Oncolytic adenovirus with controlled replication

Abstract

The present invention relates to oncolytic viruses with controlled replication which can be incorporated into carrier cells for cancer therapy.

Description

[0001] Oncolytic adenovirus with controlled replication

[0002] Field of the invention

[0003] The invention is related to the field of medicine, more particularly to the field of oncology, and specifically to virotherapy.

[0004] Background of the invention

[0005] In the last decade major advances have been made in the treatment of cancer with alternative or complementary therapies to the chemotherapy, radiotherapy and surgery. Amongst these advances are gene therapy and virotherapy, both of which use viruses with a therapeutic intention against cancer. Virotherapy uses virus that replicate and propagate selectively in tumour cells. In virotherapy the tumour cell dies by the cytopathic effect caused by the replication of the virus in its interior rather than by the effect of a therapeutic gene. The preferential replication in a tumour cell is named oncotropism and the lysis of the tumour is named oncolysis. In a strict sense, viruses that replicate selectively in tumours are named oncolytic, although in a broader sense the oncolytic word can be applied to any replication-competent virus able to lyse tumour cells, even without selectivity. In this description the oncolytic term is used in both senses.

[0006] Adenovirus are particularly suitable for virotherapy, such as serotypes 2 and 5, and other serotypes also commonly used in the field of oncolytic adenovirus (Zhao, Y. et al., Front Microbiol. 2021 Jul 21 ; 12:707290). However, the efficient delivery of the adenovirus to the tumor cells remains a challenge.

[0007] An advanced therapy called Celyvir has shown clinical benefit in patients suffering from cancer (Ruano D. et al. 2020. Molecular Therapy, 28(4): 1033-1042). This product uses mesenchymal stem cells (MSCs) as cellular vehicles to deliver the oncolytic adenovirus ICOVIR5 directly to the tumor.

[0008] Despite the advantages of this virus, there was a small amount of adenoviruses reaching the tumor. Therefore, improved viruses and therapeutic methods are required to deal with this disadvantage.

[0009] Summary of the invention

[0010] The inventors have developed a new expression system to control the replication of an oncolytic adenovirus at will that is based on two main modifications. Firstly, the genome of the adenovirus has been modified by introducing Tet operator (TetO) repeats in the promoter of the adenoviral early region 1A (E1A) gene to control its replication. Secondly, the system involves the use of carrier cells as vehicles that incorporate the gene that expresses the tetracycline repressor (TetR). The administration of tetracycline allows controlled replication by releasing TetR, thus enabling viral replication. Therefore, the clinical application of this system will involve administering tetracycline to the patient only when it is considered that the cellular product has accumulated optimally within the tumor. The invention allows the virus to behave as normal replicative oncolytic adenovirus once it is released from the carrier cell, as tumor cells do not express TetR.

[0011] In a first aspect, the invention relates to a carrier cell comprising: i) an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and ii) a recombinant replication-competent oncolytic human adenovirus genome comprising the sequence of at least one tetracycline operator (TetO) element, wherein the expressible nucleic acid sequence of item (i) is not present in the recombinant replication-competent oncolytic human adenovirus genome of item (ii) , and wherein the at least one TetO element is located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene.

[0012] In a second aspect, the invention relates to a method for obtaining the carrier cell of the first aspect of the invention, said method comprising the steps of: i) introducing the expressible nucleic acid sequence encoding the tetR element or a functionally equivalent variant thereof into the carrier cell; and ii) infecting the carrier cell obtained in step i) with a recombinant replication- competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element or a functionally equivalent variant thereof.

[0013] In a third aspect, the invention relates to an expression system comprising: i) a carrier cell; ii) an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and iii) a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element nor a functionally equivalent variant thereof.

[0014] In a fourth aspect, the invention relates to a pharmaceutical composition comprising the carrier cell according to the first aspect of the invention and a pharmaceutically acceptable excipient.

[0015] In a fifth aspect, the invention relates to a carrier cell according to the first aspect of the invention, an expression system according to the third aspect of the invention or a pharmaceutical composition according to the fourth aspect of the invention for use in medicine.

[0016] In a sixth aspect, the invention relates to a carrier cell according to the first aspect of the invention, an expression system according to the third aspect of the invention or a pharmaceutical composition according to the fourth aspect of the invention for use in the prevention and / or treatment of solid tumors.

[0017] In a further aspect, the invention relates to a method for the prevention and / or treatment of solid tumors in a subject comprising: i) administering to the subject the carrier cell of the first aspect of the invention or the pharmaceutical composition of the fourth aspect of the invention, and then ii) administering to the subject a tetratcycline antibiotic.

[0018] In a further aspect, the invention relates to a kit selected from the group consisting of: i) a kit comprising the carrier cell according to the first aspect of the invention; and ii) a kit comprising an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element nor a functionally equivalent variant thereof.

[0019] In a further aspect, the invention relates to a recombinant replication-competent oncolytic human adenovirus genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising the sequence encoding the tetR element or a functionally equivalent variant thereof.

[0020] In a further aspect, the invention relates to the adenovirus comprising the recombinant human adenovirus genome of the invention.

[0021] Brief description of the figures

[0022] Figure 1. A) Oncolytic adenoviruses ICOVIR15, ICOVIR15-R7, ICOVIR15-Luc and ICOVIR15-R7-Luc. B) Dose-dependent citotoxicity assay on A549 cells. C) Dose dependent cytotoxicity assay on MenSCs. D) Infected A549 luciferase kinetic expression at different time-points post-infection.

[0023] Figure 2. A) Western blot of E1a and late viral proteins expressed from MenSCs and MenSCs-TetR infected with ICOVIR15-Luc or ICOVIR15-R7-Luc. B) Luciferase activity at different time-points post-infection. C) Viral production at different time-points postinfection.

[0024] Figure 3. A) Viral production (viral genomes / ml) at day 10 post-infection. B) Production of viral infective particles (Infection Units / ml) by MenSCs expressing the Tet-repressor without tetracycline treatment at day 10 post-infection. C) Visual phenotype of MenSCs and MenSCs-TetR (observed by phase-contrast imaging), either non-infected (control) or infected with ICOVIR15 or ICOVIR15-R7, in the presence or absence of tetracycline at day 10 post-infection.

[0025] Figure 4. A) Cell biodistribution in the tumor at different time points post-treatment. B) Luciferase activity at different time-points post-treatment. C) Tumor volume at different time-points post-treatment. D) Tumor growth at different time points post-treatment. E) Phase contrast image of the phenotypic evolution of infected cells.

[0026] Figure 5. A) Tumor volume at different time-points post-treatment. B) Tumor growth at different time-points post-treatment.

[0027] Detailed description of the invention

[0028] The present invention solves the problem of low counts of oncolytic virus reaching the tumors by providing a new expression system to control the replication of an adenovirus at will, allowing the carrier cell to reach and accumulate in the tumour before the onset of virus replication.

[0029] Carrier cell of the invention, recombinant adenovirus of the invention In a first aspect, the invention relates to a carrier cell, from here onwards the carrier cell of the invention, comprising: i) an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and ii) a recombinant replication-competent oncolytic human adenovirus genome comprising the sequence of at least one tetracycline operator (TetO) element, wherein the expressible nucleic acid sequence of item (i) is not present in the recombinant human adenovirus genome of item (ii), and wherein the at least one TetO element is located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene.

[0030] As used herein, the term “carrier cell,” used interchangeably with “cell”, “cell carrier”, “cell vehicle,” “carrier vehicle,” cell-based delivery vehicle” and “cell-based vehicle” refers to any cell that can be or is infected with virus or otherwise associated with virus, such as through chemical or physical interaction between the virus and a surface protein, or by infection of the cytoplasm or nucleus of the cell with the virus. As used herein, a carrier cell refers to a cell that can be infected with a virus, such as an oncolytic virus, and in which a virus / oncolytic virus can replicate. In a particular embodiment of the carrier cell of the invention, the cell is permissive to viral infection and replication.

[0031] The expression “permissive to viral infection and replication”, as used herein, refers to the fact that the carrier cells support viral infection and replication. Permissive cells are also productive cells since the virus which infected the cell, is able to reproduce and produce viral progeny. In a particular embodiment of the carrier cell of the invention, the cell is a stem cell. The term “stem cell”, as used herein, refers to cells which have the capacity to differentiate into a number of different cell types, especially those of a closely related family of cells. The term “multipotent” is also used to refer to “stem cells”. Examples of stem cells (autologous or allogeneic) that can be used as carrier cells include: adult stem cells; embryonic stem cells; fetal stem cells; neural stem cells; mesenchymal stem cells; totipotent stem cells; pluripotent stem cells; induced pluripotent stem cells; multipotent stem cells; oligopotent stem cells; unipotent stem cells; adipose stromal stem cells; endothelial stem cells (for example, endothelial progenitor cells, placental endothelial progenitor cells, angiogenic endothelial cells, pericytes); adult peripheral blood stem cells; myoblasts; small juvenile stem cells; skin fibroblast stem cells; tissue / tumor-associated fibroblasts; epithelial stem cells; and embryonic epithelial stem cells, for example. In a particular embodiment of the carrier cell of the invention the cell is selected from the group consisting of: adult stem cells, embryonic stem cells, fetal stem cells, neural stem cells, mesenchymal stem cells, totipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipose stromal stem cells, endothelial stem cells, adult peripheral blood stem cells, myoblasts, small juvenile stem cells, skin fibroblast stem cells, vascular stem cells, epithelial stem cells, and embryonic epithelial stem cells. In a more particular embodiment of the carrier cell of the invention, the cell is a mesenchymal stem cell.

[0032] The term “mesenchymal stem cell”, or “MSC”, as used herein, refers to multipotent progenitor cells derived from the mesoderm. Examples of mesenchymal stem cells include, but are not limited to, mesenchymal stem cells isolated / derived from: adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placenta, placenta-derived adherent stromal cells, placenta-derived decidual stromal cells, endometrial regenerative cells, placental bipotent endothelial / mesenchymal progenitor cells, amniotic membrane or fluid mesenchymal stem cells, amniotic fluid derived progenitors, Wharton's Jelly mesenchymal stem cells, pelvic girdle stem cells, Chorionic Villus Mesenchymal Stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, adventitial reticular stem cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, exfoliated deciduous teeth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, stem cells from apical papilla, muscle satellite cells and other such cells. In a particular embodiment of the carrier cell of the invention the cell is any of the cells disclosed in Kim J, et al., (2015, Viruses. 2015 Nov 27;7(12):6200-17). In a particular embodiment of the carrier cell of the invention the mesenchymal stem cell is obtained from menstrual blood. Techniques and methods to obtain mesenchymal stem cells form the menstrual blood are well known in the art and an example of such techniques and methods is provided in the Examples of this description. In a further particular embodiment of the carrier cell of the invention, the cell is a human cell.

