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    • 专利摘要:
    Treatment of glioma or glioblastoma with parvovirus mvm, by viral particles containing dna with a nucleotide sequence consisting essentially of the sequence of mvmp and mvmi viruses, or by pharmaceutical compositions containing said viral particles. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2561906A1
    申请号:ES201431151
    申请日:2014-07-30
    公开日:2016-03-01
    发明作者:Jose María ALMENDRAL DEL RÍO；Jon GIL-RANEDO；Marta IZQUIERDO
    申请人:Universidad Autonoma de Madrid；
    IPC主号:
    专利说明:

    TREATMENT OF GLIOMA OR GLIOBLASTOMA WITH PARVOVIRUS MVM
    The present invention is encompassed in the fields of molecular biology and medicine, and refers to the use of a non-pathogenic mouse virus in humans, called mouse minute virus (MVM), belonging to the Parvoviridae family, for the treatment of glioma, especially glioblastoma.
    STATE OF THE PREVIOUS TECHNIQUE Glioma
    Glioma is a type of neoplasm that occurs in the brain or spinal cord. Hecalled glioma because it arises from glial cells. Your most frequent locationIt is the brain.There are different classifications for gliomas; specifically, they can be classified bythe type of affected cells, by their degree, ie their pathological evaluation, and by theirLocation.By cell type, gliomas are named according to the specific cell typethat more resemble the affected cells. According to this classification, theMain types of gliomas are:-Ependimomas: ependymal cells.-Astrocytomas: astrocytes; Glioblastoma multiforme is the most common astrocytoma.-Oligodendrogliomas: oligodendrocytes.By grade, gliomas are classified based on the pathological evaluation of the tumor.Low-grade, well-differentiated (that is, non-anaplastic) gliomas are referenced,benign and predict a better prognosis for the patient.High grade, are referred to undifferentiated or anaplastic gliomas, malignant and worseforecast.The most common system for the classification of astrocytomas is established by theWorld Health Organization (WHO), which assigns a rating from 1 to 4, being 1the least aggressive and 4 is the most aggressive:-OMS Grade 1-for example, pilocytic astrocytoma.-OMS Grade 2-for example, diffuse or low-grade astrocytoma.-OMS Grade 3-for example, anaplastic astrocytoma (malignant).-OMS Grade 4-for example, glioblastoma multiforme (the most common glioma in adults).By location, gliomas can be classified according to whether they are located above orbelow a meningeal structure called tentorio, which covers the surface


    superior of the cerebellum and supports the occipital lobe of the brain, that is, it delimitssections inside the cranial cavity.Supratentorial (above the tentorium): 70% occurs in adults;Infratentorial (below the tentorium): 70% occurs in children.
    Glioblastoma and glioma treatments.
    The worst prognosis of gliomas corresponds to grade 4. Of these, glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor in adults. The life expectancy of patients with GBM is between 12 and 15 months, despite multidisciplinary treatment, with a 5-year survival rate lower than 5% (Nature reviews 10 (5): 319-331). The conventional treatment of glioblastoma involves surgery, radio-and chemotherapy, and depends on the location, the type of cells and the degree of malignancy. The first therapeutic strategy is complete surgical resection, although this is limited by the presence of eloquent areas and high infiltration in the cerebral parenchyma. Surgery is combined with the subsequent or concomitant administration of radio-and / or chemotherapy, mainly with temozolomide (Expert Rev Neurother (2010) 10 (4): 507514), an alkylating chemotherapeutic drug, which is able to cross the blood-brain barrier effectively. Notwithstanding the above, today there are still no specific or general treatments available for these types of cancer that demonstrate clear clinical benefits.
    Tumor stem cells and glioblastoma therapy.
    Traditionally it has been considered that all cancer cells have the same ability to generate new tumors, but this view has recently changed with the hypothesis of tumor stem cells (CSC). This idea establishes that exclusively a subpopulation of cancer cells with aberrant stem cell properties, CSCs, is capable of generating and maintaining the tumor (Cancer research (2006) 66 (19): 93399344). The existence of CSC populations has been demonstrated in a multitude of tumor types, including GBM (Nature (2004) 432 (7015): 396-401), based on their capacity for self-renewal, differentiation and ability to induce tumors in animal models. Specifically, the glioblastoma stem cells obtained from patients (hereinafter hGSC), have demonstrated their truncal and tumorigenic character. The properties of hGSC explain the poor efficacy of current treatments (Nat Biotechnol (2007) 25 (2): 193-194), since the expression of CSC markers in GBM


    correlates with tumor progression, recurrence and survival of patients (Clin Cancer Res (2008) 14 (1): 123-129). Current therapies are designed conceiving the tumor as a homogenous population of highly proliferative cells, ignoring that hGSC are relatively quiescent and have radio and chemoresistance mechanisms (Nature (2006) 444 (7120): 756-760; Semin Radiat Oncol (2009 ) 19 (2): 78-86). This fact explains the invariable recurrences suffered by patients, who have experienced strong reductions in tumor volume after a treatment, due to the elimination of the bulk of the tumor, but without the elimination of the subpopulation of hGSC, which subsequently repopulates the tumor. Therefore, only a therapy capable of eliminating hGSC could achieve a definitive decrease in tumor volume. Due to the above, it is currently considered that the models of cancer therapies have to demonstrate their efficacy primarily against CSC. Those studies based on established cell lines are insufficient and biased, since these cells adapt gradually to a non-physiological environment due to the clonal selection to which they are subjected by in vitro culture, distancing their phenotypes and genotypes from their tumors of origin (Nature reviews (2003) 3 (12): 895-902). In contrast, CSC-based models faithfully reproduce the phenotypic and genotypic properties of the original tumor, possessing a predictive power over the efficacy of the therapies assayed in them much higher than the established lines (Cancer cell (2006) 9 (5): 391 -403). In fact, there are clear examples of therapies that were successfully applied in models based on established cell lines (Eur J Cancer Clin Oncol (2006) 19 (6): 799-805; Nat Med (1995) 1 (9): 938- 943), but failed both in clinical practice (Gene Ther (2000) 7 (10): 867-874; PLoS Med (2008) 5 (1): e8) and in models based on CSC (Clin Cancer Res (2008) 14 (5): 1571-1580; Journal of neuro-oncology (2012) 108 (1): 53-58).
    Parvoviruses
    Parvoviruses are icosahedral viruses of single-stranded DNA of 5 kb, which have ahuge potential interest in cancer therapy due to its natural oncotropism, its cyclevital lytic, and be essentially non-pathogenic for humans.Parvoviridae is a family of infective viruses for insects and vertebrates (dogs, cats,pigs, chickens, geese, rabbits, horses, etc.), including humans (Tijssen, P. et al,2011, Virus taxonomy 9th Report of the International Committee on Taxonomy of Viruses.ICTV Ninth Report). These viruses have a genome with single-stranded DNA like acidnucleic, approximately 5 kb. They are characterized by a capsid of 25 nm in diameter,of 60 protein subunits structurally defined by an icosahedral symmetry andlacking viral wrap (naked). The capsid is made up of two proteins


    primary structures, a majority, VP2, of 64-85 kD and another minority, VP1, of 8096 kD, but essential for infectivity. The virions assemble their capsid and mature in the nucleus as a cellular compartment (see Figure 2).
    Parvoviruses as anti-cancer agents.
    The natural oncotropism of parvoviruses explains that they were and continue to be isolated from tumors, tumor cell lines, preparations from other oncogenic viruses, or from experimental animals subjected to carcinogenic treatments (Proc Natl Acad Sci USA (1960) 46 (9): 1256 -1258). The productive infection of the Parvovirus requires functions expressed during the S phase of the cell cycle (Adv Virus Res (1987) 33: 91-174), so they only multiply in proliferative tissues, and therefore the hyperplasia of the pre-existing tissues. cancerous and cancerous favors its multiplication and lithic effects. In addition, these viruses meet very important biosecurity requirements from the perspective of their clinical use, since they are very selective in their host range: under physiological conditions, rodent parvoviruses are not pathogenic at all for humans. But the genetic and phenotypic changes that are triggered in cell transformation, raise the barriers that limit the host range of these viruses and allow mouse parvovirus like the tiny mouse virus (MVM, the prototype strain (p) deposited in the mouse). ATCC as: ATCC® VR-1346 ™), one of the models used in this invention, can be multiplied in human neoplastic cells (Nature (1982) 300 (5892): 537-539).
    Parvovirus in glioblastoma therapies.
    The use of Parvovirus in oncolytic therapy of Glioblastomas has a double interest, on the one hand the difficulties of treating these tumors by conventional therapies (discussed above), and the fact that these tumors develop in the environment of the brain, whose differentiated cells Non-proliferative drugs are not permissive to parvovirus infection. In this regard, the use of Parvovirus in different trials related to glioblastoma cells or gliomas has been described. The behavior of two strains (p, i) of mouse parvovirus MVM (J Virol (1988) 62 (8): 2605-2613) in established human glioblastoma lines (U87 and U373) in culture (J) has been studied in detail. Virol (2001) 75 (23): 11573-11582; J Virol (2010) 84 (4): 2090-2099; J Virol (2010) 84 (10): 5043-5051 Note that neither of these two cell lines are stem cells.


    In J Virol (2001) 75 (23): 11573-11582 it is described that the MVMi strain did not complete the productive cycle in any of the cells tested. In the U373 cells of glioblastoma, although the macromolecular synthesis of the virus was complete, the production of mature infectious virions was low, and their exit from the cells to the inefficient medium, preventing the proliferation of the infection in the culture. In contrast to the infection with MVMp, the U87 cells of glioblastoma behaved essentially as resistant to this virus. No experimental data are provided for U373 cells. Likewise, preclinical tests have been described in culture and in animals (J Biomed Biotechnol 2010: 350748, Geletneky, K. et al, Regression of advanced rat and human gliomas by local or systemic treatment with oncolytic parvovirus H-1 in rat models. Oncol, Kiprianova, I. et al, 2011, Regression of glioma in rat models by intranasal application of parvovirus H-1, Clin Cancer Res), with rat parvovirus H-1. Similar assays are described in patent publication WO2012 / 003932, although established and non-hGSC primary glioblastoma cell lines are employed. In addition, it is very important to point out that, although they belong to the same Parvovirudae family, the biological properties of the different species and even strains is very different, especially as regards pathogenesis and tropism, so the characteristics of a species are not extrapolated. strain to another. On the other hand, recruitment has begun for clinical trials in patients with glioblastoma with the rat parvovirus H-1 (http://clinicaltrials.gov/). Although some of the publications mentioned above describe a cytotoxic activity of parvoviruses in cells derived from glioblastoma, none of the documents describe or suggest the possibility that parvoviruses MVMp or MVMi infect hGSC of glioblastoma, which are the characteristics of the life cycle of the virus in these cells, or if they possess cytotoxic activity that could be used in therapies. Furthermore, none of the tests performed and described describes or provides an effective treatment of human tumors generated by hGSC, in particular of glioblastoma, thus maintaining the need for such treatment.
    SUMMARY OF THE INVENTION
    A first object of the present invention relates to a viral particle comprising anucleotide sequence consisting essentially of:a) the sequence SEQ ID NO 1 (GenBank: J02275.1); orb) the sequence SEQ ID NO 2 (GenBank: M12032.1);for use as a medicine.


