WO2019104449A1 - Composition for intratumoral injection comprising a dna vector encapsulated in chitosan nanoparticles and use thereof in cancer treatment - Google Patents

Abstract

The present invention is related to the pharmaceutical field, and particularly to a pharmaceutical composition for intratumoral injection comprising nanoparticles of natural origin (chitosan), which are used as vectors for encapsulation of genes for proteins that are able to control tumour growth, generate an immune response, or both. In particular, the pharmaceutical composition for intratumoral injection comprises chitosan nanoparticles encapsulating DNA from the fusion protein from avian reovirus (REO), which slows tumour growth and finally leads to elimination thereof by generation of syncytia, and the use thereof in cancer treatment by in vivo transfection.

Description

 i

COMPOSITION FOR INTRATUMORAL INJECTION COMPRISING

VECTOR OF

ENCAPSULATED DNA IN QUITOSAN NANOPARTICLES AND ITS USE IN THE

TREATMENT OF CANCER

FIELD OF THE INVENTION

 The present invention relates to the pharmaceutical area, and especially, to a pharmaceutical composition for intratumoral injection comprising nanoparticles of natural origin (chitosan) that are used as vectors for the encapsulation of protein genes that are capable of controlling tumor development , create immune response or both. In particular, the pharmaceutical composition for intratumoral injection comprises nanoparticles of chitosan that encapsulates DNA from the fusion protein of Avian Reovirus (REO), which delays tumor growth and eventually leads to its elimination via generation of syncytia, and its use in the treatment of cancer by transfection in vivo.

BACKGROUND

Cancer is the second leading cause of death worldwide according to the World Health Organization (WHO, World Health Organization - ENT Country Profiles, 2014. http: //VAyw.who.int/nhh/studies/en/ #C). This disease is caused by a dysfunction in the cell cycle, which leads to the uncontrolled growth of transformed cells, leading to the formation of a cell mass or tumor. Once the primary lesion is established, the tumor cells eventually acquire the ability to invade and colonize the surrounding tissue, leading to even metastasis, which corresponds to secondary tumors at distant points in the body. This dissemination, in addition to other characteristics, has allowed the classification of cancer, in different stages. The TNM system is the most widely used cancer staging system, comprising 5 main stages: stage 0 contains localized abnormal cells that have not spread to nearby tissue. In stage I and II the cancer affects nearby tissues, in stage III the cancer has invaded organs close to the primary tumor affecting the lymph nodes. Finally, in stage IV cancer has spread to distant parts of the primary tumor invading multiple organs of the body (metastasis) (National Cancer Institute / What Is Cancer? Http://www.cancer.gov/about-cancer/what -is-cancer, American Cancer Soclety, Colorectal Cancer Facts & Figures 2014-2016, Atlanta, Ga: American Cancer Soclety, 2014. http://www.cancer.org/espanol/cancer/colonyrecto/guladetallada/spanlsh-colon- detalled-toc-df).

Despite the significant progress made in cancer therapies, mortality rates for most malignancies remain alarmingly high. Therefore, the inhibition of the growth and progression of cancer, turns out to be one of the great challenges facing modern medicine. As an example, a study conducted by the National Cancer Institute (http://www.lncancer.cl/), Indicates that 21% of the Chilean population dies from this disease, the conventional therapies used have the disadvantages that have a narrow therapeutic index (chemotherapy), high secondary effects (radiotherapy and chemotherapy) and that are often unable to completely eliminate the disease because of the microchromatástasls, and above all, have a high cost. At the therapeutic level, the treatment against this disease has generated a protocol that incorporates mechanical, radiological, chemical means and recently the search of cellular and molecular targets has been added (A. Urrutlcoechea, R. Alemany, J. Balart, A. Vlllanueva, F Vlnals and G. Capella, Recent advances in cancer therapy: an overview. Current Pharmaceutical Design. 2010, Vol. 16, No.1, p. 3-10. DOI: 10.2174 / 138161210789941847). Conventional anti-cancer therapies are surgery in the first instance, which is resolutive in primary stages, and is associated with radiotherapy when near metastasis is suspected. On the other hand, when metastasis is suspected or is characterized in sentinel lymph nodes or by the physiopathological characteristics of the tumor, a systemic treatment called chemotherapy is applied. The latter has a series of disadvantages as the narrow therapeutic index in advanced stages, which is further reduced as the treatment progresses when the tumor acquires resistance to the drugs (Jia-Hui Xu, Shl-Llan Hu, Guo-Dong Shen, Tumor suppressor genes and their underlylng interactions in paclitaxel resistance in cancer therapy, Cancer Cell Int. 2016, Vol.20, No. 16:13. Doi: 10.1 186 / s12935-016-0290-9).

A recurring problem of cancer therapy is the incomplete eradication of the tumor mass or the lack of effect when it has already occurred mlcrometástasis, which will eventually lead to recurrence of the disease. That is why, in order to develop a powerful and long-lasting tumor therapy, it is desirable to eradicate the primary tumor, together with the induction of a mechanism that allows the eradication of non-detectable metastatic foci or micrometastases. Although chemotherapy has tried to be the mechanism to achieve this process through its antiproliferative or cytotoxic effects, it has been extensively characterized that tumor cells acquire the ability to resist the effect of drugs through tumor editing, leading to the selection of cells increasingly resistant to treatments (Hanahan D., Weinberg RA. (2011). Hallmarks of Cancer: The Next Generation, Cell., 2011, 144, No. 5, pp. 646-674, DOI 10.1016 / j.cell. 2011.02.013). Another problem with this therapy is that it not only destroys rapidly growing cancer cells, but also destroys and / or slows the growth of healthy cells that grow and divide rapidly. In recent decades, it has been shown that tumor cells are capable of eluding immunological surveillance through different strategies, either modulating antigenic processing and presentation mechanisms, or negatively regulating the proliferation, activation and signaling of specific T lymphocytes ( Hanahan D., Welnberg RA (2011) Hallmarks of Cancer: The Next Generatlon, Cell., 2011, 144, No. 5, pp. 646-674, DOI 10.1016 / J. cell.2011 .02.013). Taking this into account, the search is now under way for strategies that allow the specific recognition of these cancer foci not only by drugs but also by the body's own cells. Based on the foregoing, the search for therapies has focused on stimulating the Individual's immune response to allow the body itself to respond to the disease, which is currently known as immunotherapy. It seeks to stimulate the Individual's own immune system to allow an effector response against highly specific and specific tumor cells (Farkona, Sofía y cois, Cancer, mmunotherapy: the begining of the end of cancer, BMC Medicine, 2016, Vol .14, No. 73. DOI: 10.1186 / s12916-016-0623-5). Taking this into account, it is that the search for strategies that allow specifically recognizing these cancer foci is being carried out not only by drugs but also by the body's own cells. Therefore, the use of the Immune system itself has been proposed as an alternative, with the concept of immunoblot, which refers to the processes by which the cells of the Immune system seek and recognize foreign pathogens, such as bacteria and viruses, or pre-cancerous and cancerous cells in the body (Dunn Gavln, Bruce Alien, Ikeda Hlroakl, Old Lloyd and Schrelber Robert.) Cancer mmunoedltlng: from immuno-survelllance to tumor scape, Nature mmunology, 2002, Vol. 3, No. 11. pp. 991 -998 DOI: 10.1038 / n1102-991). Other antecedents derived from the effect of chemo-therapeutic agents, have determined that the regression of the tumor growth induced by some of these, is due to the specific activation of the immune response against the tumor (Haynes N., Van der Most, Lake, RA and Smyth, MJ Immunogencc anti-cancer chemotherapy as an emergent concept, Current Opinion, Immunology, 2008, Vol.20, No. 5, pp. 545-557 DOI: 10.1016 / j.coi.2008.05.008). The process of induction of the immunological response as an antitumor strategy takes advantage of the characteristics of this response, such as immunological memory and the ability to migrate and homing to find the antigens, both in order to prevent the spread and the recurrence of the tumor cells or finding them in places where they have settled. Some strategies based on the activation or induction of the immunological response against the tumor propose vaccination as a treatment against cancer. For this, tumor antigens are required and a series of antigen carriers have been used, including used tumors, purified tumor antigens, whole tumor cells or dead tumor cells. All these can be used in combination with adjuvants, clots, in addition to the same antigens (Yaddanapudl K, Mitchell RA, Eaton JW Vaccine Cancer: Looking to the Future, Oncolmmunology, 2013. Vol. 2, No. 3. DOI: 10.4161 /onci.23403).