[0033] When the carrier cell is used for delivery of oncolytic viruses to a cancer or to cancer cells (see below), the type of cell selected as carrier cell will be preferably a cell type which has a preference for targeting the cancer cell type, i.e., has cell tropism for the cancer cell or cancer tissue. The term “tropism”, as used herein, refers to the preferentiality that the carrier cell has for targeting or binding to or localizing next to cells of a specific type, in particular tumoral cell types or cancer cell types. In a particular embodiment of the carrier cell of the invention, the carrier cell has tropism for cancer cells, in particular lung cancer cells, and neuroblastoma cells, more particularly wherein the lung cancer is adenocarcinoma.

[0034] In the context of the present invention, the carrier cell comprises a first element: an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof.

[0035] The expression “expressible nucleic acid sequence”, as used herein, refers to all nucleic acid sequences which encode polypeptides and which can be expressed in a cell including those encoding small peptides. The nucleic acid sequence encoding said expressible polypeptide may comprise merely coding sequence or may comprise additional non-coding sequences. In the context of the present invention the expressible nucleic acid sequence encodes a “tetracycline repressor (TetR) element” or a “functionally equivalent variant thereof”.

[0036] In the present invention, the term "nucleic acid" refers to a repetition of monomers called nucleotides, linked by phosphodiester bonds. There are two types of nucleic acids: DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). In the present invention, "DNA" means both the genetic material of living organisms that controls heredity and is located in the nucleus or mitochondria of cells as well as recombinant genetic material, such as an expression vector, codifying for the expressible nucleic acid sequence according to the invention. In the present invention "RNA" means the molecule resulting from the transcription of a DNA sequence. The nucleic acid of the invention may contain one or more modifications to nucleobases, sugars and / or nucleotide bonds. In a particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence encoding the TetR element or a functionally equivalent variant thereof is an RNA sequence or a DNA sequence.

[0037] The term “tetracycline” or “tetracycline antibiotic”, as used herein, refers generally to a class of broad-spectrum antibiotics with perhydro-phenanthrene core, preferably corresponding to the subgroup of the Anatomical Therapeutic Chemical Classification System having ATC code J01 AA. These antibiotics are widely used for infections caused by Gram-positive and Gram-negative bacteria, intracellular mycoplasma, chlamydia, and Rickettsia. In a preferred embodiment, the tetracycline antibiotic is tetracycline. The chemical formula of the tetracycline may be C22 H24 N2 08, preferably having ATC code J01AA07. In the present context, the term tetracycline further includes “tetracycline analogs”, a term which is intended to include compounds which are structurally related to tetracycline and which bind to the Tet repressor element referred to herein. Tetracycline analogs include, without limitation, demeclocycline, doxycycline, chlortetracycline, lymecycline, metacycline, oxytetracycline, minocycline, rolitetracycline, penimepicycline, clomocycline, tigecycline, eravacycline, sarecycline, omadacycline and combinations thereof. Preferred tetracycline analogs are anhydrotetracycline (ate), doxycycline (dox), chlorotetracycline, oxytetracycline, hydrochloride tetracycline, or deoxytetracycline. In a more preferred embodiment, the tetracycline antibiotic is doxycycline.

[0038] The terms "tetracycline repressor element" or “TetR element”, also known as "tet repressor protein" and " tetracycline repressor protein", which are all used interchangeably herein, refers to a polypeptide that exhibits specific binding to an inducing agent; in particular tetracycline or a tetracycline analog, exhibits specific binding to at least one tet operator sequence when the tetracycline repressor protein is not bound by an inducing agent; and / or is capable of being displaced or competed off from a tetracycline operator by an inducing agent. The term “tetracycline repressor element" includes naturally-occurring (i.e., native) tetracycline repressor protein polypeptide sequences and functional derivatives thereof. In a particular embodiment of the carrier cell of the invention the TetR element is selected from a group consisting of: Tet A, Tet B, Tet C, Tet D, Tet E, Tet G, Tet H, Tet J, Tet 30 as described in Levy, S. B. et al., Antimicrob. Agents Chemother. 1999 43, 1523-1524 or a functionally equivalent variant thereof. In a more particular embodiment of the carrier cell of the invention the TetR element comprises a sequence according to SEQ ID NO: 1. In an even more particular embodiment of the carrier cell of the invention the TetR element consists of a sequence according to SEQ ID NO: 1.

[0039] SEQ ID NO: 1

[0040] MSRLDKSKVINSAL E LLNEVGI EGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAI EMLDRHHTHFCPL EGESWQDF LRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAF LCQQGFSLENALYALSAVGHF TLGCVLEDQEHQVAKE ERETPTTDSM

[0041] In another particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element encodes a sequence according to SEQ ID NO: 1. In another particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence comprises a sequence according to SEQ ID NO: 2. In another particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence consists of a sequence according to SEQ ID NO: 2.

[0042] SEQ ID NO: 2 ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAG GTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAA TAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTA GAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATC GCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATT AGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTT ACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTG ATAGTATG

[0043] The terms “functional variant” and “functionally equivalent variant” are interchangeable and are herein understood as all those peptides derived from TetR elements or peptides by means of modification, insertion and / or deletion of one or more amino acids, provided that the function of the TetR element, i.e. exhibiting specific binding to an inducing agent; exhibiting specific binding to at least one Tet operator sequence when the tetracycline repressor protein is not bound by an inducing agent; being displaced or competed off from a tetracycline operator by an inducing agent, is substantially maintained.

[0044] In one particular embodiment of the carrier cell of the invention, the functionally equivalent variants of the TetR element are those showing a degree of identity with respect to the SEQ ID NO: 1 or with respect to the sequence encoded by SEQ ID NO: 2, greater than at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. In another particular embodiment of the carrier cell of the invention, the functionally equivalent variants of the TetR element are those showing a degree of identity with respect to the sequence encoded by the expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element greater than at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the sequence encoded by the expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element is the sequence according to SEQ ID NO: 1.

[0045] The terms “identity”, “identical” or “percent identity” in the context of two or more amino acid or nucleotide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Publicly available software programs can be used to align sequences. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity “X” of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y / Z), where Y is the number of nucleotide residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the second sequence is longer than the first sequence, then the global alignment taken the entirety of both sequences into consideration is used, therefore all letters and null in each sequence must be aligned. In this case, the same formula as above can be used but using as Z value the length of the region wherein the first and second sequence overlaps, said region having a length which is substantially the same as the length of the first sequence.

[0046] For instance, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0047] In a particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence encoding the TetR element or a functionally equivalent variant thereof is a DNA sequence, wherein the DNA sequence is an expression vector. In another particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence encoding the TetR element or a functionally equivalent variant thereof is comprised in an expression vector.

[0048] The term “vector”, as used herein, refers to a construct capable of delivering, and optionally expressing, one or more polynucleotides of interest into a host cell, such as a plasmid. A person skilled in the art will understand that there is no limitation as regards the type of vector which can be used because said vector can be a cloning vector suitable for propagation and for obtaining the polynucleotides or expression vectors in different heterologous organisms suitable for purifying the fusion proteins of the invention. The term “cloning vector”, as used herein, refers to a vector suitable for propagation and to obtain the adequate polynucleotides or gene constructs or expression vectors in different heterologous organisms suitable for the purification of the vector. The term “expression vector”, as used herein, refers to a vector suitable for expression of a polynucleotide in a target cell. Thus, suitable vectors according to the present invention include expression vectors in superior eukaryotic cells based on viral vectors (adenoviruses, viruses associated to adenoviruses as well as retroviruses and lentiviruses) as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg pHCMV / Zeo, pCR3.1, pEFI / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6A / 5- His, pVAXI, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTI. In a particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence encoding the TetR element or a functionally equivalent variant thereof is a DNA sequence, wherein the DNA sequence is an expression vector selected from a group consisting of a plasmid, a retrovirus, an adenovirus, and a lentivirus. In a particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence encoding the TetR element or a functionally equivalent variant thereof is comprised in a lentiviral vector.

[0049] The vector of the invention can be used to transform, transfect, or infect cells which can be transformed, transfected or infected by said vector. Said cells can be prokaryotic or eukaryotic. By way of example, the vector wherein said DNA sequence is introduced can be a plasmid or a vector which, when it is introduced in a host cell, is integrated in the genome of said cell and replicates together with the chromosome (or chromosomes) in which it has been integrated. Said vector can be obtained by conventional methods known by the persons skilled in the art (Sambrook et al., 2001 , “Molecular cloning, to Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3 a). In a particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence encoding the TetR element is stably integrated into the genome of the cell.

[0050] In order for the TetR element or a functionally equivalent variant thereof to be expressed in the carrier cell, the TetR element expression is controlled by transcriptional elements which promote the transcription of the nucleic acid sequence encoding the T etR element. Examples of transcriptional elements which regulate the transcription are, without limitation, promoters and enhancers. The term “promoter” generally refers to a DNA sequence that is located upstream the coding region of a coding sequence, can be specifically identified and bound to by an RNA polymerase, and is required by transcription. The term “enhancer” refers to a DNA sequence that increases transcription frequency of the coding sequence interlocked therewith. The enhancer enhances the transcription by increasing the activity of a promoter. An enhancer may be located either at the 5' or the 3'end of a gene, and even may exist as an intron within a coding sequence. An enhancer might significantly affect gene expression, which might increase the gene transcription by 10-200 folds, or even by thousand times. In a particular embodiment of the carrier cell of the invention, the expressible nucleic acid sequence encoding the TetR element or a functionally equivalent variant thereof is a DNA sequence, wherein the DNA sequence is an expression vector, or is comprised in an expression vector, wherein the expression vector comprises a constitutively active promoter operably linked to the TetR element or to the functionally equivalent variant thereof. The expression “constitutively active”, as used herein, refers to a promoter that functions to continually activate the expression of the encoding sequence to which it is “operably linked”.

[0051] The carrier cell of the invention, as stated, comprises a recombinant replication- competent oncolytic human adenovirus genome comprising the sequence of at least one tetracycline operator (TetO) element.

[0052] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. In the context of the present invention the replication-competent oncolytic human adenovirus genome is recombinant due to the fact it comprises nucleic acid sequences which are not found in nature.

[0053] The term “replication-competent”, as used herein, refers to a virus that is capable of infecting and replicating within a particular host cell, in the present case the virus genome is capable of replicating and produce viral progeny in the carrier cell of the invention.