    A second object of the present invention relates to the use of a viral particle thatcomprises a nucleotide sequence consisting essentially of:a) the sequence SEQ ID NO 1; orb) the sequence SEQ ID NO 2;in the preparation of a medication for the treatment of glioma.
    Another object of the present invention relates to a viral particle comprising anucleotide sequence consisting essentially of:a) the sequence SEQ ID NO 1; orb) the sequence SEQ ID NO 2;for use in the treatment of glioma.
    A further object of the present invention is a pharmaceutical composition thatit comprises as an active ingredient a viral particle as it has been definedpreviously.
    Another additional object of the present invention is the use of the pharmaceutical composition as such andas defined above for the preparation of a medication for theglioma treatment.
    According to another object, the present invention relates to a compositionpharmaceutical as defined above, for use in the treatment ofglioma
    DESCRIPTION OF THE FIGURESFigure 1. Characterization of human glioblastoma stem cells.A, Light-field microscopy photographs of neurospheres generated by cellsTrunks (CS5 to CS9) obtained from four glioblastoma patients.B, Phenotypic characterization of CSs by staining with specific antibodies against theCD133 core markers, human nestin, Sox2 and GNL3. The stains werequantified by flow cytometry and immunofluorescence.C, Growth kinetics of crops CS5-9. The number of viable cells is showndetermined every five days by staining with trypan blue.D, Generation of tumors in immunodeficient rats after orthotopic xenotransplantation ofthe human glioblastoma stem cells of patient # 7. Showing images of


    MRI representative of two tumors in axial and sagittal view.
    Figure 2. Main properties of parvovirus MVM.
    A. Structure of the parvovirus MVM capsid.
    B. Genetic organization of the MVM. The disposition of the non-structural (NS) and structural (VP) genes that form the capsid is illustrated. The NS1 protein is the main cytotoxic component of the virus.
    C. Life cycle of the MVM (based on Valle et al., 2006). The capsids are assembled in the nucleus and in the nuclear replication of the genome the NS1 protein participates.
    Figure 3. Parvovirus MVM (p, i) replicates its genome and expresses the cytotoxic protein NS1 in human glioblastoma stem cells (hGSC).
    A. Immunofluorescence of NS1 protein expression in hGSC cells, expressing the CD133 backbone marker, analyzed by confocal microscopy.
    B. Percentages of hGSC that express NS1 and replicate viral DNA after infection with MVMp
    or with MVMi.
    C. Blot analysis of the accumulation of the NS1 protein, at the indicated times, in hGSC cultures infected with MVMp. The samples were 15 μg of protein in each case, and an actin loading control was also shown.
    D.  Southern blot analysis of MVMp and MVMi replication in hGSC cultures, and in U373 and human neural stem cell (hNSC) controls. The replication species of the viral genome (dRF, mRF, ss) are shown.
    Figure 4. The two strains of parvovirus MVMp and MVMi efficiently kill human glioblastoma cells in culture.
    A. Proportion of viable hGSCs cells, derived from patients # 5, 7, 8 and 9, after infection with MVMp and MVMi, and analysis of the production of infectious virus, determined over several days post-inoculation.
    B. Suppression of self-maintenance of hGSC, measured as clonogenic potential, after infection with MVMp and MVMi. The colonies were counted at 15 d.p. sowing (** p <0.05; *** p <0.001).
    Figure 5. The two strains of parvovirus MVMp and MVMi express structural proteins and produce capsids in human glioblastoma stem cells (hGSC).
    A. Blot analysis of the accumulation of structural proteins (VP1, VP2 and VP3) of MVM in infected hGSCs, on the indicated days. The samples were 15 μg of protein


    by lane. C, uninfected control. B Analysis by flow cytometry of the formation of capsids in the indicated hGSC cultures, as well as in the control lines NB324K and U87, after infection with MVM. Representative results demonstrating the expression of VPs and capsid formation in CS5, 8 and 9 are shown.
    Figure 6. Parvovirus MVM induces a DNA damage response by the expression of NS1 in human GSCs.
    A. Induction of a DNA damage response (DDR) in oncoespheres of hGSCs by infection with MVM. The colocalization by confocal microscopy of the viral NS1 protein with DDR and the replication of the virus (vDNA-Cy5) is shown in the infected cells.
    B. DDR in hGSC and established lines of human glioblastoma induced by the two strains of MVM. Representative results of colocalization of the markers indicated by confocal microscopy are shown. C. Blot analysis of the induction of RPA32 (a major DDR marker) in the lines and hGSC indicated in response to infection with MVM. Load control by tubulin.
    Figure 7. Electrical pulses increase MVM's ability to express NS1 and kill hGSC.
    A. Effect of electroporation of hGSC cells infected by MVMp on the relative expression of NS1.
    B. Increased death of hGSC infected with MVMp by electroporation. Average values are shown with standard deviations.
    C. Confocal microscopy images showing the increase in the percentage of hGSC expressing the NS1 protein of MVM after treatment by electroporation (*** p <0.001).
    Figure 8. Parvovirus MVM damages and inhibits the growth of human glioblastoma based on GSC implanted in mouse brain.
    Glioblastoma cells (patient # 7) were implanted in immunodeficient rat and mouse model and the generated glioblastomas treated with MVM.
    A. Tissue damage caused by intratumoral inoculation of MVMp at 14 dpi. The structural differences of the tumors between the inoculation of saline control (above) with respect to MVMp (below) monitored by NMR, hematoxylin-eosin staining (H & E), human nestin staining, MVM VP protein expression and staining of nuclei.
    B. The structural damage caused by MVM in the tumors colocalizes with the expression of NS1, the induction of DDR (ATM marker), and the viral genome replication (vDNA-Cy5).


    C. Above: mouse brain diagram showing the place of implantation of the hGSC. Below: Representative images of the development of tumors in mice treated with saline control (PBS), or inoculated with MVMp or MVMi at 60 dp transplant.
    D. Volumes measured by nuclear magnetic resonance (NMR) of tumors
    5 developed in mice transplanted with hGSC and subjected to treatmentsindicated. The viruses were administered: intratumoral MVMp, intranasal MVMi.
    E. Kaplan-Meyer analysis of the survival of the mice subjected to the indicated treatments (salt control n = 7, MVMp n = 5, MVMi n = 6).
    DETAILED DESCRIPTION OF THE INVENTION The present invention provides a useful treatment against glioma, preferably of the glioblastoma type, by the use of viral particles comprising a nucleotide sequence consisting essentially of the nucleotide sequence SEQ ID NO 1, corresponding to the genome. of the tiny mouse virus strain MVMp, or essentially
    15 in the nucleotide sequence SEQ ID NO 2, corresponding to the genome of the tiny virus strain of the MVMi mouse.
    The examples show that these strains destroy the human glioblastoma stem cells (hGSC) recently obtained from glioblastoma patients, as well as their effect
    20 on tumors caused by hGSC in mice.
    MVMp is the primitive strain of tiny mouse parvovirus. Its genome corresponds to the sequence SEQ ID NO 1.
    25 MVMi is the immunosuppressive strain of mouse minute parvovirus. Its genome corresponds to the sequence SEQ ID NO 2.
    Within the framework of the present intention, the term "consists essentially of the sequence of
    nucleotides SEQ ID NO 1 or 2 ", includes not only sequences SEQ ID NO 1 and 2, but
    30 also includes all mutants and variants of the nucleotide sequence of the parvovirus MVM that could be obtained naturally or artificially, but that do not affect the essential characteristics of the parvovirus of interest in the present invention, especially that they do not alter any property biological activity related to tropism or anticancer activity, of the original MVM strains (p, i).


    The genome of both strains of mouse miniature parvovirus MVMi and MVMp has been sequenced in its entirety. These two strains have nucleotide sequences with 97% identity to each other (J. Virology 1992, 66 (5), 3118-3124; J. Virology 1986, 57, 656669). According to the reference publication "Springer Index of Viruses" (2nd Edition, 2011, Editorial Springer), the MVMp strain has a genome of 5149 nucleotides, detailed in
    J. Virol 1986, 57, 656-669, the MVMi strain of 5087 nucleotides, detailed in Nucl Acids Res 1985, 13, 3617-3633. In the present invention they are respectively referenced as SEQ ID NO 1 and SEQ ID NO 2. Such sequences SEQ ID NO 1 and SEQ ID NO 2 correspond to the genetic information, in the form of DNA, of the parvoviruses MVMp and MVMi ie they are nucleotide sequences.
    In the context of the present invention, "viral particle" refers to a structural unit formed by a protein shell or a more complex shell containing carbohydrates, lipids and / or proteins, and at least one nucleic acid molecule, either ARN
    or DNA. Thus, "viral particle" includes virions, viruses, etc.
    Also, by "viral particle" within the framework of the present invention are understood to include
    plasmids comprising a nucleotide sequence consisting essentially of:
    a) the sequence SEQ ID NO 1; or
    b) the sequence SEQ ID NO 2.
    "Viral particle" is also understood in the context of the present invention as those vectors comprising the plasmids defined above.
    In this way, a first object of the present invention refers to a viral particle
    comprising a nucleotide sequence consisting essentially of:
    a) the sequence SEQ ID NO 1; or b) the sequence SEQ ID NO 2; for use as a medicine.
    A second object of the present invention relates to the use of a viral particle that
    comprises a nucleotide sequence consisting essentially of:
    a) the sequence SEQ ID NO 1; or
    b) the sequence SEQ ID NO 2; in the preparation of a medication for the treatment of glioma.


    A third object relates to a viral particle comprising a sequence ofnucleotides consisting essentially of:a) the sequence SEQ ID NO 1; orb) the sequence SEQ ID NO 2;
    5 for use in the treatment of glioma.
    In the above defined second and third objects, according to a preferred embodiment, the viral particle comprises a nucleotide sequence consisting of the sequence SEQ ID NO 1.
    According to another preferred embodiment, the viral particle comprises a nucleotide sequence consisting of the sequence SEQ ID NO 2.
    In all of the above, preferably the glioma is a glioblastoma.
    According to a preferred embodiment, the glioblastoma is a relapse, that is, the patient with glioma or glioblastoma has been treated with conventional treatments, but has not been eliminated in its entirety and reoccurs.
    Preferably, the medicament for the treatment of glioma comprises a vehicle suitable for the drug to be administered intracerebrally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intracutaneously, intracecally, intraventricularly, orally, enterally, parenterally, intranasally or dermally, more preferably a
    25 suitable vehicle so that it can be administered intracranially or nasally.
    According to a preferred embodiment, glioma or glioblastoma develops from human cancer stem cells.
    Another object of the present invention is a pharmaceutical composition comprising as active ingredient a viral particle as defined above.
    Preferably, the viral particles will be contained in the above-defined drug or pharmaceutical composition in a therapeutically active amount.


    Preferably, in said pharmaceutical composition or in said medicament the viral particle is in a concentration of between 106 and 1013 units pfu / ml (pfu = lysis plate forming unit), more preferably between 107 and 1013 pfu / ml, furthermore preferably between 108 and 1013 pfu / ml.
    This concentration of viral particles in the pharmaceutical composition can be referred, within the framework of the invention, to the concentration of a single type of viral particle, the pharmaceutical composition not containing any other type of viral particle. For example, the composition may contain only viral particles comprising a nucleotide sequence consisting essentially of the sequence SEQ ID NO 1 at the indicated concentration. Another example would be a viral particle comprising a nucleotide sequence consisting of the sequence SEQ ID NO 1, at the indicated concentration.
    Alternatively, the indicated concentration can be achieved by mixtures of various types of viral particles as defined above, together reaching the indicated concentration. All possible combinations that will be apparent to one skilled in the art are included within the scope of the present invention.
    Pfu / ml is a quantitative measure usually used in virology, and corresponds to the number of infectious viral particles capable of forming lysis plaques in monolayers of susceptible cells per volumetric unit. It is a functional measure rather than a measure for the absolute number of particles: virus particles that are defective or that fail to infect their target cell will not produce a plaque and therefore will not be counted. For example, a composition comprising MVM at a concentration of 106 pfu / ml indicates that 1 milliliter of the composition contains enough virus particles to produce 106 infectious plaques in a cell monolayer, but it is not possible to establish a relationship between pfu and the number of virus particles by this assay. By means of complementary methods, for example by haemagglutination (see Methods below) it is possible to determine the total number of viral particles in a preparation whether or not they are infectious.
    The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, the age, weight, sex, tolerance, immunocompetence, etc., of the mammal. In the sense used in this description, the term "therapeutically effective amount" refers to the concentrations of viral particles comprising a nucleotide sequence consisting essentially of: a) the sequence SEQ ID NO 1;