 Another methodology that has begun to be thoroughly studied to promote its clinical use is Viroterapla, which is based on the use of modified non-pathogenic viral strains that are capable of infecting tumor cells and replicating selectively causing direct death of these It can lead to the activation of an antitumor immune response (Pol, Jonathan et al .. Trial Watch- Oncolytlc viruses and cancer therapy, Oncolmmunology, 2016, Vol. 5, No. 2. DOI: 10.1080 / 2162402X.2015.1117740; Workenhe ST and cois.The role of oncolytlc virus

Immunotherapies to subvert cancer immune evasion. Future Oncology. 2015, Vol. 11, No. 4, pgs. 675-689, DOI: 10.2217 / fon.14.254). The selectivity towards the tumor has been achieved through the use of attenuated viruses that preferentially replicate in tumor cells, this through the inactivation of viral genes that are necessary for replication in normal cells, but dispensable in tumor cells, or by the use of modified viruses by means of genetic engineering in which the genes essential for repliccation have been disposed in tumor-specific promoters (Barzón L. et al., Cllnlcal triais of gene therapy, vlrotherapy, and immunotherapy for major gonomas, Cancer Gene Therapy, 2006, Vol. 13, pp. 539-554. DOI: 10.1038 / sj .cgt.7700930).

In general, this therapy is divided into two types depending on the characteristics of the viruses used, being fusogenic or lytic viruses. Oncolytic virus therapies use their ability to replicate and use tumor cells by specifically taking advantage of the molecular alterations of tumors that do not prevent the replication of the virus (Russell S. et al., Oncolytlc vlrotherapy, Nature blotechnology, 2012, Vol. 30, No 7, pp. 658-670, DOI: 10.1038 / nbt.2287). On the other hand, there is the use of fusogenic viruses, which are capable of killing their target cells by induction of fusion between the infected and uninfected cells (Klasse, PJ et al., Mechanlsms of enveloped virus entry in animal cells. Advanced Drug Dellvery Revolves, 1998, Vol. 34, No. 1, pp. 65-91, DOLI O.1016 / S0169-409X (98) 00002-7). The characteristics of these can be specific to certain virus species or a result of directed genetic engineering. Although viral therapy has been widely used, it has disadvantages inherent to the use of active viruses, such as immunogenicity, toxicity, viruses can spread in the treated patient and mutate, recovering their pathogenic potential; the malignant cells can become resistant to the vlroterapla product of its genomic instability; in addition to heterogeneous and relatively Incomplete diffusion of virus in neoplastic lesions. In addition, the antiviral immune response (either innate or adaptive) is often an obstacle to the efficient application of vlroterapla in patients with cancer, mainly because it sequesters or neutralizes viral particles before they reach the tumor (Pol Jonathan, Buqué Aitziber, Aranda Fernando, Bloy Norma, Cremer Isabelle, Eggermont Alexande, Erbs Phlllppe, Fuclkova Jltka, Galón Jéróme, Llmacher Jean-Marc, Prevllle Xavier, Sautés-Frldman Catherlne, Splsek Radek, Zltvogel Laurence, Kroemer Guido & Galluzzl Lorenzo, Trlal Watch- Oncolytlc vlruses and cancer therapy, Oncolmmunology, 2016, Vol. 5, No. 2. DOI: 10.1080 / 2162402X.2015.1117740).

There are several viruses that kill their target cells by induction of fusion between cells (Klasse, PJ and co., Mechanicsms of enveloped virus entry in animal cells, Advanced Drug Dellvery Revolves, 1998, Vol. 34, No. 1, p. 65-91, DOI: 10.1016 / S0169-409X (98) 00002-7). This process is carried out by specific viral proteins, such as the fusion protein (F) of Orthomyxovlrus (W. Welssenhorn, A. Dessen, LJ Calder, SC Harrlson, JJ Skehel and DC Wlley.) Structural basls for membrane fusion by enveloped vlruses, Molecular Membrane Blology, 1999, Vol.16, No. 1, pp. 3-9, http: // crysta i.harvard.edu / iib-

Kempf. Entry and uncoatlng of enveloped vlruses. Blochemlcal Journal. 1994, Vol. 302, No. 2, pgs. 313-20. http://www.biOchemj.Org/content/302/2/313. long16). In orthomlxovlrus, we find the Infectious salmon anemia virus (ISAV), this virus possesses genes encoding membrane fusion (F) glycoproteins, which mediates the virus-cell fusion to allow the virus to enter the target cell (Aspehaug Vldar, Mlkalsen Aase, Snow Mlchael, Biering Eirik and Vlllolng Stéphane. Characterlzatlon of the Influenza Salmon Anemia Virus Fusion Proteln, Journal of Vlrology, 2005, Vol. 79, No. 19, pp. 12544-53 DOI: 10.1128 / JVI .79.19.12544- 12553.2005). This viral fusion protein (ISAV-F) is classified as a type 1 fusion protein, which is characterized by being synthesized as an inactive precursor molecule, which requires a proteolytic cut to acquire its conformation. active fusogenic, which is triggered by a conformational change induced by a decrease in pH (ISAV-F).