[0054] The term “oncolytic”, as used herein, refers to virus capable of selectively replicating in dividing cells (e.g., a proliferative cell such as a cancer cell) with the aim of slowing the growth and / or lysing said dividing cell, either in vitro or in vivo, while showing no or minimal replication in non-dividing cells. Typically, an oncolytic virus contains a viral genome packaged into a viral particle or virion and is infectious (i.e. capable of infecting and entering into a host cell or subject). As used herein, the term encompasses oncolytic adenoviruses as well as viral particles generated thereof. In some embodiments, oncolytic viruses are mammalian viruses that are designed and / or selected for their ability to selectively infect and kill transfected cancer cells, and by their ability to activate the host immune system against not only the virus, but also tumor antigens.

[0055] Therefore, in the context of the present invention, the expression “recombinant replication-competent oncolytic adenovirus genome” refers to a genome of an adenovirus which has been altered by genetic engineering and does not occur naturally, and which is capable of producing adenovirus progeny when the reproductive cycle is initiated and which is capable of selective replicating in dividing cells.

[0056] In a particular embodiment of the carrier cell of the invention, the recombinant replication- competent oncolytic adenovirus is a human adenovirus. The term “human adenovirus”, or “HAdV”, as used herein, encompasses any of the species of adenovirus known, from A to G, and its serotypes, in particular HAdV-A serotype 12, HAdV-A serotype 18, HAdV- A serotype 31 , HAdV-B serotype 12, HAdV-B serotype 3, HAdV-B serotype 7, HAdV-B serotype 11 , HAdV-B serotype 14, HAdV-B serotype 16, HAdV-B serotype 21 , HAdV-B serotype 34, HAdV-B serotype 35, HAdV-B serotype 50, HAdV-B serotype 55, HAdV-C serotype 1 , HAdV-C serotype 2, HAdV-C serotype 5, HAdV-C serotype 6, HAdV-C serotype 57, HAdV-D serotype 8, HAdV-D serotype 9, HAdV-D serotype 10, HAdV-D serotype 13, HAdV-D serotype 15, HAdV-D serotype 17, HAdV-D serotype 19, HAdV-D serotype 20, HAdV-D serotype 22, HAdV-D serotype 23, HAdV-D serotype 24, HAdV-D serotype 25, HAdV-D serotype 26, HAdV-D serotype 27, HAdV-D serotype 28, HAdV-D serotype 29, HAdV-D serotype 30, HAdV-D serotype 32, HAdV-D serotype 33, HAdV-D serotype 36, HAdV-D serotype 37, HAdV-D serotype 38, HAdV-D serotype 39, HAdV-D serotype 42, HAdV-D serotype 43, HAdV-D serotype 44, HAdV-D serotype 45, HAdV-D serotype 46, HAdV-D serotype 47, HAdV-D serotype 48, HAdV-D serotype 49, HAdV-D serotype 51 , HAdV-D serotype 53, HAdV-D serotype 54, HAdV-D serotype 56, HAdV-D serotype 58, HAdV-D serotype 59, HAdV-D serotype 60, HAdV-D serotype 62, HAdV-D serotype 63, HAdV-D serotype 64, HAdV-D serotype 65, HAdV-D serotype 67, HAdV-D serotype 69, HAdV-D serotype 70, HAdV-D serotype 71 , HAdV-D serotype 72, HAdV-D serotype 73, HAdV-D serotype 74, HAdV-D serotype 75, HAdV-E serotype 4, HAdV-F serotype 40, HAdV-F serotype 41 , and HAdV-G serotype 52.

[0057] In a particular embodiment of carrier cell of the invention, the recombinant replication- competent oncolytic human adenovirus is from a serotype selected from the group consisting of serotype 5, serotype 7 and serotype 12. In a more particular embodiment of carrier cell of the invention, the recombinant replication-competent oncolytic human adenovirus is from serotype 5.

[0058] The term “adenovirus genome” in the context of the present invention relates to linear, non-segmented double stranded DNA molecules of about 26-46 kilobases (Kbp) long containing between about 23-46 protein-coding genes. The genes present in the adenovirus genome are organized in transcription units, containing between 1-8 coding sequences. The transcription units are successively transcribed in the early, intermediate and late reproductive viral cycle. In particular the transcription units E1A, E1 B, E2A, E2B, E3 and E4 are transcribed early in the viral reproductive cycle.

[0059] The recombinant replication-competent oncolytic human adenovirus genome of the carrier cell of the invention is characterized by comprising a sequence of at least one tetracycline operator (TetO) element. The term “tetracycline operator” or “TetO”, as used herein, refers to nucleic acid sequence which is recognized by the TetR element, or a functionally equivalent variant thereof, and to which the TetR binds inhibiting the transcription of amino acid encoding sequences downstream of said TetO elements. In the present context the term TetO is meant to include all classes of TetO operators, such as, without limitation, operators from the classes Tet A, Tet B, Tet C, Tet D, Tet E, Tet G, Tet H, Tet J and Tet 30. In a particular embodiment of the carrier cell of the invention, the TetO element is selected from the group consisting of the TetO1 element (SEQ ID NO: 3), and the TetO2 element (SEQ ID NO: 4), preferably is the TetO2 element. In a particular embodiment of the carrier cell of the invention, the TetO element is the TetO1 element comprising a sequence according to SEQ ID NO: 3, and / or the TetO2 element comprising a sequence according to SEQ ID NO: 4. In yet another particular embodiment of the carrier cell of the invention, the TetO element is the TetO1 element consisting of a sequence according to SEQ ID NO: 3, and / or the TetO2 element consisting of a sequence according to SEQ ID NO: 4. In yet another particular embodiment of the carrier cell of the invention, the plurality of TetO elements comprises a combination of the TetO1 element and the TetO2 element, preferably both elements are alternating. The expression “elements are alternating” refers to the case wherein if the first element is TetO1 the second element will be TetO2, and subsequently, or vice-versa.

[0060] SEQ ID NO: 3

[0061] ACTCTATCATTGATAGAGT

[0062] SEQ ID NO: 4

[0063] TCCCTATCAGTGATAGAGA

[0064] According to the carrier cell of the invention, the at least one TetO element is located in the “promoter of the recombinant human adenovirus early region 1A (E1A) gene”. The expression is meant to encompass promoters for the E1A gene of all the variants of adenovirus previously described, as well as, cases wherein the promoter of a variant replaces the endogenous promoter of the recombinant adenovirus being used in the carrier cell of the invention. For example, a carrier cell comprising the genome of the recombinant replication-competent oncolytic human adenovirus from serotype 5 wherein the promoter of the E1A gene corresponds to the human adenovirus from serotype 7. In a particular embodiment of the carrier cell of the invention the promoter of the recombinant human adenovirus early region 1A (E1A) gene is the endogenous or wildtype promoter of the human adenovirus early region 1A (E1A) gene.

[0065] In another particular embodiment of the carrier cell of the invention the promoter of the recombinant human adenovirus early region 1A (E1 A) gene is a tissue specific promoter. The term “tissue-specific” is intended to mean that the promoter to which the gene essential for replication is operably linked functions specifically in that tissue so that replication proceeds in that tissue. This can occur by the presence in that tissue, and not in non-target tissues, of positive transcription factors that activate the promoter. It can also occur by the absence of transcription inhibiting factors that normally occur in nontarget tissues and prevent transcription as a result of the promoter. Thus, when transcription occurs, it proceeds into the gene essential for replication such that in a target tissue, replication of the vector and its attendant functions occur.

[0066] Tissue specificity is particularly relevant with respect to targeting an abnormal counterpart of a particular tissue type while avoiding the normal counterpart of the tissue, or avoiding surrounding tissue of a different type than the abnormal tissue, while treating the abnormal tissue. In a particular embodiment, the promoter is “tumour-specific”, which means that the promoter functions specifically in tumoural tissues. For example, the recombinant adenoviruses of the carrier cell of the invention can be useful for treating metastases to the liver. One specific example is colon cancer, which often metastasizes into the liver. It has been found that even when colon cancer metastasizes into the liver, the CEA promoter is active in the cells of the metastases but not in normal liver cells. Accordingly, normal human adult liver should not support replication of a virus that has viral genes essential for replication linked to the colon cancer CEA-specific promoter. Replication should occur in the primary cancer cells. Another example is the alphafetoprotein promoter, which is active only in hepatocellular carcinoma. A further example is the tyrosinase promoter, which is active only in melanoma and not in normal skin. In each case, replication is expected in the abnormal but not the normal cells.

[0067] Examples of tissue-specific promoters are, without limitation, alphafetoprotein promoter, DE3 promoter, tyrosinase promoter, carcinoembryonic antigen (CEA) promoter, surfactant protein promoter, E2F promoter, telomerase hTERT promoter, prostatespecific antigen promoter, COX-2 promoter, albumin gene promoter, the core promoter of hepatitis virus, the promoter of the globulin-binding protein which binds to thyroxine and ErbB2 promoter. In a particular embodiment of the carrier cell of the invention, the promoter of the recombinant human adenovirus early region 1A (E1A) gene is replaced from the group consisting of an E2F promoter, a telomerase hTERT promoter, a tyrosinase promoter, a prostate-specific antigen promoter, an alphafetoprotein promoter, and a COX-2 promoter.

[0068] In another particular embodiment of the carrier cell of the invention, the promoter of the recombinant human adenovirus early region 1A (E1A) gene is an altered endogenous promoter of the recombinant human adenovirus early region 1A (E1A) gene, wherein said promoter has been altered by the insertion of elements into the promoter nucleotide sequence which allow the promoter to gain specific characteristics such as a tissuespecific promoter characteristics and / or a tumour-specific promoter characteristics. Examples of said elements are specific several transcription factor binding sites such as binding sites for the hypoxia induced factor (HIF-1), the Ets transcription factor, the T- cell factor / lymphoid enhancer factor (tcf / lef) (as disclosed in Fuerer C and Iggo R. 2002. Gene Ther, 9(4):270-81), the E2F transcription factor or the Sp1 transcription factor. In a particular embodiment of the carrier cell of the invention the promoter of the recombinant human adenovirus early region 1A (E1A) has been altered by the insertion of at least one, at least two, at least three, at least four or more copies of a nucleic acid element selected from a group consisting of: hypoxia induced factor (HIF-1), the Ets transcription factor, the T-cell factor / lymphoid enhancer factor (tcf / lef), the E2F transcription factor or the Sp1 transcription factor. In a more particular embodiment the promoter of the recombinant human adenovirus early region 1 A has been altered by the insertion of four E2F-binding site hairpins and one Sp-1 binding site following nucleotide 415 in the E1A promoter. In a particular embodiment of the carrier cell of the invention the promoter of the recombinant human adenovirus early region 1A (E1A) has been altered by the insertion of at least one, at least two, at least three, at least four or more copies of the nucleic acid element according to SEQ ID NO: 14.