    b) the sequence SEQ ID NO 2;
    that produce the desired effect and, in general, will be determined, among other causes, bythe characteristics of said pharmaceutical compositions and the therapeutic effect to5 get.
    According to a preferred embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.
    According to a preferred embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient
    According to another preferred embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable adjuvant.
    Preferably, in the pharmaceutical composition the vehicle or excipient is such as to allow the administration of said composition intracerebrally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intracutaneously, intracecally, intraventricularly, orally, enterally, parenterally, intranasally or dermally. More preferably, administration is intracerebrally,
    Intravenous, or intranasal.
    According to a preferred embodiment, the pharmaceutical composition comprises at least one additional active ingredient.
    As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological or other different effect in diagnosis , cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
    The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a planned modified form, which provides the specific activity or effect.
    The composition of the invention may be administered with electric pulses in order to
    35 increase the expression of the NS1 cytotoxic viral protein. Such electrical pulses may be applied by techniques similar to electrochemotherapy or electroporation


    irreversible, which are currently being used for other therapeutic purposes both in preclinical studies and in clinical trials against various cancers (ESJO 34 (2): 232240; Technol Cancer Res Treat 10 (1): 73-83; http: // clinicaltrials. gov /).
    In the composition of the invention one or more such principles may be contained.assets, to provide a combination therapy.
    Preferably, the "active ingredient" will be an ingredient with antitumor activity, ie, an antitumor agent. Such active ingredient with antitumor activity can be selected,
    10 for example, but not limited to, an active ingredient selected from docetaxel, rituximab, carboplatin, cisplatin, paclitaxel, vinorelbine, gemcitabine, irinotecan, doxorubicin, dacarbazine, cytarabine, temozolomide or bevacizumab, 5-fluorouracil, folinic acid, irinotecan or paclitaxel . More preferably, it will be a drug commonly used for the treatment of glioma or glioblastoma, for example temozolomide or bevacizumab.
    15 Temozolomide is a triazene, indicated for the treatment of brain tumors, more specifically glioblastoma multiforme. Bevacizumab is a humanized monoclonal antibody, indicated for the treatment of some neoplastic diseases such as colon cancer, breast cancer, non-small cell lung cancer, renal cell carcinoma and glioblastoma.
    Another preferred embodiment relates to a pharmaceutical composition as defined above, for use in the treatment of glioma in combination with any type of electrical pulse caused in tumors.
    According to another object of the invention, this refers to the pharmaceutical composition as it has been defined above, for its use as a medicine. Preferably, the medicament is for the treatment of glioma, more preferably glioblastoma.
    Thus, another object of the invention is the use of a pharmaceutical composition as defined above, for the preparation of a medicament for the treatment of glioma, more preferably glioblastoma.
    The term "medicament", as used in the context of the present invention, refers to any substance used for prevention, diagnosis, relief, treatment or


    cure of diseases in man and animals.
    Both the viral particles and the compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art. Thus, they may be, without being limited, in sterile aqueous solution or in biological fluids, such as serum. The aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate the ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, and the like, and nutrients including glucose, dextrose, vitamins and minerals. Alternatively, the compositions can be prepared for administration in solid form. The compositions may be combined with various inert carriers or excipients, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate. The viral particles or pharmaceutical compositions of the invention can be administered by any route, for which said composition will be formulated in the appropriate pharmaceutical form and with pharmacologically acceptable excipients to the chosen administration route.
    Such compositions or combination preparations and / or their formulations may be administered to an animal, including a mammal and, therefore, man, in a variety of ways, including, but not limited to, intracerebral, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.
    Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.


    EXAMPLES
    In the present invention the mechanisms explaining the destruction of human glioblastoma stem cells (hGSC) obtained recently from patients, produced by infection with two strains of parvovirus MVM (p, i) are shown. This conclusion was demonstrated in four groups of experiments:
    (i)  Expression of cytotoxic (NS1) and structural (VP) proteins of the virus in hGSC;
    (ii)  death of hGSC due to infection with MVM;
    (iii) synergy of MVM with electric pulses to kill hGSC;
    (iv) Effectiveness of MVM in orthotopic human GBM models in mice xenotransplanted with hGSC. In the mice treated with MVMi or MVMp the progress of the tumors was slowed and the animals survived more times than the untreated ones.
    Example 1
    Cells from glioblastoma patients, in the form of selective in vitro cultures of hGSC in spheres, allowed a direct analysis of their permissiveness and susceptibility to infection with the MVM. As shown in Figure 3, both strains tested for this virus (MVMp, MVMi) were able to express in hGSC of the different patients, the main non-structural protein NS1 (figure 3A), which is cytotoxic and regulates replication and other stages of the life cycle of the virus. The expression of the viral NS1 occurred in a variable percentage in the different hGSC, although it is important to emphasize that the NS1 was detectable by immunofluorescence in all cases and with the two MVMp strains, MVMi (Figure 3B). The expression of NS1 in all infected hGSCs was also clearly demonstrated in blot (Figure 3C) and viral replication by quantitative hybridization in southern-blots, demonstrating the synthesis of viral replication intermediates in hGSC (Figure 3D).
    Example 2
    The MVMp and MVMi viruses demonstrated their ability to kill hGSC cells in culture. In particular, this trial was conducted in two different conditions. In a first test, the cells were cultured in liquid medium in the presence or absence of MVM, and a marked drop in viable cells (measured by trypan blue) could be observed in the cultures due to the MVM infection (Figure 4A). In a second trial, the cells were infected with viruses and their ability to form colonies in later days was measured. Both MVMp and MVMi viruses prevented the formation of colonies in very significant proportions (Figure 4B), demonstrating the ability of both viruses to inhibit the self-maintenance of hGSC, an essential property to generate tumors.


    Example 3
    The synthesis of structural proteins and the formation of capsids of MVMp and MVMi viruses in hGSCs have been determined. As shown in Figure 5, this assay allowed to conclude that all hGSC cells are productively infected by the MVM, since they produce viral capsids.
    Example 4
    We demonstrate that MVMi and MVMp viruses induce a response to DNA damage (DDR) in hGSC (Figure 6). This assay is important to understand the toxic effect of these viruses on these cells, since the DDR response distorts the cellular viability and the oncogenic potential of hGSC.
    Example 5
    It has been studied whether electrical pulses, which commonly disrupt cell gene expression, could increase the expression of parvovirus MVM cytotoxic proteins in hGSC. As shown in Figure 7, electrical pulses transmitted in milliseconds to the hGSC by electroporation, significantly increase the expression of the cytotoxic protein NS1 of the MVM. This result suggests that electrical pulses applied in situ should increase the anti-glioblastoma efficacy of MVM in patients.
    Examples 6 and 7: The real anti-glioblastoma efficacy of the two MVMp and MVMi viruses was analyzed in two mouse models xenotransplanted in brain with hGSC:
    Example 6
    When the MVMp virus was applied by controlled intratumoral injection (convective enhanced inoculation, CED) in rat brain, it was observed that it produced significant damage to the tissue structure of glioblastomas that had been induced by hGSC implantation 30 days earlier (Figure 8A ). In damaged regions, expression of NS1, structural VP protein, replication of the MVMp genome, and a DDR response (Figure 8B) are detected. Therefore, MVMp is an effective toxic agent against human glioblastoma in rats.
    Example 7
    The MVMp and MVMi viruses were inoculated to immunodeficient mice implanted with hGSC in the brain several days before. MVMp was administered by convection-enhanced inoculation, while MVMi was administered intranasally. The growth of


    The tumors were monitored in all cases continuously by NMR (Figure 8C), comparing with control mice to which a saline solution (PBS) was administered. The growth rate of the tumors (Figure 8D) showed a clear inhibition in the mice inoculated with the MVM viruses, whose inhibitory effect was slightly higher in the intranasal inoculation of MVMi. The therapeutic effect of both viruses against human glioblastoma was also evident when the survival rate of the mice was observed (Figure 8E), since while in administrations with PBS the animals died of glioblastoma between 60-67 days post-implantation, Survival of infected animals was around 50% (MVMp) or 90% (MVMi) higher. In fact, the infected animals died of the known symptoms induced by MVM in immunodeficient mice (J Virol 73 (3): 1774-1784; J Virol 75 (23): 11573-11582; J Virol 82 (3): 1195-1203) and not of symptoms attributable to glioblastoma. Therefore, MVMp and MVMi viruses are very effective therapeutic agents against human glioblastoma based on hGSC in vivo.
    In the following, the materials and methods used in the realization of the examples described above are detailed.
    MATERIALS AND METHODS1.-Human glioblastoma stem cells.
    1.1. Obtaining. The samples of human tumor tissue were donated by the Neurosurgery service of the Ramón y Cajal Hospital in Madrid, diagnosed as gliomas by the Pathology service of the same hospital. In all cases, the informed consent of the patients was obtained and the approval of the Institutional Ethics Committee of the Ramón y Cajal Hospital in Madrid and the Severo Ochoa Molecular Biology Center (CSICUAM). The biopsies were collected at the foot of the operating room under aseptic conditions, kept in PBS supplemented with 0.6% glucose at 4ºC and processed during the two hours following its reception. The tissue was mechanically washed and disintegrated to obtain fragments smaller than 1 mm3. Subsequently, it was enzymatically disintegrated by incubating with 0.1% trypsin (Difco) and 0.04% DNase type I (Sigma) for 45 minutes at 37 ° C. It was washed three times with PBS 0.04% DNase and mechanically disintegrated again, passing the tissue through decreasing diameter Pasteur glass pipettes until a homogenous cell suspension was obtained. Finally, the cell suspension was filtered (40 μm pore diameter filter, Becton-Dickinson).


    1.2 Culture of glioblastoma stem cells. Human glioblastoma stem cells were obtained by culturing cell suspensions obtained from each biopsy in chemically defined growth medium composed of DMEM: F12 (1: 1) with glutamax (Invitrogen), supplemented with 0.5% albumax I (Invitrogen), 5 mM HEPES (Invitrogen), 0.915% glucose (Sigma), N2 1x (Invitrogen), 20 ng / ml FGF-2 (Peprotech), 20 ng / ml EGF (Peprotech), 2 μg / ml heparin (Sigma), amino acids non-essential (44 mM L-Ala, 45 mM L-Asn, 40 mM L-Asp, 40 mM L-Glu, 30 mM L-Pro) and, alternatively, antibiotic mixture (penicillin 63.2 mg / ml and 0.1 mg / ml streptomycin) or gentamicin (0.055 mg / ml). 20% fresh medium was added twice a week. In this way proliferation is selected positively as stem cell neurospheres derived from neural tissue. When the size of the neurospheres reached approximately 250 μm, they were enzymatically digested with trypsin and EDTA (Merck) and mechanically, until a homogeneous unicellular suspension was obtained. After the process, the typical cell viability, analyzed by trypan blue exclusion, was greater than 90%. The expansion of the cultures was carried out at a density of 1 · 10 5 cells / ml. The experiments were carried out with cells in passage between 6 and
    10The cells obtained showed the characteristics indicated in Figure 1.
    1.3. Flow cytometry. In all the experiments, a minimum of 30,000 events per sample was acquired in a FACSCalibur cytometer (BD Biosciences). These events were pre-selected by a region based on their size and complexity (FSC and SSC parameters, respectively) in order to exclude cell debris or other contaminating particles. The CellQuest software (BD Biosciences) was used to acquire the events and the FlowJo software (Tree Star) was used to analyze the data. Cells were resuspended in PBS (pH 7.2) with 0.5% BSA and 2 mM EDTA and 5 μl of anti-CD133 antibody or 10 ml of isotype control antibody (IgG2b-APC, Immunostep) was added for each 1 · 106 of cells. It was incubated for 15 minutes at 4 ° C or 15 minutes at room temperature, respectively, and two washes were performed before analyzing the samples on the flow cytometer, exciting the fluorophore with the 488 nm laser and collecting the emission at 661/16 nm (FL4). For capsid staining (B7) the cells were permeabilized with PBS + 0.1% triton for 10 ml at RT. Subsequently they were blocked with PSB + 0.1% triton + 1% FCS for 20 min at RT. The primary and secondary antibodies were incubated under the conditions described (see below), washing with PBS between them. Samples were analyzed in the flow cytometer, exciting the fluorophore with the 488 nm laser and collecting the emission at 530/30 nm (FL1)