Another type of protein with fusogenic activity is the p10 protein of Avian Reovirus (REO) belonging to the Reovirus family. This protein is classified as a small non-structural fusion-associated membrane protein (FAST), classifying this virus as one of the few non-enveloped viruses that induce cell-cell fusion (Clechonska M. and cois.) Reovirus FAST protelns: virus- encoded cellular fusogens, Trends in Microblology 2014, Vol. 22, No. 12, DOI: 10.1016 / j .tim .2014.08.005). These proteins are capable of generating the virus-cell fusion process (necessary for the entry of a virus enveloped into the target cell) or cell-cell fusion, which leads to the formation of cellular agglomerates that are biologically, structurally and biochemically highly unstable, known as syncytia. The cell death mediated by syncytia occurs through non-apoptotic mechanisms, through a process of recruitment, fusion and disintegration. The increase in the size of a syncytium, the constant signals associated with the cell cycle and / or the severe depletion of the cellular metabolism that occurs as a result of trying to maintain the large cellular structure, would lead to the self-digestion of the syncytia, causing death (Bateman Andrew R., Harrlngton Kevln J., Kottke Tlm, Ahmed Atique, Melcher Alan A., Gough Mlchael J., Llnardakls Emmanouela, David Riddle, Dletz Alian, Lohse Chrlstine M., Strome

Scott Peterson Tlm, Simarl Robert and Vlle, Richard G. Viral fusogenlc membrane glycoproteins klll solid tumor cells by nonapoptotic mechanlsms that promote cross presentation of tumor antigens by dendritic cells. Cancer Research. 2002, Vol. 62, No. 22, p. 6566-78. hitp: //cancerres.aacriournals.orq/content/62/22/6566.! onq).

In addition to the above, it is proposed that the expression of viral fusogencal membrane glycoproteins (FMGs) would be highly immunogenic per se (independent of any effect of induction of syncytia), promoting the generation of immunity specific tumor (Bateman Andrew, Bullough Francis, Murphy Stephen, Emlllusen Lisa, Lavlllette Dimitri, Cosset Frangois-Loic, Cattaneo Roberto, Russell Stephen and Vlle Richard .Fusogenlc membrane glycoproteins as a novel class of genes for the local and Immune-mediated control of tumor growth, Cancer Research, 2006, Vol 60, No. 6, pp. 1492-7, http://cancerres.aacrjournals.Org/coriient/60/6/1492, canvas).

The expression of fusion proteins would be highly immunogenic by itself, and its ability to generate syncytia and cell death would generate a strategy to combat tumor development. However, for this to occur, the tumor cells must express these fusion proteins, so they must be transfected in situ or in vitro with the genes that code for these fusion proteins.

The use of genes coding for viral proteins as gene therapy demonstrated that the expression of these proteins alone results in a significant increase in the control of tumor growth (Bateman, Andrew R. et al., Viral fusogenlc membrane glycoproteins klll solid tumor cells by nonapoptotic mechanlsms that promote cross presentation of tumor antigens by dendritic cells, Cancer Research, 2002, Vol 62, No. 22, pp. 6566-78, http://cancerres.aacrlournals.org/content/62/22/6566.long) . On the other hand, work carried out by Hoffman and cois in the year 2008 (Hoffmann D et al., Immune-mediated anti-neoplastic effect of intratumoral RSV envelope glycoprotein expression is related to apoptotic death of tumor cells but not to the size of syncytia. World Journal Gastroenterology, 2008, Vol.14, No. 12, pp. 1842-1850, DOLI O.3748 / wjg.14.1842), perform the transfection of tumor lines with genes that encode proteins from the respiratory syncytial virus using adenovlrus as transfection vector, demonstrated that generates responses associated with cytotoxic T lymphocytes and that it produces an infiltration in the tumor by natural cells "klller" and macrophages (Hoffmann D, Grunwald T, Bayer W, Wlldner O. Immune-medlated antl-neoplastlc effect of ntratumoral RSV envelope glycoproteln expresslon s related to apoptotlc death of tumor cells but not to the slze of syncytla. World Journal Gastroenterology. 2008, Vol.14, No. 12, pages: 1842-1850.

DOI: 10.3748 / wjg.14.1842).

The main problem in applying these treatments lies in the transfection methodologies, in their efficiency and in how they could be harmful to the cells. For example, the use of biobalistics is highly toxic, since it causes the death of cells by mechanical damage (O'Brlen John and Sarah Lumrnls, Nanobiolistics: a method of biolistic transfectlon of cells and tlssues uslng a gene gun wlth novel nanometer -slzed projectlles, BMC Blotechnology, 2011, Vol. 1 1, No. 66. DOI: 10.1186 / 1472-6750-11 -66). On the other hand, electroporation, which consists of small electrical pulses to induce the formation of pores, is an effective method, but unfortunately generates cell destruction due to the heat produced by the application of high voltage. Other types of transfection use viruses as vectors for the introduction of a gene of interest, but are limited by the pathogenicity of the virus and its high genetic instability (Peer, D., Karp, JM, Hong, S., Farokhzad, OC, Margallt , R., Langer, R. Nanocarriers as an emerging platform for cancer therapy, Nature Nanotechnology, 2007, Vol.2, 751-760, DOI: 10.1038 / nnano.2007.387).

Among the highly used methods for the transfection of cells in vitro, is the use of cathodic cells such as llpofectamna, which are associated with DNA molecules forming complexes that are able to cross the cell membrane. Although these methods have high levels of transfection efficiency, these vectors have a high cost and high toxicity, which is not applicable for a tumor transfection in vivo. That is why there is a need to find alternative vectors for the incorporation of exogenous genetic material by tumor cells. Considering the above, nanotechnology is used as a transfection method. Nanotechnology is tradclclonally defined as submicron-sized molecular devices or nanoparticles that are predominantly found between 5 to 500 nm in at least one dimension. Research has led to methods for incorporating therapeutics into blocompatible nanoparticles that include polymeric nanoparticles, liposomes, mlcelar systems, inorganic nanoparticles, nanotubes, and dendrimers (Khodabandehloo H., Zahednasab H, Ashrafi hafez A. Nanocarriers Usage for Drug Delivery Cancer Therapy, Iranlan Journal of Cancer Prevention, 2016, Vol.9, No. 2, pp. 65-91, DOI: 10.17795 / ijcp-3966).

Polymeric nanoparticles are capable of protecting nucleic acids from endonucleases, in addition to condensing the negative phosphate of nucleic acids with cationic polymers in polypeptides. After escaping from the vascular space in the tumors, the particle must traverse the interstitial space towards the target cell. Therefore, the effectiveness of nanoparticles depends on a variety of physicochemical bio-characteristics of the particle and the cell surface, so that the uptake of the vector in the cells depends both on the opportunity for interaction and on the dynamics of the particles (Khodabandehloo H., Zahednasab H, Ashrafi hafez A. Nanocarriers Usage for Drug Delivery in Cancer Therapy, Iranlan Journal of Cancer Prevention, 2016, Vol. 9, No. 2, pp. 65-91. DOI: 10.17795 / ijcp- 3966).

Within the polymeric nanoparticles it is possible to mention a linear polysaccharide called chitosan (CH) consisting of residues of D-glucosamine (2-amino-2-deoxl-glucose) and N-acetyl-glucosamine (2-N-acetyl-2) deoxy glucose) linked by a b bond (1-4). Chitosan is the most important derivative of chitin obtained from crustaceans, shrimps and crabs, it can be solubilized in acid medium (pH <6.5) due to the protonation of its NH- _{2 group} . This polymer is inexpensive, has a strong affinity for DNA by charge interaction, and has a low toxicity (Sun, Y. and cois, Preparation, characterization and transfection efficacy of chitosan nanoparticles containing the intestinal trefoil gene factor Molecular Biology Report, 2012, Vol. 39, No. 2, pp. 945-52 DOI: 10.1007 / s1 1033-011 -0820-4; and Carrillo, Carolina et al., Chitosan nanoparticles as non-viral gene delivery Systems: Determinenation of loading efficiency, Blomedicine & Pharmacotherapy, 2014, Vol. 68, No. 6, pp. 775-783, DOI: 10.1016 / j.biopha.2014.07.009). In addition, the positive charge of the CH allows it to interact with the negatively charged proteoglycans of the plasma membrane, inducing the normalization of chitosan nanoparticles by endocytosis.