[0069] SEQ ID NO : 14

[0070] AGATCAAAGGG

[0071] In a further particular embodiment of the method of the invention, the TetO element present in the promoter of the recombinant human adenovirus early region 1A (E1A) gene is flanked by an insulator. The term "insulator" refers to a genetic sequence which blocks the interaction between promoters and enhancers. In a further embodiment, the insulator is a chromatin insulator. In a further particular embodiment of the carrier cell of the invention, the promoter of the recombinant human adenovirus early region 1A (E1A) gene comprises at least one insulator. This helps to prevent promoter interference (i.e. where the promoter from one transcription unit impairs or promotes expression of an adjacent transcription unit) between adjacent viral nucleic acid sequences. This is also thought to help minimise the risk of recombination between viral sequences to generate replication-competent virus. Furthermore, it is also thought to help reduce silencing of nearby expression cassettes when they are stably integrated into the cells genome (Moriarity et al., (2013) Nucleic Acids Res. 41 : e92; Yahata et al., (2007). In addition, the presence of insulators further prevents the leakage expression of the promoter of the recombinant human adenovirus early region 1A (E1A) gene. The term “leakage expression” refers to the basal expression, or non-regulated expression, present in a promoter which leads to very low levels of expression of the gene which is controlled by the promoter. The term is meant to further include the expression of the gene due to the action of other promoters or enhancers which interact in cis or trans leading to the undesired expression of the gene. Examples of such insulators are, without limitation, the CTCF insulator, the gypsy insulator, and the p-globin locus. In a particular embodiment of the carrier cell of the invention, the at least one TetO element is flanked by at least two insulators, wherein the insulator is selected from the CTCF insulator, the gypsy insulator, and the p-globin locus. In another particular embodiment of the carrier cell of the invention, the at least TetO element is flanked by at least two insulators wherein the insulators have the sequence according to SEQ ID NO: 13, preferably wherein the insulator according to SEQ ID NO: 13 has been inserted at position 385 of the recombinant human adenovirus genome, wherein the positions refers to the nucleotide positions defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007. In a particular embodiment of the carrier cell of the invention, the promoter of the recombinant human adenovirus early region 1 A (E1A) gene comprises at least one insulator, wherein the insulator is selected from the CTCF insulator, the gypsy insulator, and the p-globin locus. In another particular embodiment of the carrier cell of the invention, the promoter of the recombinant human adenovirus early region 1A (E1A) gene comprises at least one insulator wherein the insulator has the sequence according to SEQ ID NO: 13, preferably wherein the insulator according to SEQ ID NO: 13 has been inserted at position 385 of the recombinant human adenovirus genome, wherein the positions refers to the nucleotide positions defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007.

[0072] SEQ ID NO : 13 tcgagaccctgaaactgtcttcgactccggggccccgttggaagactgagtgcccggggcacggcacaga agccgcgcccaccgcctgccagttcacaaccgctccgagcgtgggtctccgcccagctccagtcctgtga tccgggcccgccccctagcggccggggagggaggggccgggtccgcggccggcgaacggggctcgaaggg tccttgtagccgggaatgctgctgctgctgctggggggatcacagaccatttctttctttcggccaggct gaggccctgacgtggatgggcaaactgcaggcctgggaaggcagcaagccgggccgtccgtgttccatcc tccacgcacccccacctatcgttggttcgcaaagtgcaaagctttcttgtgcatgacgccctgctctggg gagcgtctggcgcgatctctgcctgcttactcgggaaatttgcttttgccaaacccgctttttcggggat cccgcgcccccctcctcacttgcgctgctctcggagccccagccggctccgcccgcttcggcggtttgga tatttattgacctcgtcctccgactcgctgacaggctacaggacccccaacaaccccaatccacgttttg gatgcactgagaccccgacattcctcggtatttattgtctgtccccacctaggacccccacccccgaccc tcgcgaataaaaggccctccatctgcccc

[0073] In a particular embodiment of carrier cell of the invention, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve TetO element is located in the region comprised after the last viral packaging sequences and before the E1A TATA box or in the region comprised after the E1A TATA box and before the transcription start site of the E1 A gene. In another particular embodiment of the carrier cell of the invention, the recombinant human adenovirus genome comprises the sequence of a plurality of TetO elements, preferably between 2 and 14 TetO elements, more preferably 7 TetO elements. In another particular embodiment of the carrier cell of the invention, the recombinant replication-competent oncolytic human adenovirus genome comprises 7 TetO elements according to the sequence SEQ ID NO: 5. In a particular embodiment of the carrier cell of the invention, the seven TetO elements comprise a sequence according to SEQ ID NO: 5. In a further particular embodiment of the carrier cell of the invention, the seven TetO elements consist of a sequence SEQ ID NO: 5.

[0074] SEQ ID NO: 5

[0075] GCCCTTTCGTCTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG AGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCC TATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAG AAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG AGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCC TATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACC

[0076] The term “viral packaging sequence”, also known as “signal i ” or “encapsulation sequence”, as used herein, refers to the sequences of viral nucleic acid which direct packaging comprising between about 200 and 400 nucleotides and are situated at the N-terminus of the viral genome before the gene E1A of the genome.

[0077] The term “E1A gene”, as used herein, refers to the immediate early gene of the adenovirus genome firstly transcribed following infection and replication. The term includes both endogenous E1A genes, i.e. wild-type E1A gene, and recombinant E1A genes, wherein the gene has been modified and does not correspond to the wild type genome sequence. The E1A gene coding region spans the nucleotides positions of adenovirus genome 560-1542, wherein the positions refers to the nucleotide positions defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007. In a particular embodiment of the carrier of the cell, the E1A gene of the adenovirus is the endogenous E1 A adenovirus gene, preferably wherein the endogenous E1 adenoviral gene is under the control of the E1A endogenous adenoviral promoter. In another particular embodiment of the carrier of the cell, the E1A gene comprises a sequence according to the region positions of adenovirus genome 560- 1545, wherein the positions refers to the nucleotide positions defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007.

[0078] In a particular embodiment the E1A gene of the adenovirus is modified to render the adenovirus oncolytic. In a more particular embodiment of the carrier cell of the invention, the E1A gene of the adenovirus comprises a deletion of 24 nucleotides of the region responsible for binding the Rb protein. In another particular embodiment of the carrier cell of the invention, the E1A gene of the adenovirus comprises a deletion of 24 nucleotides which spans the region between positions 923 to 946 of the recombinant human adenovirus genome, wherein the positions refers to the nucleotide positions defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007.

[0079] The term “TATA box” according to the invention, refers to a sequence of nucleotides that serves as the main recognition site for the attachment of RNA polymerase in the promoter region of eukaryotic genes. Located at around 25 nucleotides before the start of transcription, it consists of the seven-base consensus sequence TATAAAA. In particular embodiment of the carrier cell of the invention, the recombinant replication- competent oncolytic adenovirus genome comprises a TATA box located at about positions 468 to 475 of the genome of the human adenovirus type 5 wherein the positions of the nucleotide sequence of the genome are defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007. In particular embodiment of the carrier cell of the invention, the recombinant replication- competent oncolytic adenovirus genome comprises a TATA box with a sequence according to TATTTATA. In another particular embodiment of the carrier cell of the invention, the recombinant replication-competent oncolytic adenovirus genome consists of a TATA box with a sequence according to TATTTATA.

[0080] In a particular embodiment of the carrier of the cell, the at least one TetO element is located between positions 378 and 467, or between positions 476 and 559 of the genome of the human adenovirus type 5 wherein the positions of the nucleotide sequence of the genome are defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007. Located at the region of the positions 468 to 476 of the genome of the human adenovirus type 5 is the TATA box sequence, while from position 560 until position 1545, as previously mentioned is the gene E1A, wherein the positions of the nucleotide sequence of the genome are defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007

[0081] In a particular embodiment of the carrier of the cell, the at least one TetO element is placed immediately after the E1A TATA box. In another particular embodiment of the carrier cell of the invention, wherein the at least one TetO element replaces the 24 base pairs immediately after the E1A TATA box, preferably replaces the nucleotides 476 to 499 of the genome of the human adenovirus type 5 wherein the positions of the nucleotide sequence of the genome are defined according to the GenBank database entry with accession number AY339865, version of 13 August 2007.

[0082] In a particular embodiment, the recombinant replication-competent oncolytic human adenovirus genome comprises a fiber modified by incorporating an integrin binding RGD- 4C motif into the HI loop.

[0083] In a particular embodiment of the carrier cell of the invention, the recombinant replication- competent oncolytic human adenovirus genome comprises a sequence according to SEQ ID NO: 8. In another particular embodiment of the carrier cell of the invention, the recombinant replication-competent oncolytic human adenovirus genome consists of a sequence according to SEQ ID NO: 8.

[0084] A further aspect of the invention relates to a recombinant replication-competent oncolytic human adenovirus genome, from here onwards the recombinant replication-competent oncolytic human genome of the invention, comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising the sequence encoding the TetR element. In a particular embodiment of the recombinant replication-competent oncolytic human genome of the invention, the genome comprises a sequence according to SEQ ID NO: 8. In another particular embodiment of the recombinant replication-competent oncolytic human genome of the invention, the genome consists of a sequence according to SEQ ID NO: 8.

[0085] A further aspect of the invention relates to an adenovirus comprising the recombinant replication-competent human oncolytic adenovirus genome of the invention. All the definitions and embodiments previously described in relation to the first aspect of the invention are equally valid for this further aspect.

[0086] Methods of the invention

[0087] All the definitions and embodiments previously described in relation to other aspects are equally valid for the current aspects.

[0088] A further aspect of the invention refers to a method for obtaining the carrier cell according to the invention, from here onwards the method of the invention, comprising the steps of: i) introducing the expressible nucleic acid sequence encoding the TetR element, or a functionally equivalent variant thereof, into the carrier cell; and ii) infecting the carrier cell obtained in step i) with a recombinant replication- competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant replication-competent oncolytic human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element nor a functionally equivalent variant thereof.

[0089] The expression “introducing the expressible nucleic acid sequence encoding the tetR element into the carrier cell”, as used herein, refers to the process of introducing into a cell the mentioned sequence by any method which allows said introducing such as transformation, transfection or infection by conventional methods known by the persons skilled in the art (Sambrook et al., 2001 , “Molecular cloning, to Laboratory Manual”, 2nded., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3 a).

[0090] The expression “infecting the carrier cell obtained in step i) with a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element”, as used herein, refers to a process of infecting a cell obtained from step i) with an adenovirus as described according to conventional methods known by the skilled person in the art (Sambrook et al., 2001 , “Molecular cloning, to Laboratory Manual”, 2nded., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3 a).