    1.4. Clonogenic analysis of hGSC. The clonogenic capacity of self-renewal of the tumor stem cells was estimated by the soft agar colony formation test. The agar base layer was prepared by mixing equal volumes of 1% sterile agar and 2x proliferation medium, obtaining a final solution of 0.5% agar in 1x proliferation medium. For the upper layer, 0.7% agar and 2x proliferation medium were mixed in equal proportions and, immediately, the cells were added. In this way, a 0.35% agar solution was obtained in 1x proliferation medium, containing
    10,000 cells / ml. The cells were incubated at 37 ° C and 5% CO2 for 14 days and the viable colonies were stained with 1 ml / well of 600 μg / ml MTT in proliferation medium, photographed and counted using the ImageJ software (http: // rsbweb .nih.gov / ij /). To do this, regions of interest (ROIs) of the same size were determined between the different conditions and the number of colonies within each of them was quantified using the Nucleus Counter plugin.
    1.5. Induction of tumors by orthotopic xenotransplantation of hGSC. All the tests with animals were carried out following the rules of the Spanish Royal Decree 1201/2005 BOE. The animals were maintained and handled in a pathogen-free environment, ensuring the containment of level 2 (P2) risk. All the trials that required the use of surgery were carried out in horizontal laminar flow cabin of biosecurity level P2. The immunodeficient animal models used for orthotopic xenotransplantation were the nude rat (named Crl: NIH-Foxn1rnu by Charles River and Hsd: RH-Foxn1rnu by Harlan) of approximately 4-5 weeks and 150 grams of weight, or the Scid mouse (Charles River or Harlam) of 6-8 weeks and 20 g of weight. The hGS cells were inoculated by stereotaxic surgery in anesthetized animals by inhalation of a constant flow (2-3 l / min) of isoflurane (Nicholas Piramal), O2 (2 l / min) and N2O (1 l / min), provided by a gaseous anesthesia team (McKinley). The animals were immobilized in a stereotaxic apparatus (Kopf) and a longitudinal incision was made on the skin of the skull using an electric scalpel (Diatermo MB122). Subsequently, a craniotomy was performed with a dentist's lathe (Zénit Mod. 5750) in the area of interest and 1-5 · 105 cells in 10 μl of PBS were injected into the caudate putamen of the right hemisphere, at 3.5 mm to the bregma right and 4 mm deep, at a constant flow of 1 μl / min through a motorized injector (Stoelting Mod.310). The hole in the skull was covered with dentist cement (TAB 2000, Kern Pharma) and the incision was sutured with surgical staples (Stoelting). Antibiotic ointment and sulfanilamide powder (Kern Pharma) were applied to the wound.
    1.6. Monitoring the development of tumors by magnetic resonance (MRI). TO
    21 10


    each animal was injected with 0.4 ml (mouse) to 0.7 ml (rat) of the gadolinium contrast agent (Magnevist, Schering) intraperitoneally. The animals were anesthetized in the manner described above and placed prone in a radiofrequency probe adapted to their size. A Biospec BMT 47/40 spectrometer (Bruker Ettlingen) was used, equipped with a 12 cm shielded gradient system, operating at 4.7 Teslas. After the overall homogeneity adjustment and the acquisition of a gradient echo image in the transverse direction, the direction of the posterior cuts was selected and images of 1 mm thickness of axial and sagittal projections of the brain of the T1-weighted animals were obtained. (700 msec repeat time and 15 msec echo time). The size of the images was 256x256 pixels and the field of view was 4 cm. To calculate the volume, the tumor contour of each section was determined manually using the software ParaVision (Bruker Ettlingen) and its areas were added to obtain the total volume. The images obtained were processed with the ImageJ program. In certain cases, the three-dimensional reconstruction of the tumor was performed by obtaining a series of images in which the entire brain is included along the Z axis.
    2. Parvovirus MVM. In our laboratory, plasmids containing the complete genome of the MVMp and MVMi strains, including the palindromic sequences of the ends, have been constructed, which allows obtaining viral particles by transfection of cells. The strains have the characteristics indicated in Figure 2.
    2.1. Production of Parvovirus MVM in culture. The parvovirus MVM preparations were generally obtained in NB324K cells. Ten confluent P100 plates (5x107 cells) were infected at a multiplicity of infection (MOI) of 0.005 plaque-forming units / cell (PFU / cell) in 1 ml of complete PBS (PBSc, PBS with 0.9 mM CaCl2 and MgCl2 0). , 5 mM) with 0.1% FCS. After 1 h at 37 ° C, the inoculum was removed and incubated in DMEM with 5% FCS for 5 h to allow internalization of the virus. Subsequently the adhered cells were raised with trypsin-EDTA, diluted in 400 ml of DMEM with 5% FCS and seeded in 40 P100 plates, incubated until the appearance of cytopathic effect (approximately 5 days).
    2.2. Purification of parvovirus MVM. The virus present in the medium was recovered by precipitation with 3.4% polyethylene glycol 6000 and 0.5 M NaCl overnight at 4 ° C and subsequently centrifuged at 5000 rpm for 30 min in a Sorvall GSA angular rotor. To recover the intracellular virus, the cell pellet was resuspended in Tris-HCl 50


    mM pH 7.5, 1 mM EDTA (TE) and subjected to three consecutive freeze / thaw cycles, after which 0.2% final SDS was added and clarified at 8000 rpm, 10 min at 4 ° C in the rotor floating Sorvall HB4. Virus from the medium and recovered intracellular virus were pooled and centrifuged through a 20% sucrose cushion (Merck) in 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1 M NaCl and 0.2% of SDS for 18 h at 16000 rpm in a TST 28.38 rotor and the pellet was resuspended in TE with 0.2% Sarkosil (Sigma) and centrifuged by equilibrium gradient of CsCl (ni = 1.371) until equilibrium, for 24 h to 50000 rpm and 15ºC, in a TFT 80.13 rotor. From the top of the gradient, fractions of 0.5 ml were collected by syringe and the presence of empty capsids and DNA-filled viruses was determined by haemagglutination (see below). Finally, the fractions containing the viruses on one side and the capsids on the other were collected and dialysed against PBSi.
    2.3. Virus evaluation by Hemagglutination. For the hemagglutination (HA) assay of MVM, adult mouse blood was used. This blood was washed three times with PBSi, collecting the erythrocytes by centrifugation in a tabletop centrifuge at 1500 rpm for 5 min. After washing, the 50% (v / v) erythrocyte sediment was resuspended in PBSi and stored at 4 ° C until the moment of use. The HA was carried out in U-profile microtest plates (Nunc). The samples to be evaluated were applied in a final volume of 100 μl in PBSi and serial dilutions 1: 2 in PBSi were made. Finally, 50 μl of 2% erythrocytes in PBSi were added to each well, the plate was gently shaken and kept at 4 ° C in the dark for at least two hours. The title was obtained from the inverse of the highest dilution that maintains the haemagglutinating capacity.
    2.4. Parvovirus MVM assay by formation of lysis plates. Monolayers of NB324K cells seeded 24 h before were infected in P60 plates at a density of 220000 cells / dish. The culture medium was removed, washed with complete PBS (PBSc) and the viral inoculum was added in 400 μl per P60, diluted in PBSc with 0.1% FCS. After one hour of adsorption at 37 ° C with gentle agitation, the inoculum was removed and 7 ml of plating medium (DMEM 10% FCS, 0.6% agarose LM-GQT of Pronadisa) equilibrated at 37 ° C was added. After a 6-day incubation, the plates were fixed in 10% formaldehyde (Panreac) in PBSi and stained with 0.2% violet crystal (Panreac) in 10% formaldehyde in PBSi. The count of the number of lysis plates multiplied by the dilution allows to obtain the titer in plaque forming units per milliliter (PFU / ml).


    2.5. Obtaining viral DNA. The low molecular weight DNA from the extracts of the electroporated cells was obtained by the Hirt method adapted for MVM with 20 μg / ml carrier tRNA to ensure recovery. Briefly, the transfected cells were lysed in Hirt's solution (50 mMTris pH 7.5, 0.5% SDS, 10 mM EDTA) and digested with proteinase K (100 μg / ml) (Merck) for 2 hours at 37 ºC. The reaction was adjusted to 1M NaCl and the genomic DNA was precipitated overnight at 4 ° C. The enriched fraction of viral DNA was obtained in the supernatant after centrifuging the sample at 4 ° C and 14 K rpm for 30 min in a mini-fugue. This DNA was precipitated with 2.5 volumes of absolute ethanol, washed with 70% ethanol to remove salts and resuspended in water or in 50 mM Tris pH 7.5 and 10 mM EDTA (TEADN).
    2.6. DNA electrophoresis and membrane transfer. Samples were electrophoresed 2-3 hours at 60V on a 0.8% agarose gel (Gibco) in Tris-Boronic-EDTA buffer (45 mM Tris-borate, 1 mM EDTA) with 5 μg / ml bromide. ethidium (Boehringer) together with molecular weight markers (HindIII, Ø29) and controls of replicative forms and viral DNA. The transfer was made to a Nylon membrane (Hybond-N +, Amersham Pharmacia) in a 0.4 M NaOH solution overnight. Finally, the membrane is washed in a 2X solution of SSC for 10 minutes at room temperature (20X SSC is: 3 M NaCl, 0.3 M sodium citrate) and allowed to dry.
    2.7. Hybridization with specific MVM probe. The membrane is incubated in prehybridization solution (5X SSC, 5X Denhardts's [Ficoll (Ty400), Polyvinylpyrrolidone, BSA], 10 mM Tris 7.5, 0.5% SDS, 50% Formamide), to eliminate possible nonspecific binding, for four hours at 42 ° CA the hybridization solution is then added, which is formed by the same components of the pre-hybridization solution in addition to the probe, which is total DNA of the MVM virus marked in vitro at high specific activity with 32p per "Random priming" It is incubated at 42º C for one or two days. Finally, the filter is washed with a solution of 0.1X SSC and 0.5% SDS at 50 ° C for three hours, before exposure.
    3.  Eukaryotic cells established in culture.
    3.1. Culture. The following mammalian cell lines have been used: NB324K: newborn human kidney fibroblasts transformed with SV40 large T antigen. A9: line derived from mouse L fibroblasts selected by the HGPRT phenotype. U-373 MG: glioblastoma-human astrocytoma (ATCC: HTB 17). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) buffered with 0.3% NaHCO3 and in a 5% CO2 atmosphere. The medium was supplemented with antibiotics (75 U / ml of


    streptomycin, 75 μg / ml penicillin G), 2 mM L-Glutamine and 5% fetal bovine serum (FCS) decomplemented at 56 ° C for 30 minutes.
    3.2. Treatment of cells by electric pulses. The cells were trypsinized,
    5 centrifuged in a tabletop centrifuge for 5 min at 1200 rpm, resuspended in DMEM with 5% FCS at a concentration of 2x107 cells / ml and kept on ice for 30 min. For electric pulses (or electroporation), 150 μl of the cell suspension was added to the mixture of 20 μg of salmon carrier "carrier" DNA in 26 μl of DMEM with 5% FCS and 10 μl of 1.5 M NaCl. After a gentle stirring,
    10 transferred to the electroporation cuvette (Biorad, 0.4 cm wide) and a pulse was given at 250 V and 450 μF in a Biorad Generator electroporator with capacity expander. After the electrical pulse, the cells were seeded in the culture medium specified above, composed of DMEM: F12 (1: 1) with glutamax (Invitrogen), supplemented with 0.5% albumax I (Invitrogen), 5 mM HEPES ( Invitrogen), 0.915% glucose (Sigma), N2 1x (Invitrogen), 20
    15 ng / ml FGF-2 (Peprotech), 20 ng / ml EGF (Peprotech), 2 μg / ml heparin (Sigma), non-essential amino acids (L-Ala 44 mM, L-Asn 45 mM, L-Asp 40 mM , 40 mM L-Glu, 30 mM L-Pro) and, alternatively, mixture of antibiotics (penicillin 63.2 mg / ml and streptomycin 0.1 mg / ml) or gentamicin (0.055 mg / ml), pre-heated to 37 ºC.
    20 4. Immunological techniques
    4.1. Antisera. The primary and secondary antibodies used were:
    Antibody DilutionIncubationComercial HouseReference
    Immunofluorescence and Flow Cytometry p- (S / T) AMT / ATR Substrates B7 CD133 CD133 stem cell marker pHistone H2A.X pAc mAc pAc pAc pAc1: 100 1:50 1: 100 1: 100 1: 1001 h RT 1 h RT 12 h 4 º C 12 h 4 º C 1 h RTCell Signaling Non-Commercial Abcam Abcam Cell2851S C.R. Parrish ab16518 ab19898 2577S