In the prior art it is possible to mention the scientific publication entitled "Preparation, characterization and transfection efficacy of chitosan nanoparticles containing the intestinal trefoil factor gene", Yong Sun et al Mol Biol Rep (2012) 39: 945-952 DOI 10.1007 / s1 1033- 011 -0820-4 which refers to a polypeptide with potential pharmacological value for the prevention and healing of damaged tissues but which has low production capacity, which limits its application. Chitosan is chosen as a non-viral vehicle for the release of genes due to its intrinsic characteristics. Chitosan nanoparticles are prepared to wrap ITF cDNA and their size, zeta potential, stability, release profile and efficiency and loading capacity are studied. The ability to transfer genes in HEK293 cells was evaluated. The data revealed that the chitosan / DNA nanoparticles were successfully prepared with sizes less than 500 nm and positive zeta potentials. The nanoparticles would protect the DNA from nuclease degradation and have DNA release profiles dependent on the N / P ratios. In addition, the transfection efficiency of chitosan / DNA nanoparticles was equivalent to Llpofectamlna (TM). It proposes the chitosan / DNA nanoparticles for ITF gene therapy. "Enhanced intracellular uptake and endocytic pathway selection mediated by hemocompatible ornithine grafted chitosan polycationfor gene delivery", Susan. M. Alex et al Collolds and Surfaces B: Blolnterfaces 122 (2014) 792-800, refers to the creation of gene vectors that would facilitate the transfer of genes to cells, with greater efficacy and safety. For the design of the vector, the preferred polymers are non-viral colloidal systems as they are susceptible to chemical modification. Chitosan is chosen for chemical conjugation with the amino acid ornithine to generate the chitosan-ornithine (CON) conjugate to release the gene. FTIR and ¹ H NMR spectrum were used to confirm the chemical composition of the chitosan derivative. An improved cushioning capacity was found in the synthetic chitosan derivatives when compared to the original unmodified chitosan. The cationic derivative formed nanoparticles when mixed with negatively charged DNA. The derivative in interaction with blood plasma showed a negligible adsorption of protein and did not cause hemolysis or RBC aggregation in the blood. In an in vitro culture it was confirmed that the CON derivative was not toxic to the cells and was capable of transfecting with an explicit increase in the cellular uptake of nanoparticles. In addition, it was shown that clatrin mediates the route and plays a dynamic role in the internalization of nanoparticles. "Improved stability and efficacy of chitosan / pDNA complexes for gene delivery", Noemi Cifani and cois Biotechnol Lett (2015) 37: 557-565 DOI 10.1007 / s10529-014-1727-7 describes polymeric polycations being chitosan the most powerful vehicle for release of genes. Study the stability of the chitosan / DNA complex under storage. Polyelectrolyte complexes are synthesized at a loading ratio of 10 using 50 kDa of chitosan and plasmid (p) DNA encoding a reporter GFP.

The preparations were stable up to 3 months at 4 ^S C and showed reproducible transfection efficiencies in vitro in HEK293 cells. In addition, a methodology was developed that increased the efficiency of in vitro transfection of complexes of Chitosan / 150% pDNA. Notably, the intracellular pDNA release and the transfected cells reached maximum 5 days after the transfixion of active cells mltotically, thus improving the potential of gene therapy mediated by polymeric nanoparticles.

"Chltosan nanoparticles as non-viral gene dellvery Systems: Determlnatlon of loadlng efflclency" Carolina Carrillo and cois available at SclenceDIrect www.sciencedirect.com Blomedlclne & Pharmacotherapy (2014) discloses that qultosan is used in particle release systems for therapeutic purposes, being One of its most important applications in non-viral vectors for gene therapy. Due to its positive charge, it is capable of forming DNA complexes (polyplexes) that protect nucleic acids. Two methods are used to obtain the nanoparticles of nucleic acid-qultosan: Simple complexation (of depollution of qultosan or of different salts of qultosan with plasmid) and ionic gelaclon (by adsorption of plasmid in the nanoparticles or by encapsulation of the plasmid in the nanoparticles) . The determination of the loading efficiency of nanoparticles of qultosan with the plasmid was conducted by electrophoretic mobility of the samples in agarose gel. The nanoparticles were characterized according to morphology, size and surface charge. The polyplexes were found to be spherical and of manometric size (between 100-230 nm) with a zeta potential between 37 and 48 mV. Positive results were obtained in the agarose gel electrophoresis in all the studied cases: a concentration between 20 and 30 mg / mL of qultosan salts was required while for the remaining samples of qultosan studied, 100% efficiency did not occur. Load up to a concentration equal to 100 mg / mL (regardless of previous depollution and the method performed).

"Chltosan nanoparticles as a potential nonvlral gene dellvery for HPV-16 E7 mammalian cells", Allreza Tahamtan et al, Artificial Cells, Nanomedlclne, and Biotechnology, 2014; DOI: 10.3109 / 21691401.2014.893522, prepares chitosan nanoparticles as a vehicle for the E7 gene of human papillomavirus type 16 (HPV-16) and its gene transfection capacity was evaluated in vitro. The expression of the green fluorescent protein (pEGFP) was used as the reporter gene. The gel electrophoresis showed complete binding to the CS NPs with pDNA. The transfection of the CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was observed. The expression of the E7 proteins was confirmed under SDS-PAGE and western blot analysis. In conclusion, CS NPs can serve as an effective non-viral vehicle for the release of eukaryotic cells.

In summary, cancer is one of the diseases with the highest incidence and mortality rates worldwide. In recent times, a type of therapy has emerged that takes advantage of the pathogenic activity of viruses in eukaryotic cells, using lytic and fusogenic viruses, called vlroterapla. The fusogenic viruses are able to carry out cell-cell fusion by fused membrane glycoproteins (FMGs), producing highly unstable tumors of tumor cells, which leads to their cell death. This only occurs in cells that express FMG and, therefore, the incorporation of the gene has been used as anti-tumor therapy. Currently the transfection is carried out using catholic lines which, although efficient, are highly toxic, and their use in vivo is not feasible.

The present invention relates to a pharmaceutical composition for intratumoral injection comprising nanoparticles of a blocompatible, bloodegradable, non-immunogenic and non-toxic polymer, of natural origin, which can be used as vectors for the encapsulation of genes whose proteins are capable of control tumor development, create an immune response against the tumor or both, by direct injection into the tumor. In particular, the present invention is relates to an intratumoral injection composition comprising nanoparticles for encapsulating the gene (DNA, SEQ ID No.:1) of the Avian Reovirus fusion protein (REO, SEQ ID No.:2), inserted into an expression vector ( plRES2), which allows the expression of the gene, in mammalian cells.