[0091] In a particular embodiment of the method of the invention, step ii) is carried out at a multiplicity of infection (MOI) of at least 50 infectious units per cell (lU / cell). The term multiplicity of infection (MOI) or concentration of transducing units (TU) are used interchangeable herein and refers to the number of viruses or virus particles that infect a single cell on average.

[0092] One example of carrying out the method of the invention is described in the Examples present in this description.

[0093] Expression system of the invention

[0094] All the definitions and embodiments previously described in relation to other aspects are equally valid for the current aspects.

[0095] A further aspect of the present invention relates to an expression system, from here onwards the expression system of the invention, comprising: i) a carrier cell; ii) an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and iii) a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element nor a functionally equivalent variant thereof.

[0096] In a particular embodiment of the expression system of the invention if further comprises a tetracycline antibiotic, preferably doxycycline.

[0097] Compositions and therapeutic uses of the invention

[0098] All the definitions and embodiments previously described in relation to other aspects are equally valid for the current aspects.

[0099] The carrier cell of the invention can be part of a composition comprising further elements which are useful in a composition used for treatment. Therefore, another aspect of the present invention relates to a pharmaceutical composition comprising the carrier cell of the invention and a pharmaceutically acceptable excipient, from herein after the pharmaceutical composition of the invention. The term “excipient” refers to a substance that aids the absorption of the carrier cell or any of the components or compounds of the pharmaceutical composition of the invention, or the components or compounds and / or aids the preparation of the pharmaceutical composition in the sense of giving it consistency or flavours to make it more palatable. Thus, excipients may have the function, by way of example, but not limited to, binding the components (e.g. starches, sugars or cellulose), sweetening, colouring, protecting the active substance (e.g. to insulate it from air and / or moisture), filling a pill, capsule or any other presentation or a disintegrating function to facilitate the dissolution of the components, not excluding other excipients not listed in this paragraph. The term ‘excipient’ is therefore defined as a material which, included in the dosage forms, is added to the active substances or their associations to enable their preparation and stability, to modify their organoleptic properties or to determine the physical and chemical properties of the pharmaceutical composition and their bioavailability.

[0100] The expression “pharmaceutically acceptable excipient”, as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible with the carrier cell of the invention.

[0101] The “dosage form” is the configuration to which the active ingredients and excipients are adapted to provide a pharmaceutical composition or medicinal product. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered.

[0102] The carrier cell of the invention or the pharmaceutical composition of the invention may be specially formulated for administration in liquid form, including those adapted for the following: (a) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes, mouthwash or hydrogels, (b) parenteral administration, for instance, by subcutaneous, intramuscular or intravenous injection of, for example, a sterile solution or suspension, (c) intracavity administration (e.g. intraperitoneal instillation), intravesical (i.e. urinary bladder) instillation, (d) intraorgan administration (e.g. intraprostatical administration), I topical application (i.e. cream, ointment or spray applied to the skin), (f) intravaginal or intrarectal administration (e.g. as a pessary, cream, foam, enema or suppository) or (g) aerosol (e.g. as an aqueous aerosol, liposomal preparation or solid particles containing the agent (s)). In a particular embodiment, the carrier cell of the invention or the pharmaceutical composition of the invention may be specially formulated for intravenous administration, intramuscular administration and / or intraperitoneal administration.

[0103] It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0104] In addition to what is described above, the present invention also covers the possibility that the pharmaceutical composition of the invention may be administered to a subject together with other components or compounds, even if these do not form part of the pharmaceutical composition of the invention.

[0105] The carrier cell of the invention, the expression system of the invention, or the pharmaceutical composition of the invention find use as a medical product for the treatment of a disease such as cancer. Hence, a further aspect of the present invention relates to a carrier cell of the invention, an expression system of the invention or a pharmaceutical system of the invention for use in medicine.

[0106] Another aspect of the present invention relates to a carrier cell of the invention, an expression system of the invention or a pharmaceutical system of the invention for use in the prevention and / or treatment of solid tumors, from here onwards the treatment use of the invention.

[0107] As used herein, the term “treating” (or “treat” or “treatment”) refers to processes involving a slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related symptoms, conditions, or disorders. The treatment of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The “treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject / patient suffering from the disorder or disease. Accordingly, the “treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject / patient may experience a broad range of responses to a treatment.

[0108] The terms “prevention”, “to prevent”, “preventing” or “prophylaxis”, as used herein, include, without limitation, the capacity to prevent, decrease, reduce, minimize, or delay the onset or development of a disease or condition before its onset. It also refers ameliorating the risk of a symptom, clinical sign, disorder, condition, or disease. The term prevention also includes protecting the subject from a symptom, disorder, condition, or disease. In the context of the present invention the prevention refers to the prevention of cancer.

[0109] The term "solid tumor", as used herein, refers to a malignant solid tumor. Although the term includes various types of solid tumors named for the cell types that form them, namely sarcomas, carcinomas, and lymphomas, the term does not include leukemia. In various embodiments, the term "solid tumor" refers to cancers (called sarcomas) that arise from connective or supporting tissue (e.g., bone or muscle), cancers that arise from the body's glandular cells and the epithelial cells that line the body, cancers of lymphoid organs such as the lymph nodes, spleen, and thymus (called lymphomas). Lymphoid cells are present in almost all tissues, and therefore lymphomas can develop in a variety of organs. As used herein the term “solid tumor” includes, but is not limited to, colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma, hepatocellular carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, basal cell carcinoma, breast cancer, cutaneous squamous cell carcinoma, chondrosarcoma, Includes cancers including angiosarcoma, cholangiocellular carcinoma, soft tissue sarcoma, colorectal cancer, melanoma, Merkel cell carcinoma, and glioblastoma multiforme.

[0110] The term “cancer”, as used herein, refers to a disease characterized by uncontrolled cell division (or by an increase of survival or apoptosis resistance) and by the ability of said cells to invade other neighbouring tissues (invasion) and spread to other areas of the body where the cells are not normally located (metastasis) through the lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body. Depending on whether or not they can spread by invasion and metastasis, tumors are classified as being either benign or malignant: benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they only grow locally; whereas malignant tumors are tumors that are capable of spreading by invasion and metastasis. Biological processes known to be related to cancer include angiogenesis, immune cell infiltration, cell migration and metastasis. As used herein, the term cancer includes, but is not limited to, the following types of cancer: breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; haematological neoplasms including acute lymphocytic and myelogenous leukemia; T- cell acute lymphoblastic leukemia / lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia / lymphoma; intraepithelial neoplasms including Bowen’s disease and Paget’s disease; liver cancer; lung cancer; lymphomas including Hodgkin’s disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma, Kaposi’s sarcoma, basal cell carcinoma, retinoblastoma and squamous cell cancer; testicular cancer including germinal tumours such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumours, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor.

[0111] In a particular embodiment of the treatment use of the invention the solid tumor is lung cancer.

[0112] The term “lung cancer”, as used herein, refers to a neoplasm, e.g., a malignant neoplasm, of the lung within a given subject, wherein the neoplasm is of epithelial origin (i.e., carcinoma of the lung). Lung carcinomas are categorized by the size and appearance of the malignant cells and the term “lung cancer” includes both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The term “lung cancer” includes both localized and metastasized lung cancer. The term “lung cancer” can be qualified by the terms “localized” or “metastasized” to differentiate between different types of tumors, where “localized” refers to the original mother tumor, and “metastasized” refers to the tumors that have spread from the original mother tumor.

[0113] In another particular embodiment of the treatment use of the invention the lung cancer is adenocarcinoma. The term “adenocarcinoma” as used herein refers to a type of non-small cell lung cancer that forms in cells that secrete mucus and other substances, usually developing in the outer portions of the lungs.

[0114] In a particular embodiment of the treatment use of the invention, the treatment is of a subject.

[0115] The term “subject”, as used herein, refers to an individual, plant or animal, such as a human, a nonhuman primate (e.g., chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs. The term does not denote a particular age or sex. In a preferred embodiment of the invention, the subject is a human.

[0116] Methods of treatment

[0117] All the definitions and embodiments previously described in relation to other aspects are equally valid for the current aspects.

[0118] A further aspect of the present invention relates to a method for the prevention and / or treatment of solid tumors in a subject, from here onwards the method of treatment of the invention comprising: i) administering to the subject the carrier cell of the invention or the pharmaceutical composition of the invention; and then ii) administering to the subject a tetracycline antibiotic.

[0119] In a particular embodiment of the method of treatment of the invention the administration of step i) and / or of step ii) is intratumoral, intraperitoneal, intravenous, subcutaneous, oral, mucosal, or rectal administration, more preferably the administration of step i) is intravenous and the administration of step ii) is oral.

[0120] In a further particular embodiment of the method of treatment of the invention the administration of step i) and / or step ii) is a single dose administration or a multiple dose administration.

[0121] The term “multiple dose”, as used herein, refers to two, three, four, five, six, seven, eight, nine, ten or more doses administered to the subject. It will be evident to the skilled person in the art that the requirement of the number of doses will be determined by a variety of factors such as, without limitation, the subject, the stage of the disease, the requirements of the administration. In a particular embodiment of the method of treatment of the invention the administration is a two, three, four, five, six or more dose administration. In a particular embodiment of the method of treatment of the invention the dose of carrier cells is of between 1x102to 1x1O10cells / kg body weight, preferably between 1x104to 1x108cells / kg body weight, even more preferably 0.5x106cells / kg body weight.

[0122] In another particular embodiment of the method of treatment of the invention the dose of the tetracycline antibiotic is of between 2.5 mg / kg to 100 mg / kg body weight, preferably between 5 mg / kg to 50 mg / kg body weight, more preferably 25 mg / kg body weight.

[0123] In a particular embodiment of the method of treatment of the invention the tetracycline antibiotic is tetracycline or doxycycline, preferably doxycycline

[0124] As previously stated the carrier cell of the invention allows to determine the onset of the replication of the replication-competent oncolytic adenovirus genome. Therefore, step ii)) of the method of treatment of the invention can be delayed until the carrier cell reaches and accumulates in the target cells, in order to increase the therapeutic effect of the oncolytic adenovirus. Therefore, in a particular embodiment of the method of treatment of the invention, step ii) is carried out at least 72 hours after step i), preferably at least 96 hours after step i).

[0125] In a particular embodiment of the method of treatment of the invention, the subject is a mammal, preferably a human.

[0126] In a particular embodiment of the method of treatment of the invention, the solid tumor is selected from the group consisting of: lung cancer and neuroblastoma.

[0127] In a further particular embodiment of the method of treatment of the invention, the lung cancer is lung adenocarcinoma.