    Antibody DilutionIncubationComercial HouseReference
    Signaling
    Nestina mAc1: 5001 h RTChemicon NoMAB5326 J. M.
    NS1 (SP7) pAc1: 20001 h RTcommercial NoAlmendral J. M.
    NS1 (b-Gal) mAc1: 1001 h RTcommercialAlmond
    Nucleostemina (GNL3) pAc1: 5001 h RTChemiconAB5689
    Nucleostemina (GNL3) pAc1: 2001 h RTR & DAF1638
    pRPA32 (S4 / S8) pAc1:501 h RTBethylA300-245A
    Sox2 pAc1: 3001 h RTChemicon NoAB5603 J. M.
    VP (VPB) pAc1: 1001 h RTcommercial NoAlmendral J. M.
    VP (VBK) Alexa 555 Antibody pAc1:201 h RTcommercialAlmond
    against rabbit IgG Alexa 647 Antibody pAc1: 50045 min RTInvitrogenA-31572
    against rabbit IgG Antibody Alexa488 pAc1: 50045 min RTInvitrogen
    against mouse IgG Western Blot pAc1: 50045 min RTInvitrogenA-31570
    Actina pAc1: 10001 h RTSigma NoA2066 J. M.
    NS1 (SP7) pAc1: 200001 h RTcommercial NoAlmendral J. M.
    VP (VPB) pAc1: 100001 h RTcommercialAlmond
    Tubulin mAc1: 50001 h RTSigmaT9026
    Mouse Ig-HRP pAc1: 30001 h RTDAKOP0161
    Rabbit Ig-HRP pAc1: 30001 h RTDAKOP0448


    4.2.-Indirect immunofluorescence (IF). Inmucitofluorescence analysis of neurospheres and individual cells was carried out, as well as by immunohistofluorescence of brain tissue. In the case of spherical preparations, a confocal laser scanning microscope LSM510 META coupled to an inverted microscope Axiovert200 (Zeiss) or a multipurpose confocal laser scanning microscope LSM710 coupled to an inverted microscope AxioObserver (Zeiss) was used. For the preparations with individual cells, an Axiovert200 (Zeiss) inverted microscope coupled to a monochrome and color ccd camera or a multiphoton confocal laser scanning microscope LSM710 coupled to an AxioObserver inverted microscope (Zeiss) was used, as indicated in each case. The images were processed with the programs Adobe Photoshop CS4 (Adobe Systems Incorporated) and ImageJ / FIJI (http://rsb.info.nih.gov/ij/) taking as reference of zero signal the same samples without using antibodies (control of autofluorescence) or only with secondary antibodies (control of autofluorescence plus non-specificity of secondary antibodies). Complete neurospheres: the spheres were adhered to coverslips coated with a poly-lysine matrix (0.005% in PBS) and laminin (10 μg / ml) (both from Sigma) and fixed for 30 minutes with 4% paraformaldehyde. They were permeabilized for 10 minutes at room temperature with 0.1% SDS (Sigma) in PBS and blocked by incubating 20 minutes with 0.1% SDS and 10% fetal bovine serum (Sigma) in PBS. The nuclei were stained with To-Pro-3 (Invitrogen) and mounted with Fluoromount G (Southern Biotech). Before incubations of secondary antibodies, To-Pro-3 and assembly, 4 washes of 5 minutes with PBS were performed. Individual cells: to analyze hGSC as dedifferentiated individual cells, the corresponding treatment was applied, they were adhered to coverslips coated with a poly-lysine matrix (0.005% in PBS) and laminin (10 μg / ml) (Sigma) for 30 days. minutes and fixed for 30 minutes with 4% paraformaldehyde. The cell lines were seeded on coverslips, treated and fixed for 30 min with 4% paraformaldehyde. They were permeabilized for 10 minutes at room temperature with 0.1% Triton X100 (Merck) in PBS and blocked by incubating 20 minutes with 0.1% Triton X-100 and 10% fetal bovine serum (Sigma) in PBS. For nuclear staining, DAPI (Calbiochem) was used and the assembly was carried out with Fluoromount G (Southern Biotech). Before the incubations of the secondary antibodies, the DAPI and the assembly, 4 washes of 5 minutes were made with PBS FISH plus immunofluorescence: this technique was used to complement the immunodetection of proteins with the specific recognition of DNA sequences. In


    This work was used to confirm the replication of the parvovirus in the cells, so the probe used was a mixture of three DNA oligos, complementary to different sequences of the genome of the virus, marked with Texas Red. The cells adhered in the same way as before. described were permeabilized for 10 minutes with 0.2% Triton X100 (Merck) in PBS and equilibrated for 5 minutes at room temperature in 2x SSC (0.3 M NaCl; 30 mM Na2C6H5O7, pH 7) supplemented with 15% formamide 500 ng of each oligo were hybridized in FISH buffer for 2.5 hours at 37 ° C. The preparations were washed twice with 2x SSC + 15% formamide for 30 minutes. They were blocked with 0.1% Triton X-100 and 1% FBS in PBS for 20 min at room temperature and the immunofluorescence protocol described above was continued.
    5.-Protein analysis 5.1.-Electrophoresis and membrane transfer. Protein samples, once denatured by boiling for five minutes in loading buffer (10% v / v glycerol (Merck), SDS 2.3% w / v, 5% β-mercaptoethanol (Merck), bromophenol blue 0.002 % (Merck) in Tris-HCl 0.5 M pH 6.3 with SDS 0.4%), were separated by electrophoresis in denaturing gels of polyacrylamide 5% to detect the presence of oligomers or 8% to check the presence of proteins viral Electrophoresis was carried out in Tris-Glycine (25 mM Tris (Serva), 192 mM Glycine (Gibco), 0.1% SDS) overnight at 10 mA (without limiting the voltage) in the case of maxigeles (20x16x0.2 cm) or for four hours at 100 V (without limiting amperage) for the minigels (10x10x0.1 cm). The molecular mass markers used were: "Prestained SDS-PAGE Standards, Broad Range" (Biorad) and "Protein Molecular Weight Standards, Broad Range" (Amersham). The samples were transferred to nitrocellulose (Schleicher and Schuel) in transfer buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS and 20% methanol) for one hour at 100 V using a wet transfer system (Trans blot Electroforetictransfer Cell, Biorad).
    5.2.-Membrane detection ("western blot"). After transferring the samples, the membrane was hydrated in TBS-T buffer (Tris pH 7.5 20 mM, 140 mM NaCl, 0.1% Tween 20) and incubated with shaking for one hour at room temperature in TBS-T with 5% of non-fat milk powder. After washing with TBS-T it was incubated with the primary antibody diluted in TBS-T with 1% milk powder and 1% NP40 for one hour at room temperature. After thorough washing, it was treated with the secondary antibody under the same conditions as the primary one. Finally it was washed with TBS-T and TBS (without Tween 20), it was revealed with the


    ECL system ("Enhanced Chemiluminiscence", Amersham) following the manufacturer's recommendations, and the membrane was exposed in Kodak autoradiography films.
    6. Administration of parvovirus MVM for tumor therapy in animals.
    5 6.1. Intracranial inoculation of MVMp by convection-enhanced instillation (CED). Stereotaxic inoculation of 1 · 107 pfu of MVMp in 10 μl of PBS was carried out. The conditions of the operation, including the inoculation coordinates, were identical to those described above. The viruses were inoculated by convection-enhanced administration (CED) at a constant flow of 0.5 μl / min using an injector
    10 motorized (Stoelting Mod.310). The cannula used consisted of a 431 μm external diameter silica tube (319 μm internal diameter) into which another tube, of the same material, of 236 μm external diameter (99 μm internal diameter) was inserted. The inner tube protrudes 1 mm from the end of the support tube (stepped design).
    15 6.2. Intranasal inoculation by MVMi. We proceeded to the intranasal inoculation of 10 μl (1 · 107 pfu) of MVMi in mice previously xenotransplanted orthotopically with hGSC under the conditions described.


    LIST OF SEQUENCESSEQ ID NO 1
    atttttagaa ctgaccaacc atgttcacgt aagtgacgtg atgacgcgcg ctgcgcgcgc 60 gccttcggac gtcacacgtc acttacgttt cacatggttg gtcagttcta aaaatgataa 120 5 gcggttcagg gagtttaaac caaggcgcga aaaggaagtg ggcgtggttt aaagtatata 180 agcaactact gaagtcagtt acttatcttt tctttcattc tgtgagtcga gacgcacaga 240 aagagagtaa ccaactaacc atggctggaa atgcttactc tgatgaagtt ttgggagcaa 300 ccaactggtt aaaggaaaaa agtaaccagg aagtgttctc atttgttttt aaaaatgaaa 360 atgttcaact gaatggaaaa gatatcggat ggaatagtta caaaaaagag ctgcaggagg 420 10 atctttacaa acgagctgaa cgaggagcgg aaactacttg ggaccaaagc gaggacatgg 480 aatgggaaac cacagtggat gaaatgacca aaaagcaagt attcattttt gattctttgg 540 ttaaaaaatg tttatttgaa gtgcttaaca caaagaatat atttcctggt gatgttaatt 600 ggtttgtgca acatgaatgg ggaaaagacc aaggctggca ctgccatgta ctaattggag 660 gaaaggactt tagtcaagct caagggaaat ggtggagaag gcaactaaat gtttactgga 720 15 gcagatggtt ggtaacagcc tgtaatgtgc aactaacacc attaaactaa agctgaaaga 780 gagaaatagc agaagacaat gagtgggtta ctctacttac ttataagcat aagcaaacca 840 aaaaagacta taccaagtgt gttctttttg gaaacatgat tgcttactat tttttaacta 900 aaaagaaaat aagcactagt ccaccaagag acggaggcta ttttcttagc agtgactctg 960 gctggaaaac taacttttta aaagaaggcg agcgccatct agtgagcaaa ctatacactg 1020 20 atgacatgcg gccagaaacg gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg 1080 aactaaaaaa gcagaattca gaagtttcta ttaaaactac acttaaagag ctggtgcata 1140 aaagagtaac ctcaccagag gactggatga tgatgcagcc agacagttac attgaaatga 1200 tggctcaacc aggtggagaa aacctgctga aaaatacgct agagatttgt acactaactc 1260 tagccagaac caaaacagca ttttagaaaa tttgacttaa agctgaaacc agcaaactaa 1320 25 ccaacttttc actgcctgac acaagaacct gcagaatttt tgcttttcat ggctggaact 1380 atgttaaagt ttgccatgct atttgctgtg ttttaaacag acaaggaggc aaaagaaata 1440 tcatggacca ctgttttatt gccagcacag gcaaatctat tattgcacaa gccatagcac 1500 aagcagttgg caatgttggt tgctataatg cagccaatgt aaactttcca tttaatgact 1560 gtaccaacaa gaacttgatt tgggtagaag aagctggtaa ctttggacag caagtaaacc 1620 30 agtttaaagc catttgctct ttcgcattga ggtcaaacta aaaggcagca tcaaaaagga 1680 aacaga tTGA gtcatcatga accaacacca ccacaaatga gaacattaca gtggtcagaa 1740 agaaagacca taggctgcga gaacacactc aaccaatcag agacagaatg cttaacattc 1800 atctaacaca taccttgcct ggtgactttg gtttggttga tggcccatga caaaaatgaa 1860 tttgtgcttg gttggtaaag aatggttacc aatctaccat ggcaagctac tgtgctaaat 1920 35 ggggcaaagt tcctgattgg tcagaaaact gggcggagcc aaaggtgcca actcctataa 1980 atttactagg ttcggcacgc tcaccattca aagtacgcct cgacaccgaa ctcagccaga 2040