Chitosan protects DNA from degradation and facilitates the entry of this into tumor cells, thus allowing them to express the fusion protein, which produces the formation of large multinucleated cells, by the fusion of tumor cells between yes. These multinucleated cells are highly unstable, and have a short life (120 h), which makes them more sensitive to any treatment.

Brief Description of the Invention

The present invention consists of a pharmaceutical composition for Intratumoral Injection useful in the treatment of cancer, which is of direct administration. In particular, the present pharmaceutical composition for Intratumoral Injection allows the treatment of detected tumors that are in the early stages of development. The present composition comprises nanoparticles of a cathlonic polymer selected from chitosan, which is blocompatible, bloodegradable, non-immunogenic and non-toxic. This polymer encapsulates the gene (DNA) of proteins that are able to control tumor development, create an immune response against this or both, by direct injection of the tumor. In particular, this polymer encapsulates the gene (SEQ ID No.:1) of the Avian Reovirus fusion protein (REO, SEQ ID No.:2), inserted into an expression vector (plRES2), which allows expression of the gene in mammalian cells. Chitosan protects DNA from degradation and facilitates the entry of DNA, so that after Intratumoral Injection, it is captured by the cells tumors, thus allowing them to express the mentioned fusion protein, which will produce the formation of large multinucleated cells, by the fusion of the tumor cells with each other. These multinucleated cells are highly unstable, and have a short life (120 h), which produces two effects: 1) they are more sensitive to any classical treatment that seeks to eliminate the tumor and 2) being highly unstable, they are quickly eliminated by associated with necrosis and autophagy. This produces an immunogenic death, generating warning signals and the release of tumor antigens, which triggers the activation of the immune system, triggering a specific anti-tumor response for that tumor. In this way the immune response of the organism itself is strengthened so that it attacks the tumor in a specific and fast way, by making the tumor cells "more visible" for the organism. Pre-clinical trials were conducted in immunocompetent animals, using the strain of C57 mice to which Murine B16 Melanoma Indujo was induced, and with animals of the Balb / ca strain, which induced CT26 murine colon carcinoma. Once the tumor volume reached a volume of 2 mm ³ , the mouse was treated with an nanoparticle injection composition QuItosano / pDNA-REO, in an intratumoral manner and the animals were monitored daily, until they had completed 60 days off of tumor or otherwise a tumor volume of 260 mm ³ . Where it was observed that the treatment with nanoparticles was able to cause a delay in the tumor growth and / or a regression of the tumor.

Thus, the present invention proposes a pharmaceutical composition comprising nanoparticles (NPs) of qultosan (CH) for transfection in vivo, achieving high efficiency and low toxicity. The present invention proposes in particular, to prepare NPs of CH-pDNA and once characterized, subsequently, generate NPs containing separately, the gene of the FMGs of two different viruses, REO and ISAV, which were transfected in the CT26 colon carcinoma tumor model. The effect of the expression of FMG genes on tumor development was evaluated and it is demonstrated that CH-FMG NPs allow an efficient incorporation and expression of pDNA by tumor cells, both in vitro and in vivo, showing partial effects on the control of tumor development, and therefore, an alternative for treatment or therapy against cancer.

Brief Description of the Figures

 Figures 1 A-1 C. Characterization of the chitosan / pDNA complex. Figure 1A Load efficiency of qultosan at different N / P ratios (mean ± standard deviation (S.D) n = 3). Figures 1 B and 1C Analysis of migration delay in agarose gel for chitosan / pDNA nanoparticles at N / P = 4 ratios; N / P = 20; N / P = 28; N / P = 40. The + sign means that there is pDNA encapsulation. Figures 2A-2D. Distribution of the zeta potential. Chitosan / pDNA nanoparticles prepared at different N / P ratios. Figure 2A, ratio N / P = 4. Figure 2B, ratio N / P = 20. Figure 2C, ratio N / P = 28 and Figure 2D, ratio N / P = 40. Measurements with the Zetazalser Nano equipment (Malvern Inst Ltd. Malvern). Figures 3A-3D. Size. Nanoparticles formed of chitosan / pDNA prepared at different N / P ratios. Figure 3A, ratio N / P = 4, Figure 3B, ratio N / P = 20, Figure 3C, ratio N / P = 28 and Figure 3D, ratio N / P = 40. Expressed in the percentage of number of nanoparticles. The samples were measured with the Zetazalser Nano equipment (Malvern Inst. Ltd. Malvern).

Figures 4A and 4B. Atomic force microscopy. Qultosan complex / pDNA-GFP at a ratio N / P = 20, Figure 4A and N / P = 28, Figure 4B, using 2.5 pg of pDNA, measured in the Nanoscope Illa Atomlc Forcé Mlcroscope. Figures 5A-5C. Chitosan transfection efficiency. Figure 5A Representative histograms of cells expressing GFP, in black the control is observed (cells without transfecting) and in red the corresponding treatment. Figure 5B Percentage of CT26 cells transfected with pcDNA-GFP using chitosan with different N / P ratios. Three repetitions of each condition were performed, where ^* indicates the significant differences (p <0.05) with respect to the control.

Figure 6. Analysis of gel migration delay. NPs formed by chitosan with the pIRES plasmids containing the genes (SEQ ID No.: 1) for the fusion proteins (SEQ ID No.:2) with an N / P ratio = 28, using as controls the plasmids without chitosan. The + sign means that there is DNA encapsulation.

Figures 7A and 7B. Determination of gene expression of viral fusion proteins in CT26 cells. Determination by means of RT-PCR. Figure 7A REO amplex of 201 bp and Figure 7B ISAV amplex of 304 bp. In the first lane, the molecular weight standard O ^' gene ruler 100pb is observed, in the second the control of PCR without cDNA, the other conditions correspond to the cells without transfecting (negative transfection control), cells transfected with llpofectamlna (positive control of transfection) and cells transfected with chitosan.

Figures 8A and 8B. Cell viability CT26 cells transfected with chitosan nanoparticles and genes coding for viral fusion proteins Figure 8A REO (SEQ ID No.2) and Figure 8B ISAV, at 24, 48 and 72 hours post transfection Incubating with the MTT reagent. The viability of the control cells (untreated) was determined arbitrarily as 100%. Figure 9. Tumor appearance using the CT26 tumor line in Balb / c mice.

The data is represented in a Kaplan Meier survival graph. This graph shows a curve for each type of treatment and each drop indicates the tumor appearance of a mouse for the corresponding treatment.

Figures 10A and 10B. Effect of in vivo transfection with nanoparticles on tumor growth. Tumor growth graph for challenged, untreated mice treated with chitosan and chitosan-Reo nanoparticles, Figure 10A, and chitosan-ISAV, Figure 10B.

Figure 11. Animals with necrosis. Graph of tumor growth of mice that presented necrosis in the post-treatment tumor.

Figures 12A and 12B. CD4 + and CD8 + lymphocyte populations present in spleen. Figure 12A Representative histograms of each marking where in black corresponds to autofluorescence of the splenoclines and in red to the evaluated condition. Figures 12B Variation of percentage of CD8 + and CD4 + lymphocytes in spleen. Each bar represents a treatment maintaining the slmbology of previous figures. The data are plotted as mean ± standard error, ^* represents p <0.05, compared with the control.

