical phenotypic condition of stem cells (Figure 3). The cells were analyzed for the expression of stem cell genes (Figure 4). B16 and 4T1 cells were also subjected to further analysis. First, an RNAseq analysis of 4T1 cells was performed. From the comparative analysis of the molecular pathways that are differentially expressed between the 4T1-VIVIT and the 4Tl-control it is seen that the 4T1-VIVIT significantly upregulates the epithelial- mesenchymal transition pathway, demonstrating that these cells have a more stem-cell phenotype as compared to controls. Moreover, to further characterize stem cell properties of 4T1-VIVIT cells, we interrogated a web-based tool that allows to explore sternness signatures in user-defined gene sets and to investigate whether the input genes are targeted by a set of transcription factors linked to pluripotency and stem cell maintenance. As input genes we used genes which were significantly upregulated in 4T1-YIVIT as compared to 4T1 -control. Interestingly, StemChecker mainly identified in our gene set similarities with gene sets expressed by adult stem cells and embryonic stem cells (Figure 5B). The analysis of transcription factor target genes, revealed a representation of targets of the pluripotency master regulators Nanog, Sox2 and OCT4 (Figure 5C). Subsequently, 4T1-VIVIT and 4Tl-control cells were transferred in mice of the same genetic background at different concentrations and tumor growth was monitored. As can be seen from Figure 6, in both conditions used, the tumor derived from cells expressing VIVIT, show a higher growth rate in vivo as compared to tumor derived from control cells, further demonstrating a more sternness phenotype of 4T1 -VIVIT cells as compared to control cells. Furthermore, selected B16-VIVIT cells and B16-control cells were transferred in vivo into recipient mice of the same genetic background and B16-VIVIT tumors were found to be poorly vascularized compared to control tumors which had a high degree of vascularization (Figure 7).

Example 2

Materials and Methods

Generation of neurospheres from stem cells of the subventricular area of the brain

Four-month-old c57bl6 mice (ENVIGO breeding, Bresso, Italy) were euthanised by cervical dislocation. Brains were removed and placed in the PG solution (saline solution containing 30% glucose). The subventricular zone (SVZ) of the forebrain was taken by performing a 6 to 8 mm coronal cut from the olfactory bulbs and was shredded into small fragments. The tissue fragments were then digested with a solution containing papain, incubated at 45°C to 37°C under mild stirring. After centrifugation for 10 minutes at 192 x g, the tissue was resuspended in 1 ml of EBSS (Eagle’s basic salt solution free of calcium and magnesium) and mechanically dissociated with the lOOOul micropipette. After further centrifugation the cells were counted and plated at a concentration of 10 ⁴ cells/cm ² in the Neurocult ™ commercial culture medium (Stem cells technologies). At each stage of the subculture, the neurospheres were mechanically dissociated to a single-cell suspension and reinserted into the specific culture medium to regenerate secondary and tertiary neurospheres for up to 60 days of culture. Infection of SVZ neurospheres by lentiviral vector expressing VIVIT

The lentiviral vector containing the VIVIT peptide or only the BFP protein (control vector) are described in Monica Soldi et ah, “Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering”, Molecular Therapy: Methods & Clinical Development, Vol. 19, December 2020.

Neural stem cells generated from the SVZ zone of the brain of C57bl/6 mice were harvested and dissociated to single cell status, then replanted into complete culture medium (Neurocult™, Stem cells technologies). After 24 hours of dissociation, when the cells were still in the single cell phase, they were infected with 100 MOI of virus for 18 hours. After infection, the cells were left to rest for 72 hours, adding fresh culture medium each day. Subsequently, the infection was checked by assessing the expression levels of the reporter protein BFP in the flow cytometer. Analysis of adult NSC migration

Adult NSCs expressing VIVIT or only the BFP reporter protein generated as described above, were plated on transwells placed on matrigel-coated slides. After 5 days the slides were removed and stained with hematoxylin and eosin. The migrated cells on the slides were visualized and acquired using the Hamamatzu slide scanner.

In vivo expansion of NSCs

To obtain stem cell expansion directly in vivo, the lentiviral vector containing the VIVIT peptide or only the BFP protein (control vector) was injected into the subventricular area of the brain of four-month-old c57bl6 mice (ENVIGO breeding, Bresso, Italy) using stereotaxic surgery. The following stereotaxic coordinates were used: 0.6mm anteroposterior, 1.6mm mediolateral, 3-2 dorsoventral. After 30 days or 90 days from the injection, the brain was removed, frozen in OCT and cut in sections of 20um. Subsequently, immunohistochemical staining of the Nestin protein, a marker of neural stem cells, was performed.