    actatgcact aactccactt gcatcggatc tcgaggacct ggctttagag ccttggagca 2100 caccaaatac tcctgttgcg ggcactgcag aaacccagaa gctggttcca cactggggaa 2160 agatggtcaa aagcctgcca ctgagcccaa cttggtcaga gatcgaggag gatttgagag 2220 cgtgcttcgg tgcggaaccg ttgaagaaag acttcagcga gccgctgaac ttggactaag 2280 5 gtacgatggc gcctccagct aaaagagcta aaagaggtaa gggtttaagg gatggttggt 2340 ttaatgttta tggtggggta attacctgtt ttacaggcct gaaatcactt ggttttaggt 2400 ctggctacaa tgggtgcctc gtacctggga ccagggaaca aggagaacca gccttgacca 2460 accaatccat ctgacgccgc tgccaaagag cacgacgagg cctatgatca atacatcaaa 2520 tctggaaaaa atccttacct gtacttctct gctgctgatc aacgctttat tgaccaaacc 2580 10 aaggacgcca aagactgggg aggcaaggtt ggtcactact tttttagaac caagcgcgct 2640 tttgcaccta agcttgctac tgactctgaa cctggaactt ctggtgtaag cagagctggt 2700 aaacgcacta gaccacctgc ttacattttt attaaccaag ccagagctaa aaaaaaactt 2760 ctgcacagca acttcttctg aagcagtcaa accatgagtg atggcaccag ccaacctgac 2820 agcggaaacg ctgtccactc agctgcaaga gttgaacgag cagctgacgg ccctggaggc 28 80 15 tctgggggtg ggggctctgg cgggggtggg gttggtgttt ctactgggtc ttatgataat 2940 caaacgcatt atagattctt gggtgacggc tgggtagaaa ttactgcact agcaactaga 3000 ctagtacatt taaacatgcc aactattgca taaatcagaa tcacaataca gaatcagagt 3060 acagacacat cagtcaaagg aaagatgatg caacatggca aatttggaca ctcatgagca 3120 ccatggagct tggtggatgc taatgcttgg ggagtttggc tccagccaag tgactggcaa 3180 20 tacatttgca acaccatgag ccagcttaac ttggtatcac ttgatcaaga aatattcaat 3240 gtagtgctga aaactgttac agagcaagac ttaggaggtc aagctataaa aatatacaac 3300 cagcttgcat aatgacctta gtagactcaa gatggttgca acaacatttt gccatacaca 3360 actcaatgga cctgcagcaa aacacttggt ttctacccct ggaaaccaac catagcatca 3420 ccatacaggt actatttttg gatctttcag cgttgacaga tgacctacga aaatcaagaa 3480 25 ggcacagttg aacataatgt gatgggaaca tgaattctca ccaaaaggaa attttttacc 3540 attgagaaca cacaacaaat cacattgctc agaacagggg acgaatttgc cacaggtact 3600 tactactttg acacaaattc agttaaactc acacacacgt ggcaaaccaa ccgtcaactt 3660 ggacagcctc cactgctgtc aacctttcct gaagctgaca ctgatgcagg tacact tact 3720 gctcaaggga gcagacatgg aacaacacaa atgggggtta actgggtgag tgaagcaatc 3780 30 agaaccagac ctgctcaagt aggattttgt caaccacaca atgactttga agccagcaga 3840 gctggaccat ttgctgcccc gcagatatta aaaagttcca ctcaaggagt agacaaagaa 3900 gtgttagata gccaatggca cagttatggc aaacagcatg gtgaaaattg ggcttcacat 3960 ggaccagcac cagagcgcta cacatgggat gaaacaagct ttggttcagg tagagacacc 4020 aaagatggtt ttattcaatc agcaccacta gttgttccac caccactaaa tggcattctt 4080 35 acaaatgcaa accctattgg gactaaaaat gacattcatt tttcaaatgt ttttaacagc 4140 tatggtccac taactgcatt ttcacaccca agtcctgtat accctcaagg acaaatatgg 4200


    gacaaagaac tagatcttga acacaaacct agacttcaca taactgctcc atttgtttgt 4260 aaaaacaatg cacctggaca aatgttggtt agattaggac caaacctaac tgaccaatat 4320 gatccaaacg gagccacact ttctagaatt gttacatacg gtacattttt ctggaaagga 4380 tgagagcaaa aaactaacca acttagagct aacaccactt ggaacccagt gtaccaagta 4440 5 agtgctgaag acaatggcaa ctcatacatg agtgtaacta aatggttacc aactgctact 4500 ggaaacatgc agtctgtgcc gcttataaca agacctgttg ctagaaatac ttactaacta 4560 accatgcttt ttctttctgt acttcatata ttattaagac taataaagat acaacataga 4620 aatataatat tacgtataga tttaagaaat agaataatat ggtacttagt aactgttaaa 4680 aataatagaa cctttggaat aacaagatag ttagttggtt aatgttagat agaataagaa 4740 10 gatcatgtat aatgaataaa agggtggaag ggtggttggt aggttaatgt tagatagaat 4800 tgtataatga aagaagatca ataaaagggt ggaagggtgg ttggtaggta ttcccttaga 4860 aggaccaaaa cttgatgtta aaataataaa acttttttaa aactcaacca agactactgt 4920 aaccaactga ctattcagtg accattagta ttactatgtt tttagggtgg gagggtggga 4980 gatacatgtg ttcgctatga gcgaactggt actggttggt tgctctgctc aaccaaccag 50 40 15 accggcaaag ccggtctggt tggttgagcg caaccaacca gtaccagttc gctcatagcg 5100 aacacatgta tctcccaccc tcccacccta aaaacatagt aatactaat 5149
    SEQ ID NO 2
    atttttagaa ctgaccaacc atgttcacgt aagtgacgtg atgacgcgcg cttcgcgcgc 60
    20 tgccttcgga cgtcacacgt cacttacgtt tcacatggtt ggtcagttct aaaaatgata 120 agcggttcag agagtttaga ccaaggcgcg aaaaggaagt gggcgtggtt taaagtatat 180 aagcaaatgc tgaagtcagt tacttatcct ttctttcatt ctgtgagtcg agacgcgcag 240 aaagagagta accaactaac catggctgga aatgcttact ctgatgaagt tttgggaaca 300 accaactggt taaaggaaaa aagtaaccag gaagtgttct catttgtttt taaaactgag 360
    25 gatgttcaac taaatggaaa agatatcgga tggaataatt acaaaaagga gctgcaggag 420 aatctttaca gacgagctga acgaggagcg gaaactacct gggaccaaag cgaggacatg 480 gaatgggaat ctacagtgga tgaaatgacc aaaaagcaag tattcattta tgactcttta 540 gttaaaaaat gtttgtttga agtgcttagc acaaaaaata tagctcctgc tgatgttact 600 tggtttgtgc agcatgaatg ggggaaagac caaggctggc actgccatgt actaattgga 660
    30 ggcaaggact ttagtcaagc tcaaggaaaa tggtggagaa ggcagctaaa tgtttactgg 720 agcagatggt tggtaacagc ctgtaatgtg cagctaacac cagctgaaag aattaaacta 780 agagaaatag cagaagacag tgagtgggtt cttataaaca actttactca taagcaaacc 840 aaaaaggact atactaaatg tgttcttttt ggaaatatga ttgcttacta ctttttaacc 900 aaaaagaaaa taagcaccag tccgccaagg gacggaggct attttctaag cagtgactct 960
    35 ggctggaaaa ctaacttttt aaaagagggc gaacgccatc tagtgagcaa attatacact 1020 gatgacatgc ggccagaaac ggttgaaacc acagtaacca ctgcgcagga aactaagcgc 1080


    ggcagaattc aaactaaaaa agaggtttct attaaaacca cacttaaaga gctagtgcat 1140 cctcaccaga aaaagagtaa agactggatg atgatgcagc cagacagtta cattgaaatg 1200 atggctcaac caggtggaga aaacctgctg aaaaatacgc tagagatttg tacgctaact 1260 ctagccagaa caaaaacagc atttgacttg attttagaaa aagctgaaac cagcaaacta 1320 5 accaactttt cactgcctga cacaagaacc tgcaagattt ttgcttttca tggctggaac 1380 tatgttaaag tttgccatgc tatttgctgt gttctaaaca gacaaggagg caaaagaaat 1440 actgttttat ttcacggacc agccagtaca ttattgcaca ggcaaatcta agccatagca 1500 caggcagttg gtaatgttgg ttgctataat gcagctaatg tgaactttcc atttaatgac 1560 agaacttgat tgtaccaaca gaagctggta ttgggtagaa actttggaca gcaagtaaac 1620 10 cagtttaaag ccatttgctc tggtcaaact attcgcattg atcaaaaagg aaaaggcagc 1680 aaacaaattg aaccaacacc agtcatcatg accacaaatg agaacattac agtggtcaga 1740 ataggctgcg aagagagacc agaacacact caaccaatta gagacagaat gctcaacatt 1800 catctaacac atacattgcc tggtgacttt ggtttggttg acaagaatga atggcccatg 1860 atttgtgctt ggttggtaaa caatctacca gaatggttac tggcaagcta ctgcgctaaa 19 20 15 tggggcaaag ttcctgattg gtcagaaaac tgggcggagc caaaggtgcc gactcctata 1980 aattcactag gttcggcacg ctcaccattc acgacaccga aaagtacgcc tctcagccag 2040 aactatgcaa taactccact tgcatcggat ctcgaggacc tggctttaga gccttggagc 2100 ctcctgttgc acaccaaata gaaacccaga gggcactgca acactgggga agctggttcc 2160 aagatggtca aaagcctgcc actgagccca agatcgagga acttggtcag ggatttgaga 2220 20 gcgtgcttcg gtgcggaacc gttgaagaga gacttcagcg agccgctgaa cttggactaa 2280 ggtacgatgg cgcctccagc taaaagagct aaaagaggta agggtttaag ggatggttgg 2340 ttggtggggt attaatgttt aattacctgt tttacaggcc tgaaatcact tggttttagg 2400 ttgggtgcct cctggctata agtacctggg accagggaac agccttgacc aaggagaacc 2460 aaccaatcca tctgacgccg ctgccaaaga gcacgacgag gcctatgatc aatacatcaa 2520 25 atctggaaaa aatccttacc tgtacttctc tgctgctgat caacgcttta ttgaccaaac 2580 caaggacgcc aaagactggg gaggcaaggt tggtcactac ttttttagaa ccaagcgcgc 2640 aagcttgcta ttttgcacct ctgactctga acctggaact tctggtgtaa gcagagctgg 2700 taaacgcact agaccacctg cttacatttt tataaaccaa gccagagcta aaaaaaa act 2760 tacttcttct gctgcacagc aaagcagtca gatggcacca aaccatgagt gccaacctga 2820 30 cggcggaaac gctgtccact cagctgcaag agttgaacga gcagctgacg gccctggagg 2880 ctctgggggt gggggctctg gcgggggtgg ggttggtgtt tctactgggt cttatgataa 2940 tcagacgcat tatagattct tgggtgacgg ctgggtagaa attactgcac tagcaactag 3000 actagtacat ttaaacatgc ctaaatcaga aaactattgc agaataagag ttcacaacac 3060 aacagacact tcagtcaaag gcaacatggc aaaagatgat gctcatgagc aaatttggac 3120 35 gccatggagc ttagtggatg ctaatgcttg gggagtttgg ctccagccaa gtgactggca 3180 atacatttgc aacaccatga gccagcttaa cttggtctca cttgatcaag aaatatttaa 3240