Figures 13A and 13B. Effect of treatment with nanoparticles on Th1 lymphocytes in spleen. Figure 13A Representative histograms of the CD4 + TBET + populations for each labeling treatment where black corresponds to autofluorescence of splenoclines and red to the evaluated condition. Figure 13B shows the percentage of the sub-population of Th1 lymphocytes present in spleen. The data are plotted as mean ± standard error, ^* represents p <0.05, compared with the control. Figures 14A and 14B. Effect of nanoparticle treatment on Th17 lymphocytes in spleen. Figure 14A Representative histograms of CD4 + RORY + populations for each labeling treatment where black corresponds to autofluorescence of splenocytes and red to the evaluated condition. Figure 14B shows the percentage of the sub-population of Th17 lymphocytes present in spleen. The data are plotted as mean ± standard error, ^* represents p <0.05, compared against the control.

Figures 15A and 15B. Effect of treatment with nanoparticles on Treg lymphocytes in spleen. Figure 15A Representative histograms of CD4 + CD25 + FOXP3 populations. Figure 15B shows the percentage of the sub population of Treg lymphocytes present in spleen. The data are plotted as mean ± standard error, ^* represents p <0.05, compared to the control.

Figures 16A and 16B. CD4 + and CD8 + lymphocyte populations present in Tumor. Figure 16A Representative histograms of each marking where the black color corresponds to autofluorescence and in red to the evaluated condition. Figure 16B Variation of percentage of CD8 + and CD4 + lymphocytes in tumor, each bar represents a treatment maintaining the symbology of previous figures. The data are plotted as mean ± standard error, ^* represents ap <0.05, compared with the control.

Detailed description of the invention

The present invention relates to a pharmaceutical composition for intratumoral injection comprising nanoparticles of a biocompatible, biodegradable, non-immunogenic and non-toxic polymer to encapsulate the gene of proteins that are capable of controlling tumor development, create immune response against this or both . In particular, the present invention relates to a pharmaceutical composition for intratumoral injection comprising chitosan nanoparticles that encapsulate a gene (SEQ ID No.:1) of the fusion protein of Avian Reovlrus. (REO, SEQ ID No.:2), inserted into an expression vector (plRES2), which allows the expression of the gene, in mammalian cells.

The qultosano protects the DNA of the degradation and facilitates the entrance of this one in the tumor cells, allowing in this way that they express the protein of fusion, which produces the formation of multnucleated cells of great size, by the fusion of the tumor cells between yes. These multinucleated cells are highly unstable, and have a short life (120 h), which makes them more sensitive to any treatment.

Thus, the present invention relates to a pharmaceutical composition for intratumoral injection comprising viral fusion proteins expressed on the surface of mammalian cells, which are capable of inducing cell fusion. By using the OVA antigenic model, the present invention shows that the hRSV fusion protein in Hek cells (ccHek-hRSV) increases the cross-presentation of antigens.

The present invention also relates to a pharmaceutical composition for intratumoral injection comprising nanoparticles - which have emerged in recent years as a method of high efficiency and low toxicity, as a treatment against cancer (Peer, D. et al., Nanocarriers as An emerging platform for cancer therapy, Nature Nanotechnology, 2007, Vol.2, 751-760 DOI:

10.1038 / nnano.2007.387). In particular, the present invention relates to an intratumoral pharmaceutical composition comprising nanoparticles of qultosan (CH) / pDNA for the transfection of genes from the fusion proteins of Reo, which was tested in vitro in the tumor cell line CT26. The present invention also relates to a pharmaceutical composition for direct administration on the tumor mass, comprising nanoparticles composed of chitosan and the gene (SEQ ID No .: 1) of the viral fusion proteins (SEQ ID No. 2), and the antitumor and lymphocyte population response, which was tested in a CT26 tumor model.

The present invention relates to a pharmaceutical composition of intratumoral injection that allows the expression of viral fusion proteins by transfection with nanoparticles of chitosan and promotes an anti-tumor immunity, which was demonstrated in a murine colon cancer model CT26.

For the development of the present invention, chitosan NPs were prepared and characterized, and their efficiency for transfection was evaluated in the CT26 cell line. In particular, the effect of the expression of viral fusion proteins in vitro on the CT26 tumor cell line transfected with chitosan NPs was evaluated. Also, the effect of the expression of the viral fusion proteins in vivo, on the tumor development and the lymphocyte population, in the spleen and tumor of mice was evaluated.

1. Materials and Methods

1.1 Plasmidial DNA

For the characterization of the nanoparticles, plasmid pCDNA3.1 -eGFP (Addgene) containing the gene coding for the green fluorescent protein was used, this was amplified in E. coli DH5-a bacteria and purified using the Favor Prep Plasmid DNA extractlon midl Kit, according to the manufacturer's recommendations, collect bacteria and proceed to the used of these, to then purify the plasmid by means of an elution column. The purified plasmid was dissolved in molecular grade deionized water and its concentration was determined by spectroscopy, measuring the absorbance at 260 nm using the Tecan Infinite M200PRO equipment. For the transfection of the cells CT26 plasmid pIRES (Clontech) was used, with 2 different inserts coding for the viral fusion protein REO and the fusion protein ISAV; which were amplified and purified by the aforementioned methodology. 1.2 Generation of nanoparticles

 Low molecular weight chitosan (Sigma) with a degree of deacetylation greater than 75.0% was used. This was dissolved in 1% acetic acid by slightly heating the solution and adjusting the pH to 5.5 with sodium hydroxide. Subsequently, the chitosan solutions were filtered sterile with a syringe filter of 0.2 um.

Then, the chitosan solutions were diluted with deionized water to achieve the desired ratio of quatose amine to DNA / phosphate ("N / P" ratio), then 100 ml of chitosan was mixed with 100 ml of pDNA. The concentration of pDNA was kept constant 0.025 mg / mL. The nanoparticles were prepared by the coacervation technique (Turan Suna, Aral Cenk, Kabasakal Levent, Keyer-Uysal Meral and Akbuga Jülide) Co-encapsulation of two plasmlds n chltosan mlcropheres as a non-viral gene delivery vehicle J Pharm Pharmaceut Sci. 2003, Vol. 6, No. 1, pp. 27-32, https: //s¡tes.ualberta.ca/~csps/JPPS6 (1) /S.Turan/encapsulation.htm) . Briefly, before mixing the solutions were heated separately at 55 ° for 5 minutes and once mixed they were stirred immediately at maximum speed for 30 seconds. This nanoparticle solution was used 30 minutes after its preparation for transfection.

The initial formation of the complexes was evaluated by determining the delay in agarose gels. For this, the samples with the different nanoparticles were loaded on a 1% agarose gel with TAE 1 X buffer, run on the gel at 120 V for 30 minutes, the gel was stained with Gel Red (Blotium) and visualized under light UV After the formation, a sample of the solution was centrifuged at 13000g per 10 mln and the supernatant was collected to determine the amount of non-complexed pDNA using a spectrophotometer. The loading efficiency was calculated according to the following equation (Sun, 2011, Turan Suna et al, Co-encapsulatlon of two plasmlds chltosan mlcropheres as a non-viral gene dellvery vehlcle, J Pharm Pharmaceut Sel. 2003, Vol. 6, .. No. 1, pp 27-32 bttps: // sites jaibería ca / ~ CSPs / JPPS6; 1 j / STuran / encapsulation.htm)..:

 Load efficiency (%) = [(total amount of pDNA) - (non-trapped pDNA / total quantity pDNA) x 100.