[0128] Kit of the invention

[0129] An aspect of the present invention relates to a kit, from here onwards the kit of the invention, selected from the group consisting of: i) a kit comprising the carrier cell of the invention; and ii) a kit comprising an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element. The term “kit” in the context of the current invention is understood as a product of the different reagents for performing the methods and / or treatments described in the present invention, both in those cases in which the kit comprises the carrier cell of the invention and in the case where it comprises the expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element, in which the different reagents are packaged together to allow for transport and storage. Nevertheless, if the kits defined in the present invention do not comprise the reagents necessary for putting the methods of the invention into practice, such reagents are commercially available and can be found as part of a kit. Suitable materials for packaging the components of the kit include, without being limited to, glass, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, sachets and the like. Kits can additionally contain instructions for using the different components in the kit. Said instructions can be in printed format or in an electronic device capable of storing instructions such that they can be read by a person, such as electronic storage media (magnetic discs, tapes and the like), optical means (CD-ROM, DVD, USB) and the like. The media can additionally or alternatively contain Internet addresses where said instructions are provided.

[0130] In a particular embodiment of the kit of the invention, the kit further comprises a tetracycline antibiotic.

[0131] In another particular embodiment of the kit of the invention, the kit according to option ii) further comprises a carrier cell, preferably the carrier cell.

[0132] All the definitions and embodiments previously described in relation to other aspects are equally valid for the kits of the invention.

[0133] The following aspects further describe the present invention:

[0134] 1 . A carrier cell comprising: i) an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and ii) a recombinant replication-competent oncolytic human adenovirus genome comprising the sequence of at least one tetracycline operator (TetO) element, wherein the expressible nucleic acid sequence of item (i) is not present in the recombinant replication-competent oncolytic human adenovirus genome of item (ii), and wherein the at least one TetO element is located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene.

[0135] 2. The carrier cell according to claim 1 , wherein the cell is selected from the group consisting of: adult stem cells, embryonic stem cells, fetal stem cells, neural stem cells, mesenchymal stem cells, totipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, adipose stromal stem cells, endothelial stem cells, adult peripheral blood stem cells, myoblasts, small juvenile stem cells, skin fibroblast stem cells, vascular stem cells, epithelial stem cells, and embryonic epithelial stem cells.

[0136] 3. The carrier cell according to claim 2, wherein the cell is a mesenchymal stem cell.

[0137] 4. The carrier cell according to claim 3, wherein the mesenchymal stem cell is obtained from menstrual blood.

[0138] 5. The carrier cell according to any one of claims 1 to 4, wherein the cell is a human cell.

[0139] 6. The carrier cell according to any one of claims 1 to 5, wherein the cell is permissive to viral infection and replication.

[0140] 7. The carrier cell according to any one of claims 1 to 6, wherein the expressible nucleic acid sequence encoding the TetR element or the functionally equivalent variant thereof is comprised in an expression vector.

[0141] 8. The carrier cell according to claim 7, wherein the expression vector is a lentiviral vector.

[0142] 9. The carrier cell according to any one of claims 1 to 8, wherein the recombinant replication-competent oncolytic human adenovirus genome is from a serotype selected from the group consisting of serotype 5, serotype 7 and serotype 12.

[0143] 10. The carrier cell according to claim 9, wherein the recombinant replication- competent oncolytic human adenovirus genome is from serotype 5.

[0144] 11 . The carrier cell according to any one of claims 1 to 10, wherein the at least one TetO element is located in the region comprised after the last viral packaging sequences and before the E1A TATA box or in the region comprised after the E1A TATA box and before the transcription start site of the E1A gene.

[0145] 12. The carrier cell according to claim 11 , wherein the at least one TetO element is located between positions 378 and 467, or between positions 476 and 559 of the genome of the human adenovirus type 5 wherein the positions of the nucleotide sequence of the genome are defined according to the Genbank database entry with accession number AY339865, version of 13 August 2007.

[0146] 13. The carrier cell according to any one of claims 11 or 12, wherein the at least one TetO element is placed immediately after the E1A TATA box.

[0147] 14. The carrier cell according to claim 13, wherein the at least one TetO element replaces the 24 base pairs immediately after the E1A TATA box, preferably replaces the nucleotides 476 to 499 of the genome of the human adenovirus type 5 wherein the positions of the nucleotide sequence of the genome are defined according to the GenBank database entry with accession number AY339865, version of 13 August 2007.

[0148] 15. The carrier cell according to any one of claims 1 to 14, wherein the recombinant replication-competent oncolytic human adenovirus genome comprises the sequence of a plurality of TetO elements, preferably between 2 and 14 TetO elements, more preferably 7 TetO elements.

[0149] 16. The carrier cell according to any one of claims 1 to 15, wherein the TetO element is selected from the group consisting of the TetO1 element (SEQ ID NO: 3), and the TetO2 element (SEQ ID NO: 4), preferably is the TetO2 element.

[0150] 17. The carrier cell according to any one of claims 15 to 16, wherein the plurality of TetO elements comprises a combination of the TetO1 element and the TetO2 element, preferably both elements are alternating.

[0151] 18. The carrier cell according to any one of claims 1 to 17, wherein the E1A gene of the adenovirus is the endogenous E1A adenovirus gene, preferably wherein the endogenous E1 adenoviral gene is under the control of the E1A endogenous adenoviral promoter.

[0152] 19. The carrier cell according to any one of claims 1 to 18, wherein the recombinant replication-competent oncolytic human adenovirus genome comprises a sequence according to SEQ ID NO: 8.

[0153] 20. The carrier cell according to any one of claims 1 to 19, wherein the expression vector comprises a constitutively active promoter operably linked to the TetR element or the functionally equivalent variant thereof.

[0154] 21. The carrier cell according to any one of claims 1 to 20, wherein the expressible nucleic acid sequence encoding the TetR element or the functionally equivalent variant thereof is an RNA sequence or a DNA sequence.

[0155] 22. The carrier cell according to claim 21 wherein the DNA sequence is an expression vector selected from a group consisting of a plasmid, a retrovirus, an adenovirus, and a lentivirus. 23. The carrier cell according to any one of claims 1 to 22, wherein the nucleic acid sequence encoding the TetR element or the functionally equivalent variant thereof is stably integrated into the genome of the cell.

[0156] 24. A method for obtaining the carrier cell according to any one of claims 1 to 23 comprising the steps of: i) introducing the expressible nucleic acid sequence encoding the tetR element or the functionally equivalent variant thereof into the carrier cell; and ii) infecting the carrier cell obtained in step i) with a recombinant replication- competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element nor the functionally equivalent variant thereof.

[0157] 25. The method according to claim 24, wherein step ii) is carried out at a multiplicity of infection (MOI) of 50 infectious units per cell (lll / cell).

[0158] 26. An expression system comprising: i) a carrier cell; ii) an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and iii) a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the TetR element nor the functionally equivalent variant thereof.

[0159] 27. The expression system according to claim 26, further comprising a tetracycline antibiotic, preferably doxycycline.

[0160] 28. A pharmaceutical composition comprising the carrier cell according to any one of claims 1 to 23 and a pharmaceutically acceptable excipient.

[0161] 29. A carrier cell according to any one of claims 1 to 23, an expression system according to any one of claims 26 or 27, or a pharmaceutical composition according to claim 28 for use in medicine.

[0162] 30. A carrier cell according to any one of claims 1 to 23, an expression system according to any one of claims 26 or 27 or a pharmaceutical composition according to claim 28 for use in the prevention and / or treatment of solid tumors. 31. A carrier cell, an expression system or a pharmaceutical composition for use according to claim 30, wherein the solid tumor is selected from the group consisting of: lung cancer, and neuroblastoma.

[0163] 32. A carrier cell, an expression system or a pharmaceutical composition for use according to claim 31 , wherein the lung cancer is lung adenocarcinoma.

[0164] 33. A method for the prevention and / or treatment of solid tumors in a subject comprising: i) administering to the subject the carrier cell according to any one of claims

[0165] 1 to 23 or the pharmaceutical composition according to claim 28; and then ii) administering to the subject a tetracycline antibiotic.

[0166] 34. The method according to claim 33, wherein the administration of step i) and / or of step ii) is intratumoral, intraperitoneal, intravenous, subcutaneous, oral, mucosal, or rectal administration, more preferably the administration of step i) is intravenous and the administration of step ii) is oral.

[0167] 35. The method according to any one of claims 33 or 34, wherein the administration of step i) and / or step ii) is a single dose administration or a multiple dose administration.

[0168] 36. The method according to claim 35, wherein the multiple dose administration is a two, three, four, five, six or more dose administration.

[0169] 37. The method according to any one of claims 33 to 36 wherein the dose of carrier cells is of between 1x102to 1x1O10cells / kg body weight, preferably between 1x104to 1x108cells / kg body weight, even more preferably 0.5x106cells / / kg body weight.

[0170] 38. The method according to any one of claims 33 to 37, wherein the dose of tetracycline antibiotic is of between 2.5 mg / kg to 100 mg / kg body weight, preferably between 5 mg / kg to 50 mg / kg body weight, more preferably 25 mg / kg body weight body weight.

[0171] 39. The method according to any one of claims 33 to 38, wherein the tetracycline antibiotic is tetracycline or doxycycline, preferably doxycycline.

[0172] 40. The method according to any one of claims 33 to 39, wherein step ii) is carried out at least 72 hours after step i), preferably 96 hours after step i).

[0173] 41. The method according to any one of claims 33 to 40, wherein the subject is a mammal, preferably a human.

[0174] 42. The method according to any one of claims 33 to 41 , wherein the solid tumor is selected from the group consisting of: lung cancer, and neuroblastoma.

[0175] 43. The method according to claim 42, wherein the lung cancer is lung adenocarcinoma. 44. A kit selected from the group consisting of: i) a kit comprising the carrier cell according to any one of claims 1 to 23; and ii) a kit comprising an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the TetR element nor the functionally equivalent variant thereof.

[0176] 45. The kit according to claim 44, further comprising a tetracycline antibiotic.

[0177] 46. The kit according to claim 44(H) or 45 in the context of claim 44(H), further comprising a carrier cell.

[0178] 47. A recombinant replication-competent oncolytic human adenovirus genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising the sequence encoding the TetR element nor the functionally equivalent variant thereof.

[0179] 48. An adenovirus comprising the recombinant replication-competent oncolytic human adenovirus genome of claim 47.

[0180] The following examples are provided as merely illustrative and are not to be construed as limiting the scope of the invention.

[0181] Examples

[0182] Material and Methods

[0183] Cell lines and viruses

[0184] The cell lines A549 and HEK293 were purchased from the American Type Culture Collection and maintained with Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C, 5% CO2. Cells were routinely tested for mycoplasma presence.