In vivo expansion of intestinal adult stem cells

To obtain intestinal adult stem cell expansion we generated mice that express an inducible form of VIVIT selectively in the intestinal stem cells. To do this, we crossed transgenic mice that have the VIVIT insert flanked by loxP sequences and transgenic mice expressing CRE recombinase under the LGR5 gene promoter (present only in intestinal stem cells). Thus, the LGR5-CRE- VIVIT mice generated, possess the CRE DNA-recombinase under the control of the LGR5 promoter, therefore it is expressed only in intestinal stem cells in an inactivated form. CRE activation is induced by tamoxifen treatment. Once activated, the CRE protein recognizes the loxP sites that flank the VIVIT and by cutting in correspondence with these it allows VIVIT expression. Therefore, with this model, we were able to induce the selective expression of VIVIT in intestinal stem cells after treatment with tamoxifen. LGR5-CRE-VIVIT mice were treated or not (control) for three weeks every 7 days with 1.2 mg of tamoxifen administered by oral gavage. Mice were then euthanized by cervical dislocation, the colon was extracted and frozen in OCT. Histological sections of 5um were performed and visualized with confocal microscopy. In these animals, the GFP reporter is expressed in LGR5 positive cells, while the expression of the TdTomato reporter is generated only when VIVIT is expressed.

Results

Generation of neural primary stem cells expressing VIVIT peptide

We generated neural stem cells expressing the VIVIT peptide or only the BFP reporter protein (empty control vector) to investigate the effects of inhibiting CN interaction with molecules containing the PxIxIT motif. We first derived neural stem cells from the subventricular zone of the brain of adult wild-type mice with background C57bl/6. After being cultivated in culture medium conditioned with EGF and FGF2 for 15 days and after performing two dissociations, the neurospheres were dissociated and infected with the VIVIT-expressing lentivirus (Figure 2) or with the empty lentivirus at MOI of 100. We subsequently evaluated the percentage of infected cells, analysing the expression of the BFP reporter protein in a flow cytometer (Figure 8). Analysis of neurosphere formation from SVZ neural stems

We evaluated the formation of neurospheres in SVZ-VIVIT and SVZ-BFP cultures. We observed how SVZ-VIVIT cultures formed neurospheres early after dissociation and in greater amounts than the SVZ-BFP control culture (Figure 9). Furthermore, the observation of neurospheres in culture at late times allowed us to assess how

SVZ-VIVIT neurospheres remained much more viable and were always numerically superior to SVZ-BFP cultures (Figure 9). The SVZ-VIVIT and BFP neurospheres were maintained in culture medium containing EGF and FGF2 until their natural senescence was reached, which is observed in vitro between 60 and 90 days. During this culture phase, cells were dissociated, counted, and replated every 8 days, and a growth curve was generated to evaluate a possible effect of VIVIT on neurosphere expansion (Figure 10A). This monitoring showed that chronic expression of the VIVIT peptide in SVZ stem cells keeps the neurospheres in their stem state and prevents their natural senescence. The final effect is therefore to enrich adult neural stem cultures strongly (Figure 10A). The migratory capacity of the NSCs- VIVIT compared to the NSCs-BFP was also assessed. The cells were plated in a transwell and after 5 days their ability to migrate to the slide placed under the transwell was evaluated. As shown in Figure 10B, VIVIT- expressing cells exhibit greater migratory capacity. This result shows that the expression of VIVIT induces the expansion of non-quiescent-activated adult stem cells, characterized by a greater migratory capacity. To obtain stem cell expansion directly in vivo , the lentiviral vector containing the VIVIT peptide or only the BFP protein (control vector) was injected into the subventricular area of the brain of four-month-old c57bl6 mice. After 30 days and 90 days from the injection, we evaluated by immunohistochemistry the presence of cells expressing Nestin protein, a marker of NSCs. As shown in Figure 11 A, the animals that received VIVIT show an increase in the number of Nestin positive cells both 30 and 90 days after the injection of the lentivirus. Furthermore, it can be appreciated how Nestin positive cells are mainly located in the corpus callosum, indicating a propensity of these cells to migrate along the brain. The same result is underlined by the fact that Nestin positive cells are more represented in the mice treated with VIVIT even in sections not proximal to the site of injection (Figure 11A, right panel). These results are in line with the observation of the increased migration of NSCs expressing VIVIT obtained in vitro. Finally, we also evaluated whether the selective expression of VIVIT in intestinal stem cells induced its expansion in vivo. As shown in Figure 1 IB, even in the intestine, animals expressing VIVIT in intestinal stem cells show an increase in the number of LGR5 positive cells.