 1.3 Zeta Potential v size

 The zeta potential is indicative of the surface charge that the nanoparticles possess, this was determined using the Zetazalser Nano equipment (Malvern Inst. Ltd. Malvern, UK). Qultosan nanoparticles with different N / P ratios (4, 20, 28 and 40) were synthesized and the zeta potential and the size of the particles obtained were measured. For a more accurate determination of the size and morphology of the NPs, the sample underwent atomic force microscopy. The measurement was made in the Nanoscope Illa Atomlc Forcé Mlcroscope team. For this experiment, qultosan nanoparticles were prepared at an N / P ratio of 20 and 28 with 2.5 pg of pcDNA.3.1 EGFP

1.4 In vitro transfection and determination of transfection efficiency

Cultures of Colon carcinoma cells (CT26) were cultured in sterile RPMI (Glbco) medium supplemented with 10% fetal bovine serum (FBS). For the transfection, 24-well plates (Falcon) were used in which 5 x 10 ⁵ cells / pocillus were incubated at 3713 with 5% C0 ₂ - The cells were transfected 24 hours later, at 70% confluence. Before the transfection, the cells were washed with sterile PBS (1X, pH 7.4). Cells with llpofectamlna 2000 and naked pDNA were transfected as control.

The quantification of the transfection efficiency was performed by the identification of GFP-positive cells by means of flow cltometry using the BD AccurP equipment (BD Blosclence, San Jose, California, USA). For this, the cells of the 24-well culture plate were harvested, washed with sterile 1X PBS, added 1X trypsin and collected in eppendorf tubes. Then, 500 pL of culture medium was added and centrifuged at 1200g for 10 mln. Subsequently the supernatant was removed, PBS was added and centrifuged again at 1200g for 10 minutes, the pellet was resuspended in sterile 1X PBS and analyzed by flow cltometry.

1.5 RNA extraction and RT-PCR

In order to determine the mRNA expression of the fusion proteins in the cells transfected with the qultosan nanoparticles, an RT-PCR was performed for the fusion proteins REO and ISAV. For which total RNA was extracted. The cells were homogenized in 2 mL of Trlzol® Reagent (Glbco, Life Technologies, New York, USA), using the manufacturer's recommendations, cell llsls to subsequently perform a phenol-chloroform extraction and precipitate the RNA with ¡-propanol. The total extracted RNA was resuspended in molecular grade deionized water. RNA (1 pg) was treated with RNase-out (Promega, Wlsconsln, USA) and cDNA synthesis was carried out, which was carried out using reverse transcrlptase M-MLV and OllgodT20 (both from Promega, Wlsconsln, USA). Both procedures were performed according to the supplier's protocol, which was treated with RNase 1 ug / 1 ug of RNA to then synthesize the cDNA using an activation temperature of the enzyme at 60 for one hour and then at 72 for 10 minutes. A PCR was carried out from the cDNA obtained, using gene splitters (SEQ ID No .: 1) coding for the REO fusion proteins (SEQ ID No.:2) and ISAV. For REO-F, the forward primers 5 ^' -CAG GGT CAT GTA ACG GAG CTA -3 ^' (SEQ ID No. 3) and reverse 5 ^{'were used} -GCT GCG TCA GCC TTA ATT TTG-3 ^' (SEQ ID No. : 4) with an annealng temperature of 570. For ISAV-F, the 5 ^' forward splitter was used -ATC AGC ATG GCT GGA GCA AGT A -3 ^' ((SEQ ID No .: 5) and reverse 5 ^' -TCT CCA ATC AGC CCG ATT TCC A -3 ^' (SEQ ID No .: 6) with an annealng temperature of 630. Subsequently an agarose gel electrophoresis was performed, where the samples were run at 100 V for 30 minutes. of molecular weight Gene Ruler 100bp # SM0241 (ThermoSclentlflc, Life Technologies, Massachusetts, USA), the gel was stained with Gel Red (Blotlum) and visualized under UV light.

1.6 Cell viability studies

The evaluation of cell viability was carried out by MTT assay (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole bromide). CT26 cells were cultured in 96-well plates at a density of 5x10 ³ cells / well in 100 pL of RPMI culture medium supplemented with 10% FBS, after 24 hours the cells were transfected with NPs that they possessed. the genes for the fusion proteins ISAV and REO, according to said genes are described in the previous sections. Cell viability was determined by the MTT assay at 24, 48 and 72 hours post transfection. To this they were withdrawn culture medium and the cells were added 20 pL of MTT leaving Incubate for 3 hours at 370 to 5% C0 _2. After that, 100 pL of dlmethyl sulfoxyl (DMSO) was added and the absorbance was measured at 570 nm in the TECAN Infinite 200PRO equipment. The cell viability of the control cells (without transfection) was arbitrarily determined as 100%. 1.7 Animals

Mice of strain C57 and Balb / c from 6 to 8 weeks were maintained with feeding ad libitum and under a cycle of light and dark, and a treatment was applied to them with the composition of the invention that comprised the nanoparticles and the volumes were determined of tumor size used (2 mm ³ for the time of treatment and 260 mm ³ for the sacrifice of the animal), as well as monotherapy was performed by measuring the tumor size using a foot meter.

1.8 Tumor induction and antitumor treatment

To evaluate the tumor growth 500 thousand live CT26 cells were resuspended in PBS. The Balb / c mice were injected with the CT26 tumor cells in the lumbar region subcutaneously. Once injected, the time of appearance of the tumor, the tumor size and the characteristics of the state of the mouse, including appearance, weight and behavior, are monitored. A maximum growth parameter of 261 mm ³ , or a maximum period of 60 days without tumor, was determined as the criterion of the end point of the experiment. At the end of this process, the mice were sacrificed by cervical dislocation.

When the tumor reaches a tumor volume of 2 mm ³ , the composition of the invention is administered by direct intratumoral injection. The composition of the invention comprised in particular qultosan nanoparticles in an N / P 28 ratio containing 10 pg of pDNA with the genes (SEQ ID No.:1) for the viral fusion proteins and then the animal was monotreated until which reaches a tumor volume of 261 mm ³ or 60 days free of tumor.

1.9 Extraction of splenocytes and tumor infiltrating lymphocytes

When the tumor reached the volume corresponding to the maximum criterion of end point of 261 m ³ or 60 days free of tumor, it proceeded to sacrifice it by dislocation cervical. Subsequently, the spleen and tumor were extracted. The spleens were disintegrated on a 0.150 mm (100 mesh) metal grid, then the erythrocytes were removed by differential lysls using ACK buffer (155mM NH ₄ CI, 10mM KHC0 ₃ , 1 mM Na ₂ EDTA, pH 7.3) for 5 hours. min in gentle agitation. On the other hand, the tumors were disintegrated and then incubated in 1 x trypsin under gentle agitation for 30 minutes at 3713. Subsequently, the supernatant was removed, centrifuged at 1200 xg for 10 min and the supernatant was discarded.