[0185] Isolation of human menstrual blood-derived mesenchymal stem cells: Menstrual blood (1-5ml) was collected from healthy female donors (n=7), aged 23-42 years, on the first 3 days of the menstrual phase using a menstrual cup (Mooncup, Brighton, UK). The protocol and cell donation for research purposes was approved by the Institutional Ethics Committee and written consent was signed for each donor. Blood samples were transferred to a sterile 50ml centrifuge tube with conical bottom, filled with sterile phosphate buffered saline (PBS, Life Technologies, Carlsbad, CA, USA), and centrifuged at 1 ,500 rpm for 5 minutes. The pellet was resuspended in a-MEM (Life Technologies) containing 20% Fetal Bovine Serum, 1% penicillin / streptomycin, and 1% gentamicin / amphotericin B (all from Life Technologies), and seeded overnight in culture flasks (Corning Inc., Corning, NY) at 37°C in a 5% CO2 atmosphere.

[0186] Next day, cell monolayer was washed once with PBS to remove non-adherent cells and cellular debris and replaced with fresh medium. Finally, MenSCs were amplified for 7-24 days (passage 0), substituting the culture medium every 3-4 days, until 75%-90% confluent, and then passaged periodically by detachment (0.25% trypsin-1 mM EDTA in PBS (Life Technologies)) after achieving a subconfluent monolayer.

[0187] MenSCs-TetR (cells expressing the Tet repressor) were generated by sorting A549 and MenSCs cells transduced with a lentiviral vector encoding the TetR (SEQ ID NO: 1) and GFP. This lentiviral vector is pHIV7-TetR-IRES-eGFP (Aagaard L et al. 2007. Molecular Therapy, 15(5): 938-945).

[0188] The oncolytic adenovirus ICOVIR15 had been previously described (Rojas JJ, et al., 2010, Molecular Therapy 18:1960-1971). Briefly, ICOVIR15 was created by inserting four E2F-binding site hairpins and one Sp-1-binding site following nucleotide 415 in the E1a promoter of AdA24-RGD, a recombinant adenovirus which has a 24-bp deletion in the CR2 of the E1A gene and a fiber modified by incorporating an integrin binding RGD- 4C motif into the HI loop (Bauerschmitz GJ, et al., 2002, Cancer Res 62:1266-1270). ICOVIR15-Luc (SEQ ID NO: 9) contains a splicing acceptor signal connected to the firefly luciferase gene inserted after the fiber gene of ICOVIR15 (Rincon E, et al., 2017, Oncotarget 8:45415-45431). The replication-controlled viruses ICOVIR15-R7 (SEQ ID NO: 8) and ICOVIR15-R7-Luc (SEQ ID NO: 10) were generated by incorporating 7 TetO2 repeats (TetO2 sequence: SEQ ID NO: 4, 7 TetO2 repeats: SEQ ID NO: 5) in the promoter of the E1a gene of ICOVIR15 and ICOVIR15-Luc respectively, specifically by replacing the 24 base pairs immediately after the TATAbox of E1a (bases 476-499 of the Ad5 adenovirus reference material GenBank AY339865). For this purpose, the 7 TetO2 repeats were amplified from the plasmid pTRE2Luc (Zabala, M. et al., 2004, TARGETED CANCER THERAPIES III, 9, S374,), kindly provided by Dr. Cristian Smerdou from CIMA (Navarra, Spain), and cloned by homologous recombination at position 476 of the Ad5 adenovirus reference material.

[0189] Viral plasmids were transfected into HEK293 cells, and the generated viruses amplified in A549 cells and purified on a CsCI gradient according to standard protocols.

[0190] Virus cytotoxicity assay

[0191] For in vitro cytotoxicity assay 3x104A549 or 7.5x103MenSCs were seeded in 96-well plates and infecting them with serial dilution of 1 or 1 / 3 of the indicated viruses starting from 200 Til per cell for A549 and 2.000 Til per cells for MenSCs. 96h later, plates were washed with PBS, stained for total protein content (bicinchoninic acid assay; Pierce Biotechnology, Rockford, IL) and absorbance was quantified. The inhibitory concentration 50 (IC50) was calculated with GraphPad Prism v6.02 (GraphPad Software Inc.) by a dose-response nonlinear regression with a variable slope.

[0192] Viral production and transgene expression assay

[0193] 7.5x103MenSCs or MenSCs-TetR were seeded in 96-well plates and infecting them with ICOVIR15-Luc or ICOVIR15-R7-Luc at a MOI of 50 TU / cell (6 well per condition). Four hours after infection, medium was removed, and cells were washed thrice with PBS and incubated with fresh medium. At the indicated time points post-infection (0, 24, 48, 72, 96, 120, 144, 168 and 192h), a small fraction of the supernatant (SN) was collected from each well. After 72 hours of culture, tetracycline (final concentration 10 pg / ml) was added to half of the wells of each condition and SN were also collected at the indicated timepoints. Viral DNA was obtained from the supernatants of infected cells using QIAamp DNA Mini and QIAamp DNA Blood Mini Kits (Qiagen, (QIAGEN, Valencia, CA). Adenoviral genome copies were quantified in triplicate by Quantitative Real Time-PCR using the specific set of primers targeting the hexon sequence (forward 5’-CTT CGA TGA TGC CGC AGT G-3’ [SEQ ID NO: 11]), reverse 5’-ATG AAC CGC AGC GTC AAA CG-3’ [SEQ ID NO: 12]). PCR conditions were: 95°C 10 minutes, 40x cycles of 95°C 15 seconds, 60°C 1 minute and 72°C 7 seconds. Real Time PCR was performed using LightCycler 480 SYBR Green I Master (Roche). Adenoviral genome copy numbers were calculated using a standard curve of serially diluted pAd5wt, a plasmid containing the complete Adenoviral type 5 genome and for which the genome copy number was known.

[0194] In the same experiment, luciferase expression was determined at the indicated time points by adding 20 pl / well of D-Luciferin (L-8200 (Biosynth) 1.5mg / mL in PBS) and land measuring luciferase activity with Multilabel Plate Reader Victor 5* (PerkinElmer, ref. 2030-0050). A second luciferase expression assay was performed on A549 cells. 1 x 105cells / well were infected at a MOI of 10 in 200 pL with ICOVIR15-Luc or ICOVIR15-R7-Luc in 96- well plates. At 24, 48, and 72 h of incubation luciferase activity was measured in a similar manner as described above.

[0195] A third experiment was conducted to determine the relationship between viral replication control and the phenotype of the producer cell. In this case, 1.5 x 104MenSCs or MenSCs-TetR were seeded in 48-well plates and infected with ICOVIR15-Luc or ICOVIR15-R7-Luc at a multiplicity of infection (MOI) of 100 transduction units (Til) per cell (six wells per condition). After 5 days of culture, tetracycline was added to half of the wells in each condition at a final concentration of 20 pg / mL for four consecutive days. Supernatants (SN) were collected from each well at the end of the experiment (10 days). Moreover, the phenotype of the cells was assessed at day 10 using phase-contrast microscopy.

[0196] To quantify the number of physical virus particles produced, viral DNA was extracted and analyzed as described previously. Additionally, the number of infectious particles was determined specifically for MenSCs-TetR infected with the viruses in the absence of tetracycline treatment. For this purpose, the same supernatant from the previous experiment was used. Viral titers were determined in triplicate using an anti-hexon staining-based method in HEK293 cells (Cascallo, Manel et al., Molecular Therapy, Volume 15, Issue 9, 1607-1615).

[0197] Western Blot

[0198] 3x105MenSCs or MenSCs-TetR were seeded in 6-well plates and infecting them with ICOVIR15 or ICOVIR15-R7 at a MOI of 50 TU / cell. Supernatants and whole-cell protein extracts were harvested 6, 24, 48, 72 and 96h post-infection. Protein extracts were resolved by electrophoresis on an 8% acrylamide gel and transferred to a nitrocellulose membrane by standard methods. Then, membranes were immunoblotted with polyclonal anti-E1a (Clone 13S-5, Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibody for viral early E1a protein detection and with polyclonal anti-Adenovirus 5 (Ab6582, Abeam, Cambridge, UK) primary antibody for viral late proteins. As housekeeping and monoclonal mouse anti-vinculin antibody was used (V9264, Sigma Aldrich, Bozeman, MO, USA). Membranes were incubated overnight at 4 °C and secondary labeled with correspondent anti-rabbit or anti-mouse HRP-linked (Dako Laboratories, Glostrup, Denmark) according to the manufacturer’s protocol. Finally, SuperSignal™ West Femto substrate (Thermo Fisher Scientific, Waltham, MA, USA) was added, and bands intensity were visualized using a Chemi-doc (Bio-Rad, Hercules, CA, USA).

[0199] In Vivo Tumor-Homing, OAdv tumor delivery and antitumor efficacy Studies

[0200] In vivo studies were performed at the ICO-IDIBELL Animal Facility (Barcelona, Spain) AAALAC unit 1155, and approved by I DI BELL’s Ethical Committee for Animal Experimentation.

[0201] Lung adenocarcinoma xenograft tumors were established by implanting 5x106A549 cells subcutaneously into both flanks of 8-week-old male. When tumors reached 800 mm3, mice (experimental day -1) were randomized (5 animals per group) into the following groups: MenSCs+ICOVIR15-Luc (previously infected with ICOVIR15Luc at MOI 50 for 24h and labeled with IVISence 680 Fluorescent cell labelling Dye (Perkin Elmer NEV12000) according to the manufacturer’s protocol); MenSCs-TetR+ICOVIR15- Luc (previously infected with ICOVIR15Luc at MOI 50 for 24h and labeled with IVISence 680); MenSCs+ICOVIR15-R7-Luc (previously infected with ICOVIR15-R7-Luc at MOI 50 for 24h and labeled with IVISence 680); and MenSC-TetRs+ICOVIR15-R7-Luc (previously infected with ICOVIR15-R7-Luc at MOI 50 for 24h and labeled with IVISence 680). The next day (day 0), animals received a single intraperitoneal administration of 1.8 x 106specific cells according to their group. On days 3, 4, 5 and 6 mice received an intraperitoneal administration of 50 mg / kg body weight of doxycycline.

[0202] To monitor cell biodistribution and viral replication, mice were intraperitoneally injected with 120 mg / kg body weight of firefly luciferin (Biosynth, Staad, Switzerland), 15 minutes before imaging. Both, fluorescent (using 675 nm excitation and 696 nm emission filters for IVISence 680 determination) and bioluminescent (30s exposure) imaging analysis were performed daily using I VIS Lumina bioimaging system (PerkinElmer). Images were analyzed with I VIS Living Image (PerkinElmer) software. Regions were manually drawn around the tumors.