    tgtagtgctg aaaactgtta ctcaggaggt cagagcaaga caagctataa aaatatacaa 3300 acagcttgca caatgacctc agtagactca tgatggttgc aacaacattc tgccatacac 3360 acctgcagca aactcaatgg aaacacttgg tttctaccct tggaaaccaa ctatagcatc 3420 gccatacagg tactatttct gcgttgacag agatctttca gtaacctatg aaaatcaaga 3480 5 aggcacaatt gagcataatg taatgggaac accaaaagga atgaattctc aattttttac 3540 cattgagaac acacaacaaa tcacattgct cagaactggt gatgagtttg ctactggaac 3600 ctactacttt gacacaaacc cagttaaact tacacacaca tggcaaacta accgtcaact 3660 tggacagcct ccactgctgt caacctttcc tgaagctgac actgatgcag gtacacttac 3720 tgctcaaggg agcagacatg gagcaacaca gatggaggtt aactgggtga gtgaagcaat 3780 10 tagaaccaga cctgctcaag taggattttg tcagccacac aatgactttg aagccagcag 3840 agctggacca tttgctgctc caaaagttcc agcagatgtt actcaaggag tggacagaga 3900 agccaatggc agtgttagat acagttatgg caaacagcat ggtgaaaatt gggctgcaca 3960 ccagagcgct cggaccagca acacatggga tgaaacaaac tttggttcag gaagagacac 4020 cagagatggt tttattcaat cagcacctct agttgttcca ccaccactaa atgggattct 40 80 15 tacaaatgca aaccctattg gaactaaaaa tgacattcat ttttcaaatg tttttaacag 4140 ctatggtcca ctaactgcat tttcacaccc aagtcctgta taccctcaag gacaaatatg 4200 ggacaaagaa ctagatcttg aacacaaacc tagacttcac ataactgctc catttgtctg 4260 taaaaacaat gcacctggac aaatgttggt ccaaatctaa tagattagga ctgaccagta 4320 tgatccaaac ggagccacac tttctagaat tgtgacttat ggtacatttt tctggaaagg 4380 20 aaaactaacc atgagagcaa aacttagagc taacaccact tggaacccag tgtaccaagt 4440 gacaatggca aagtgttgaa actcatacat gagtgttact aaatggctac caactgctac 4500 tggaaacatg caatctgtac cgcttataac aagacctgtt gctagaaata cttactaact 4560 aaccatgttt ttcctttctg tacttcatat attattaaga tacaacataa ctaataaaga 4620 aaatataata ttacatatag atttaagaaa tagaataata tggtacttag taactgttag 4680 25 acctttgaaa aaataataga attagttggt taacaagata tagaataaga taatgttaga 4740 taatgggtaa agattatgta aagggtggaa gggtggttgg ttggtattcc cttagacatg 4800 atgttaagga ccaaaaaaat aataaaattt tttaaaacta aaccaagact actgtctatt 4860 aactgaacca cagttgaacc tcagtatcac tatgttttta gggtgggggg gtgggag ata 4920 catgtgttcg ctatgagcga actggtactg gttggttgct ctgctcaacc aaccagaccg 4980 30 gctttgccgg tctggttggt tgagcgcaac caaccagtac cagttcgctc atagcgaaca 5040 catgtatctc ccaccccccc accctaaaaa catagtgata ctgat 5085
    权利要求:
    Claims (18)
    [1]
    1. Viral particle comprising a sequence of nucleotides:a) the sequence of SEO ID NO 1; orb) the sequence of SEO ID NO 2; ora mutant or variant of SEO ID N01 or SEO ID NO 2, which does not affect the characteristicsessential elements of MVM parvovirus, especially that they do not alter any biological propertiesrelated to the tropism or anti-cancer activity of the original MVM (p, i) strains; forits use as a medicine.
    [2]
    2. Viral particle that comprises a nucleotide sequence consisting of:a) the sequence of SEO ID NO 1; orb) the sequence of SEO ID NO 2;for use as a medicine.
    [3]
    3. Use of a viral particle comprising a nucleotide sequence:a) the sequence of SEO ID NO 1; orb) the sequence of SEO ID NO 2; ora mutant or variant of SEO ID N01 or SEO ID NO 2, which does not affect the characteristicsessential elements of MVM parvovirus, especially that they do not alter any biological propertiesrelated to the tropism or anti-cancer activity of the original MVM strains (p, i);
    in the preparation of a drug for the treatment of glioma.
    [4]
    Four. Use according to claim 3, wherein the viral particle comprises a sequence ofnucleotides consisting of the sequence of SEO ID NO 1.
    [5]
    5. Use according to claim 3, wherein the viral particle comprises a sequence ofnucleotides consisting of the sequence of SEO ID NO 2.
    [6]
    6. Use according to any one of claims 3 to 5, wherein the glioma is aglioblastoma.
    [7]
    7. Use according to claim 6, wherein the glioblastoma is a recurrence.
    [8]
    8. Use according to any one of claims 3 to 7, wherein the medicineit comprises a vehicle suitable for intracranial or nasal administration.
    [9]
    9. Pharmaceutical composition comprising as active principle a viral particle as defined in any of claims 1 and 2.
    [10]
    10. Pharmaceutical composition according to claim 9, wherein the viral particle is found in a concentration of between 106 and 1013 infectious units (pfu) per milliliter.
    [11]
    eleven. Pharmaceutical composition according to any one of claims 9 and 10, which comprises at least one pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable adjuvant.
    [12]
    12. Pharmaceutical composition according to claim 11, wherein the vehicle or excipient is such that it allows the administration of said composition by the intracerebral, intraperitoneal, intravenous, intramuscular, subcutaneous, intracutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal routes.
    [13]
    13. Pharmaceutical composition according to claim 12, wherein the administration is intracerebral, intravenous, or intranasal.
    [14]
    14. Pharmaceutical composition according to any one of claims 9 to 13, which comprises at least one additional active principle.
    [15]
    fifteen. Pharmaceutical composition according to claim 14, wherein the additional active principle is an antitumor agent.
    [16]
    16. Pharmaceutical composition according to any one of claims 14 and 15, wherein the additional active principle is temozolomide or bevacizumab.
    [17]
    17. Use of the viral particle according to any one of claims 1 and 2 for the preparation of a pharmaceutical composition according to any one of claims 9 to 16 for the treatment of glioma.
    [18]
    18. Use according to claim 17, wherein the glioma is a glioblastoma.
    Figure 1: Figure 2: Figure 3: Figure 3 continued: Figure 4: Figure 5: Figure 6: Figure 6 continued: Figure 7: Figure 7 continued: Figure 8: Figure 8 continued:
    <110> UNIVERSIDAD AUTÓNOMA DE MADRID
    <120> Treatment of glioma or glioblastoma with MVM parvovirus
    <130> 900208
    <160> 2
    <170> PatentIn version 3.5
    <210> 1
    <211> 5149
    <212> DNA
    <213> Parvovirus
    <400> 1atttttagaa ctgaccaacc atgttcacgt aagtgacgtg atgacgcgcg ctgcgcgcgc 60gccttcggac gtcacacgtc acttacgttt cacatggttg gtcagttcta aaaatgataa 120gcggttcagg gagtttaaac caaggcgcga aaaggaagtg ggcgtggttt aaagtatata 180agcaactact gaagtcagtt acttatcttt tctttcattc tgtgagtcga gacgcacaga 240aagagagtaa ccaactaacc atggctggaa atgcttactc tgatgaagtt ttgggagcaa 300ccaactggtt aaaggaaaaa agtaaccagg aagtgttctc atttgttttt aaaaatgaaa 360atgttcaact gaatggaaaa gatatcggat ggaatagtta caaaaaagag ctgcaggagg 420acgagctgaa atctttacaa cgaggagcgg aaactacttg ggaccaaagc gaggacatgg 480aatgggaaac cacagtggat gaaatgacca aaaagcaagt attcattttt gattctttgg 540ttaaaaaatg tttatttgaa gtgcttaaca caaagaatat atttcctggt gatgttaatt 600ggtttgtgca acatgaatgg ggaaaagacc aaggctggca ctgccatgta ctaattggag 660gaaaggactt tagtcaagct caagggaaat ggtggagaag gcaactaaat gtttactgga 720gcagatggtt ggtaacagcc tgtaatgtgc aactaacacc agctgaaaga attaaactaa 780gagaaatagc agaagacaat gagtgggtta ctctacttac ttataagcat aagcaaacca 840aaaaagacta taccaagtgt gttctttttg gaaacatgat tgcttactat tttttaacta 900aaaagaaaat aagcactagt ccaccaagag acggaggcta ttttcttagc agtgactctg 960gctggaaaac taacttttta aaagaaggcg agcgccatct agtgagcaaa ctatacactg 1020atgacatgcg gccagaaacg gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg 1080gcagaattca aactaaaaaa gaagtttcta ttaaaactac acttaaagag ctggtgcata 1140aaagagtaac ctcaccagag gactggatga tgatgcagcc agacagttac attgaaatga 1200tggctcaacc aggtggagaa aacctgctga aaaatacgct agagatttgt acactaactc 1260tagccagaac caaaacagca tttgacttaa ttttagaaaa agctgaaacc agcaaactaa 1320ccaacttttc actgcctgac acaagaacct gcagaatttt tgcttttcat ggctggaact 1380atgttaaagt ttgccatgct atttgctgtg ttttaaacag acaaggaggc aaaagaaata 1440ctgttttatt tcatggacca gccagcacag gcaaatctat tattgcacaa gccatagcac 1500aagcagttgg caatgttggt tgctataatg cagccaatgt aaactttcca tttaatgact 1560


    gtaccaacaa gaacttgatt tgggtagaag aagctggtaa ctttggacag caagtaaacc 1620
    agtttaaagc catttgctct ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca 1680aacagattga accaacacca gtcatcatga ccacaaatga gaacattaca gtggtcagaa 1740taggctgcga agaaagacca gaacacactc aaccaatcag agacagaatg cttaacattc 1800atctaacaca taccttgcct ggtgactttg gtttggttga caaaaatgaa tggcccatga 1860tttgtgcttg gttggtaaag aatggttacc aatctaccat ggcaagctac tgtgctaaat 1920ggggcaaagt tcctgattgg tcagaaaact gggcggagcc aaaggtgcca actcctataa 1980atttactagg ttcggcacgc tcaccattca cgacaccgaa aagtacgcct ctcagccaga 2040actatgcact aactccactt gcatcggatc tcgaggacct ggctttagag ccttggagca 2100caccaaatac tcctgttgcg ggcactgcag aaacccagaa cactggggaa gctggttcca 2160aagcctgcca agatggtcaa ctgagcccaa cttggtcaga gatcgaggag gatttgagag 2220cgtgcttcgg tgcggaaccg ttgaagaaag acttcagcga gccgctgaac ttggactaag 2280gtacgatggc gcctccagct aaaagagcta aaagaggtaa gggtttaagg gatggttggt 2340tggtggggta ttaatgttta attacctgtt ttacaggcct gaaatcactt ggttttaggt 2400tgggtgcctc ctggctacaa gtacctggga ccagggaaca gccttgacca aggagaacca 2460accaatccat ctgacgccgc tgccaaagag cacgacgagg cctatgatca atacatcaaa 2520tctggaaaaa atccttacct gtacttctct gctgctgatc aacgctttat tgaccaaacc 2580aaggacgcca aagactgggg aggcaaggtt ggtcactact tttttagaac caagcgcgct 2640tttgcaccta agcttgctac tgactctgaa cctggaactt ctggtgtaag cagagctggt 2700aaacgcacta gaccacctgc ttacattttt attaaccaag ccagagctaa aaaaaaactt 2760acttcttctg ctgcacagca aagcagtcaa accatgagtg atggcaccag ccaacctgac 2820agcggaaacg ctgtccactc agctgcaaga gttgaacgag cagctgacgg ccctggaggc 2880tctgggggtg ggggctctgg cgggggtggg gttggtgttt ctactgggtc ttatgataat 2940caaacgcatt atagattctt gggtgacggc tgggtagaaa ttactgcact agcaactaga 3000ctagtacatt taaacatgcc taaatcagaa aactattgca gaatcagagt tcacaataca 3060acagacacat cagtcaaagg caacatggca aaagatgatg ctcatgagca aatttggaca 3120ccatggagct tggtggatgc taatgcttgg ggagtttggc tccagccaag tgactggcaa 3180tacatttgca acaccatgag ccagcttaac ttggtatcac ttgatcaaga aatattcaat 3240gtagtgctga aaactgttac agagcaagac ttaggaggtc aagctataaa aatatacaac 3300aatgacctta cagcttgcat gatggttgca gtagactcaa acaacatttt gccatacaca 3360cctgcagcaa actcaatgga aacacttggt ttctacccct ggaaaccaac catagcatca 3420ccatacaggt actatttttg cgttgacaga gatctttcag tgacctacga aaatcaagaa 3480ggcacagttg aacataatgt gatgggaaca ccaaaaggaa tgaattctca attttttacc 3540attgagaaca cacaacaaat cacattgctc agaacagggg acgaatttgc cacaggtact 3600