[0203] Tumor volume was also calculated periodically according to the equation V (mm3) = TT / 6 x l / l / 2x i_, where W and L are the width and the length of the tumor, respectively. Data are expressed as tumor volume (mm3) and tumor growth (tumor size relative to the size at the beginning of the therapy).

[0204] A second in vivo experiment was performed using the same tumor and animal model but initiating the therapy when tumor reached 250mm3, without labelling cells with a fluorescent dye and including a control group treated with PBS. Moreover, doxycycline was added in the drinking water (100mg / 100ml water) instead of administering it intraperitoneally.

[0205] Results

[0206] Generation and characterization of ICOVIR15-R7 & R7-Luc

[0207] The oncolytic adenoviruses ICOVIR15 and ICOVIR15-Luc were engineered by incorporating 7 TetO2 repeats into the E1a gene promoter (Fig 1A).

[0208] To evaluate whether TetO repeats insertion affected viral oncolytic properties, a dosedependent cytotoxicity assay was performed on A549 and MenSCs cells. As shown in Fig 1 B and 1 C, the IC50 (amount of virus to reduce the cell culture viability by 50%) for ICOVIR15-R7-Luc and ICOVIR15-Luc was similar (0.0722 vs 0.1195 not significant for A549, and 102.3 vs 225.8 not significant for MenSCs), indicating that the incorporation of these TetO repeats retain the oncolytic properties of the parental virus.

[0209] Next, the capacity of both viruses to express luciferase (transgene incorporated under the control of the major late promoter of the viruses) was determined. For this purpose, luciferase expression was measured from A549 cells infected with ICOVIR15-Luc or ICOVIR15-R7-Luc at different time-point post-infection. Fig 1 D pointed out identical luciferase kinetic expression from both viruses. As a summary, these results indicate that incorporation of TetO repeats into the E1a gene promoter do not affect ICOVIR15 viral properties in non-modified cells.

[0210] Control of ICOVIR15-R7-luc viral replication in vitro

[0211] Two different in vitro experiments were performed to demonstrate the capacity to control ICOVIR15-R7-luc replication. First, MenSCs and MenSCs modified to express Tet repressor (MenSCs-TetR) were infected with ICOVIR15-Luc or with ICOVIR15-R7-Luc. Then, both, viral early (E1a) and late proteins production, were determined by western blot. As shown in Fig 2A, while viral proteins kinetics expression was similar in MenSCs infected with both viruses, ICOVIR15-R7-Luc viral protein expression is repressed in MenSCs expressing the TetR, effect not observed for ICOVIR15-Luc.

[0212] In a second experiment setting, MenSCs and MenSCs-TetR were infected with ICOVIR15-Luc or ICOVIR15-R7-Luc. Luciferase activity and viral production were measured at different time-point post-infection. As observed in Fig 2B, luciferase expression from MenSCs-TetR infected with ICOVIR15-R7-Luc is controlled during the first 72h post-infection, in contrast with the other groups which present normal luciferase expression kinetics. Only when tetracycline is added to the culture, ICOVIR15-R7-Luc start to produce luciferase in MenSCs-TetR. A similar phenomenon was observed for viral replication (Fig 2C), where production of new viral particles is controlled in the MenSCs-TetR group infected with ICOVIR15-R7-Luc during the first days post-infection and it is recovered after the administration of tetracycline.

[0213] Finally, we aimed to replicate the results obtained in the previous experiments in a third experiment, where we not only assessed the ability of MenSCs expressing TetR to control viral replication in terms of viral particle production but also their capacity to regulate the generation of infectious particles (infection units).

[0214] As shown in Figure 3A, only the group of MenSCs-TetR cells infected with the modified virus ICOVIR15-R7 was able to control viral replication 10 days post-infection (black bar). However, normal viral replication was restored in this group upon tetracycline administration starting from day 5 post-infection (gray bar).

[0215] Furthermore, the analysis of the ability to generate infectious particles in MenSCs-TetR infected with either ICOVIR15 or ICOVIR15-R7 in the absence of tetracycline demonstrated that the control of viral replication was not only in terms of total viral particles but also in the production of infectious units (Fig.3B)

[0216] Finally, phase-contrast microscopy analysis of cell phenotype across different experimental groups at the end of the experiment clearly demonstrated that MenSCs infected with ICOVIR15 or ICOVIR15-R7 (with or without tetracycline), as well as MenSCs-TetR infected with ICOVIR15 (with or without tetracycline) or ICOVIR15-R7 in the presence of tetracycline, exhibited a characteristic cytopathic effect associated with advanced viral replication, with 100% cell death. In contrast, MenSCs-TetR infected with ICOVIR15-R7 in the absence of tetracycline displayed a normal phenotype, similar to the uninfected control group. These findings confirm the ability of our system to regulate the progression of the viral replication program.

[0217] MenSCs-TetR infected with ICOVIR15-R7-Luc present viral replication control and enhanced antitumor efficacy in vivo

[0218] Two different in vivo mouse model have been evaluated to demonstrate the capacity to control ICOVIR15-R7-Luc viral replication in vivo and to determine the antitumor capacity of the combo MenSCs-TetR / ICOVIR15-R7-Luc. First, immunodeficient NSG mice bearing subcutaneous human A549 tumors (800 mm3) were treated intraperitoneally with MenSCs or MenSCs-TetR, labelled with a fluorescent dye and infected with ICOVIR15- Luc or ICOVIR15-R7-Luc. Cell biodistribution and viral replication were monitored daily using an in vivo image system, and tumor volume determined twice a week. Moreover, mice received intraperitoneal administration of doxycycline on days 3, 4, 5 and 6. As observed in Fig 4A, the number of injected cells reaching the tumor was similar between groups, with a slight trend towards greater accumulation in the ICOVIR15-Luc loaded- MSCs and ICOVIR15-R7-Luc loaded MenSCs-TetR groups. Regarding luciferase expression (Fig 4B), all groups, except MenSCs-TetR infected with ICOVIR15-R7-Luc, showed a high luciferase expression in the tumor during the first days after treatment, reflecting tumor viral replication. Notably, ICOVIR15-R7-Luc luciferase expression, and therefore viral replication, from MenSCs-TetR was observed only after treatment with doxycycline, reaching a lower tumor luminescence intensity but more extended over time. Finally, the group that showed a greater ability to control tumor volume and tumor growth was ICOVIR15-R7-Luc-loaded MenSCs, being this difference greater after doxycycline administration (Fig 4C and 4D). A small fraction of cells from the different groups were cultured in vitro and treated with doxycycline at the indicated time point. A phase contrast image was taken daily, and it was possible to confirm that cells from the MenSCs+ICOVIR15-Luc, MenSCS+ICOVIR15-R7-Luc and MenSCs-TetR+ICOVIR15- Luc groups began to show a cytopathic effect due to viral replication at 96h postinfection, while that MenSCs-TetR cells infected with ICOVIR15-R7-Luc showed a normal phenotype until the administration of doxycycline (Fig 4E).

[0219] To further confirm the greater antitumor efficacy of the viral controlled group (MenSCs- TetR+ICOVIR15-R7-Luc), a second in vivo experiment was carried out. Immunodeficient NSG mice bearing smaller subcutaneous human A549 tumors (250 mm3) were treated as previously indicated but doxycycline was administered added in the drinking water at day 4. Again, the group of MenSCs-TetR infected with ICOVIR15-R7-Luc exhibits the greater antitumor potential (Fig 5A and 5B).

[0220] Altogether these results confirm that it is possible to control the replication of the ICOVIR15-R7-Luc adenovirus from cells that express the Tet repressor, both in vitro and in vivo, and that this control allows increasing antitumor efficacy using oncolytic adenovirus in vivo.

Claims

Claims1 . A carrier cell comprising: i) an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and ii) a recombinant replication-competent oncolytic human adenovirus genome comprising the sequence of at least one tetracycline operator (TetO) element, wherein the expressible nucleic acid sequence of item (i) is not present in the recombinant replication-competent oncolytic human adenovirus genome of item (ii), and wherein the at least one TetO element is located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene.

2. The carrier cell according to claim 1 , wherein the cell is a mesenchymal stem cell.

3. The carrier cell according to any one of claims 1 or 2, wherein the recombinant replication-competent oncolytic human adenovirus genome is from serotype 5.

4. The carrier cell according to any one of claims 1 to 3, wherein the at least one TetO element is located in the region comprised after the last viral packaging sequences and before the E1A TATA box or in the region comprised after the E1A TATA box and before the transcription start site of the E1 A gene.

5. The carrier cell according to any one of claims 1 to 4, wherein the recombinant replication-competent oncolytic human adenovirus genome comprises a sequence according to SEQ ID NO: 8.

6. The carrier cell according to any one of claims 1 to 5, wherein the nucleic acid sequence encoding the TetR element or the functionally equivalent variant thereof is stably integrated into the genome of the cell.

7. A method for obtaining the carrier cell according to any one of claims 1 to 6 comprising the steps of: i) introducing the expressible nucleic acid sequence encoding the tetR element or the functionally equivalent variant thereof into the carrier cell; andii) infecting the carrier cell obtained in step i) with a recombinant replication- competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the tetR element nor the functionally equivalent variant thereof.

8. An expression system comprising: i) a carrier cell; ii) an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and iii) a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising a sequence encoding the TetR element nor the functionally equivalent variant thereof.

9. The expression system according to claim 8, further comprising a tetracycline antibiotic, preferably doxycycline.

10. A pharmaceutical composition comprising the carrier cell according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.11 . A carrier cell according to any one of claims 1 to 6, an expression system according to claims 8 or 9, or a pharmaceutical composition according to claim 10 for use in medicine.

12. A carrier cell according to any one of claims 1 to 6, an expression system according to claims 8 or 9 or a pharmaceutical composition according to claim 10 for use in the prevention and / or treatment of solid tumors.

13. A kit selected from the group consisting of: i) a kit comprising the carrier cell according to any one of claims 1 to 6; and ii) a kit comprising an expressible nucleic acid sequence encoding a tetracycline repressor (TetR) element or a functionally equivalent variant thereof; and a recombinant replication-competent oncolytic human adenovirus comprising a genome comprising the sequence of at least one TetO element located in thepromoter of the recombinant human adenovirus early region 1A (E1 A) gene and not comprising a sequence encoding the TetR element nor the functionally equivalent variant thereof.

14. A recombinant replication-competent oncolytic human adenovirus genome comprising the sequence of at least one TetO element located in the promoter of the recombinant human adenovirus early region 1A (E1A) gene and not comprising the sequence encoding the TetR element nor the functionally equivalent variant thereof.

15. An adenovirus comprising the recombinant replication-competent oncolytic human adenovirus genome of claim 14.

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