    tactactttg acacaaattc agttaaactc acacacacgt ggcaaaccaa ccgtcaactt 3660
    ggacagcctc cactgctgtc aacctttcct gaagctgaca ctgatgcagg tacacttact 3720gctcaaggga gcagacatgg aacaacacaa atgggggtta actgggtgag tgaagcaatc 3780agaaccagac ctgctcaagt aggattttgt caaccacaca atgactttga agccagcaga 3840gctggaccat ttgctgcccc aaaagttcca gcagatatta ctcaaggagt agacaaagaa 3900gccaatggca gtgttagata cagttatggc aaacagcatg gtgaaaattg ggcttcacat 3960ggaccagcac cagagcgcta cacatgggat gaaacaagct ttggttcagg tagagacacc 4020aaagatggtt ttattcaatc agcaccacta gttgttccac caccactaaa tggcattctt 4080acaaatgcaa accctattgg gactaaaaat gacattcatt tttcaaatgt ttttaacagc 4140tatggtccac taactgcatt ttcacaccca agtcctgtat accctcaagg acaaatatgg 4200gacaaagaac tagatcttga acacaaacct agacttcaca taactgctcc atttgtttgt 4260aaaaacaatg cacctggaca aatgttggtt agattaggac caaacctaac tgaccaatat 4320gatccaaacg gagccacact ttctagaatt gttacatacg gtacattttt ctggaaagga 4380aaactaacca tgagagcaaa acttagagct aacaccactt ggaacccagt gtaccaagta 4440agtgctgaag acaatggcaa ctcatacatg agtgtaacta aatggttacc aactgctact 4500ggaaacatgc agtctgtgcc gcttataaca agacctgttg ctagaaatac ttactaacta 4560accatgcttt ttctttctgt acttcatata ttattaagac taataaagat acaacataga 4620aatataatat tacgtataga tttaagaaat agaataatat ggtacttagt aactgttaaa 4680aataatagaa cctttggaat aacaagatag ttagttggtt aatgttagat agaataagaa 4740gatcatgtat aatgaataaa agggtggaag ggtggttggt aggttaatgt tagatagaat 4800aagaagatca tgtataatga ataaaagggt ggaagggtgg ttggtaggta ttcccttaga 4860cttgatgtta aggaccaaaa aaataataaa acttttttaa aactcaacca agactactgt 4920ctattcagtg aaccaactga accattagta ttactatgtt tttagggtgg gagggtggga 4980gatacatgtg ttcgctatga gcgaactggt actggttggt tgctctgctc aaccaaccag 5040accggcaaag ccggtctggt tggttgagcg caaccaacca gtaccagttc gctcatagcg 5100aacacatgta tctcccaccc tcccacccta aaaacatagt aatactaat 5149
    <210> 2
    <211> 5085
    <212> DNA
    <213> Parvovirus
    <400> 2atttttagaa ctgaccaacc atgttcacgt aagtgacgtg atgacgcgcg cttcgcgcgc 60tgccttcgga cgtcacacgt cacttacgtt tcacatggtt ggtcagttct aaaaatgata 120agcggttcag agagtttaga ccaaggcgcg aaaaggaagt gggcgtggtt taaagtatat 180aagcaaatgc tgaagtcagt tacttatcct ttctttcatt ctgtgagtcg agacgcgcag 240aaagagagta accaactaac catggctgga aatgcttact ctgatgaagt tttgggaaca 300
    accaactggt taaaggaaaa aagtaaccag gaagtgttct catttgtttt taaaactgag
    gatgttcaac taaatggaaa agatatcgga tggaataatt acaaaaagga gctgcaggag 420gacgagctga aatctttaca acgaggagcg gaaactacct gggaccaaag cgaggacatg 480gaatgggaat ctacagtgga tgaaatgacc aaaaagcaag tattcattta tgactcttta 540gttaaaaaat gtttgtttga agtgcttagc acaaaaaata tagctcctgc tgatgttact 600tggtttgtgc agcatgaatg ggggaaagac caaggctggc actgccatgt actaattgga 660ggcaaggact ttagtcaagc tcaaggaaaa tggtggagaa ggcagctaaa tgtttactgg 720agcagatggt tggtaacagc ctgtaatgtg cagctaacac cagctgaaag aattaaacta 780agagaaatag cagaagacag tgagtgggtt actttactca cttataaaca taagcaaacc 840aaaaaggact atactaaatg tgttcttttt ggaaatatga ttgcttacta ctttttaacc 900aaaaagaaaa taagcaccag tccgccaagg gacggaggct attttctaag cagtgactct 960ggctggaaaa ctaacttttt aaaagagggc gaacgccatc tagtgagcaa attatacact 1020gatgacatgc ggccagaaac ggttgaaacc acagtaacca ctgcgcagga aactaagcgc 1080ggcagaattc aaactaaaaa agaggtttct attaaaacca cacttaaaga gctagtgcat 1140aaaagagtaa cctcaccaga agactggatg atgatgcagc cagacagtta cattgaaatg 1200atggctcaac caggtggaga aaacctgctg aaaaatacgc tagagatttg tacgctaact 1260ctagccagaa caaaaacagc atttgacttg attttagaaa aagctgaaac cagcaaacta 1320accaactttt cactgcctga cacaagaacc tgcaagattt ttgcttttca tggctggaac 1380tatgttaaag tttgccatgc tatttgctgt gttctaaaca gacaaggagg caaaagaaat 1440actgttttat ttcacggacc agccagtaca ggcaaatcta ttattgcaca agccatagca 1500caggcagttg gtaatgttgg ttgctataat gcagctaatg tgaactttcc atttaatgac 1560tgtaccaaca agaacttgat ttgggtagaa gaagctggta actttggaca gcaagtaaac 1620cagtttaaag ccatttgctc tggtcaaact attcgcattg atcaaaaagg aaaaggcagc 1680aaacaaattg aaccaacacc agtcatcatg accacaaatg agaacattac agtggtcaga 1740ataggctgcg aagagagacc agaacacact caaccaatta gagacagaat gctcaacatt 1800catctaacac atacattgcc tggtgacttt ggtttggttg acaagaatga atggcccatg 1860atttgtgctt ggttggtaaa gaatggttac caatctacca tggcaagcta ctgcgctaaa 1920tggggcaaag ttcctgattg gtcagaaaac tgggcggagc caaaggtgcc gactcctata 1980aattcactag gttcggcacg ctcaccattc acgacaccga aaagtacgcc tctcagccag 2040aactatgcaa taactccact tgcatcggat ctcgaggacc tggctttaga gccttggagc 2100acaccaaata ctcctgttgc gggcactgca gaaacccaga acactgggga agctggttcc 2160aaagcctgcc aagatggtca actgagccca acttggtcag agatcgagga ggatttgaga 2220gcgtgcttcg gtgcggaacc gttgaagaga gacttcagcg agccgctgaa cttggactaa 2280ggtacgatgg cgcctccagc taaaagagct aaaagaggta agggtttaag ggatggttgg 2340ttggtggggt attaatgttt aattacctgt tttacaggcc tgaaatcact tggttttagg 2400

    ttgggtgcct cctggctata agtacctggg accagggaac agccttgacc aaggagaacc 2460aaccaatcca tctgacgccg ctgccaaaga gcacgacgag gcctatgatc aatacatcaa 2520atctggaaaa aatccttacc tgtacttctc tgctgctgat caacgcttta ttgaccaaac 2580caaggacgcc aaagactggg gaggcaaggt tggtcactac ttttttagaa ccaagcgcgc 2640ttttgcacct aagcttgcta ctgactctga acctggaact tctggtgtaa gcagagctgg 2700taaacgcact agaccacctg cttacatttt tataaaccaa gccagagcta aaaaaaaact 2760tacttcttct gctgcacagc aaagcagtca aaccatgagt gatggcacca gccaacctga 2820cggcggaaac gctgtccact cagctgcaag agttgaacga gcagctgacg gccctggagg 2880ctctgggggt gggggctctg gcgggggtgg ggttggtgtt tctactgggt cttatgataa 2940tcagacgcat tatagattct tgggtgacgg ctgggtagaa attactgcac tagcaactag 3000actagtacat ttaaacatgc ctaaatcaga aaactattgc agaataagag ttcacaacac 3060aacagacact tcagtcaaag gcaacatggc aaaagatgat gctcatgagc aaatttggac 3120gccatggagc ttagtggatg ctaatgcttg gggagtttgg ctccagccaa gtgactggca 3180atacatttgc aacaccatga gccagcttaa cttggtctca cttgatcaag aaatatttaa 3240tgtagtgctg aaaactgtta cagagcaaga ctcaggaggt caagctataa aaatatacaa 3300caatgacctc acagcttgca tgatggttgc agtagactca aacaacattc tgccatacac 3360acctgcagca aactcaatgg aaacacttgg tttctaccct tggaaaccaa ctatagcatc 3420gccatacagg tactatttct gcgttgacag agatctttca gtaacctatg aaaatcaaga 3480aggcacaatt gagcataatg taatgggaac accaaaagga atgaattctc aattttttac 3540cattgagaac acacaacaaa tcacattgct cagaactggt gatgagtttg ctactggaac 3600ctactacttt gacacaaacc cagttaaact tacacacaca tggcaaacta accgtcaact 3660tggacagcct ccactgctgt caacctttcc tgaagctgac actgatgcag gtacacttac 3720tgctcaaggg agcagacatg gagcaacaca gatggaggtt aactgggtga gtgaagcaat 3780tagaaccaga cctgctcaag taggattttg tcagccacac aatgactttg aagccagcag 3840agctggacca tttgctgctc caaaagttcc agcagatgtt actcaaggag tggacagaga 3900agccaatggc agtgttagat acagttatgg caaacagcat ggtgaaaatt gggctgcaca 3960cggaccagca ccagagcgct acacatggga tgaaacaaac tttggttcag gaagagacac 4020cagagatggt tttattcaat cagcacctct agttgttcca ccaccactaa atgggattct 4080tacaaatgca aaccctattg gaactaaaaa tgacattcat ttttcaaatg tttttaacag 4140ctatggtcca ctaactgcat tttcacaccc aagtcctgta taccctcaag gacaaatatg 4200ggacaaagaa ctagatcttg aacacaaacc tagacttcac ataactgctc catttgtctg 4260taaaaacaat gcacctggac aaatgttggt tagattagga ccaaatctaa ctgaccagta 4320tgatccaaac ggagccacac tttctagaat tgtgacttat ggtacatttt tctggaaagg 4380aaaactaacc atgagagcaa aacttagagc taacaccact tggaacccag tgtaccaagt 4440
    aagtgttgaa gacaatggca actcatacat gagtgttact aaatggctac caactgctac 4500tggaaacatg caatctgtac cgcttataac aagacctgtt gctagaaata cttactaact 4560aaccatgttt ttcctttctg tacttcatat attattaaga ctaataaaga tacaacataa 4620aaatataata ttacatatag atttaagaaa tagaataata tggtacttag taactgttag 4680aaataataga acctttgaaa taacaagata attagttggt taatgttaga tagaataaga 4740agattatgta taatgggtaa aagggtggaa gggtggttgg ttggtattcc cttagacatg 4800atgttaagga ccaaaaaaat aataaaattt tttaaaacta aaccaagact actgtctatt 4860cagttgaacc aactgaacca tcagtatcac tatgttttta gggtgggggg gtgggagata 4920catgtgttcg ctatgagcga actggtactg gttggttgct ctgctcaacc aaccagaccg 4980gctttgccgg tctggttggt tgagcgcaac caaccagtac cagttcgctc atagcgaaca 5040catgtatctc ccaccccccc accctaaaaa catagtgata ctgat 5085
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    同族专利:
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    ES2561906B1|2016-12-15|
    WO2016016506A1|2016-02-04|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP2404609A1|2010-07-07|2012-01-11|Deutsches Krebsforschungszentrum|Use of parvovirus for eliminating cancer stem cells |EP3669893A1|2018-12-20|2020-06-24|Universidad Autónoma de Madrid|Treatment of p53 mutated/modified cancer-forms with parvovirus|
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    PCT/ES2015/070597| WO2016016506A1|2014-07-30|2015-07-30|Treatment of glioma or glioblastoma with the mvm parvovirus|
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