The splenoclines and cells extracted from the tumor were resuspended in cold blocking buffer (IF; SFBal 2% in PBS) with a density of 1 x 10 ⁶ cells / mL for 30 min at 413. Then, to determine the CD8 + population I infocitaria, used the Antl-CD8 Ly-2 PE antibody (BDpharmingen, BD Biosclences, San Jose, California, USA); for CD4 + populations the FITC Anti-mouse CD4 antibody (eBioscence) was used; for the populations CD4 + CD25 + Foxp3Ant¡-mouse CD25 PE (eBiosclence) and Antl-mouse Foxp3 PCP (eBiosclence), for CD4 + RORY (t ⁺ ) (anti- human / mouse RORY (t ⁺ ) AFKJS-9 (eBioscence )) and for CD4 + populations T-betse used antl-human / mouse T-bet (eBioscence). All antibodies were used in a 1: 10 dilution. Finally, the labeled cells were resuspended in the Cell Staining Buffer solution (eBiosclence, San Diego, California, USA) and analyzed by Flow cltometry in the Accuri C6 equipment (BD, California, USA).

1.10 Statistical analysis

The Mann Whitney test was applied to compare the different treatments and all data are presented with standard error. For the analysis of cell viability assays, an anova two-way analysis was performed. The analyzes were performed using the GraphPad Prism 5.01 software (GraphPad Software, Inc., San Diego, California, USA). For all the analyzes a 95% confidence in the data was considered, to evaluate if the effect corresponded to a statistically significant effect, that is, a value of p <0.05.

2. Results

 2.1 Characterization of nanoparticles

 To determine the encapsulation capacity of pDNA by the nanoparticles under the different conditions evaluated (see section 1.2 above), the mixture generated with qultosan / pDNA was centrifuged and the amount of free pDNA in the supernatant was measured. It was determined that the charging efficiency of the nanoparticles qultosan / pDNA increases with the ratio N / P (amine groups of qultosan / DNA phosphates). As seen in Figure 1A, all the ratios N / P of qultosan / pDNA used have load efficiencies greater than 60%, being in the N / P ratios 28 and 40, greater than 80% (Table 1). Table 1: Size and zeta potential of nanoparticles of different N / P ratios

In order to characterize the protocols used from which the nanoparticles were generated and also to guarantee that these were bound non-labile to the pDNA, the generated nanoparticles were analyzed by agarose gel electrophoresis. In the qultosan / pDNA complex, the encapsulated pDNA showed no migration in the gel for all N / P ratios, as can observe in Figures 1 B and 1 C, contrary to what was observed in the migration of the soluble plasmid. The above is explained by a complete encapsulation, represented as a retention in the well because the pDNA is forming a complex with qultosan and this is not able to migrate through the gel.

Once the encapsulation efficiency of the generated nanoparticles was characterized, the Z-potential measurement was performed. For this, the nanoparticles qultosan / pDNA were evaluated at different N / P ratios, as shown in Figure 2. The hlstograms with respect to the potential, indicate that all the nanoparticles evaluated had a positive Z potential (range between +10 and + 58mV), without a direct relationship between the N / P ratio of the nanoparticles with qultosan and the zeta potential (Flg. 2). The size analysis of the qultosan NPs / pDNA, showed that they also have a size range between 25-56 nm radius (Flg. 3), compatible with a nanoparticle. The complexes formed with an N / P ratio = 4 have a size of around 25 nm in radius, it is observed in Figure 3a that the nanoparticles generated with an N / P ratio of 4 are not homogeneous. The complexes with a ratio N / P = 20 have a size of 39 nm radius, the complexes with a ratio N / P = 28 have a size of 45 nm radius and the complexes with a ratio N / P = 40 a size of 40 nm radius (table 2). In order to confirm the size and determine the morphology of the nanoparticles, an analysis was performed by AFM (Flg. 4), from which an image of the complex formed by quitosan / pDNA-GFP was obtained for the N / P = 20 and N / P = 28, using 2.5 pg of pDNA. Where it was obtained that the diameters of these complexes are approximately 70-88 nm. In Figure 4B, the nanoparticles with an N / P = 28 ratio show the presence of qultosan in a linear manner, which was not able to encapsulate the pDNA, probably due to excess qultosan. Table 2: DNA loading efficiencies of NPs

2.2 Determination of transfection efficiency of NPs

 To evaluate whether the nanoparticles are capable of allowing DNA transfection, it was evaluated using the cDNA coding for GFP cloned in the pcDNA plasmid, as described in section 1 .4 above. The efficiency of transfection of the GFP gene by chitosan nanoparticles was evaluated in the CT26 colon carcinoma cell line. After 48 hours post-transfection, the expression of GFP was evaluated by flow cltometry, in all cases it was expressed as the percentage of transfected cells of the total cells. Untransfected cells, a commercial transfection agent Llpofectamlna 2000 and the naked plasmid were used as controls. For qultosano / pDNA different N / P ratios were evaluated (4, 20, 28 and 40), where it was obtained that all the ratios N / P of qultosano / pDNA are capable of transfecting, being the ratio N / P = 28 the one that it has a higher efficiency of transfection (9%) than llpofectamlna (3%) (Figure 5).

2.3 Electrophoretic migration assay of chitosan-qen complexes or NPs from fusion proteins.

Once it was determined that the N / P ratio that is capable of efficiently transfecting the CT26 cell line is N / P = 28, we proceeded to evaluate the ability to encapsulate the pDNA (pIRES) containing the genes (SEQ ID No. : 1) of the viral fusion proteins REO and ISAV. As described in section 1.4 above, the complete encapsulation would be observed as retention in the well. He observed that the complexes formed by qultosan with the plasmids containing the genes (SEQ ID No.:1) for the fusion proteins are able to encapsulate the pDNA, since no migration is observed in the agarose gel (Flg. 6) . 2.4 Characterization of the expression of viral proteins in CT26

 In order to evaluate if the cells transfected with the NPs are expressing the genes (SEQ ID No.:1) for the viral fusion proteins used, an RT-PCR was performed for each cDNA, this due to the absence of the antibodies to be able to measure the direct expression of the protein. The transfected CT26 cells were used as negative transfection control and, as a positive control, the cells transfected with llpofectamna (commercial reagent) and also the PCR control which does not contain the genetic material. Figure 7A shows the PCR performed for the cDNA of the REO protein, where there is a band around 202 bp which corresponds to the size of the expected amplicon for the codon coding for this protein. Figure 7B shows the PCR performed for the cDNA coding for the ISAV protein where a 304 bp amplicon is observed both in the positive control (LF) and in the cDNA coming from cells transfected with qultosan (CH-ISAV).

2.5 Determination of the effect on cell viability after transfection with NPs of chitosan v genes of viral proteins

Cell viability was studied by determining the percentage of viable cells using an MTT reduction assay. In this experiment, the viability value of the untreated cells was considered as 100% viability. The NPs were prepared with an N / P = 28 ratio forming the chitosan / DNA complex with the genes (SEQ ID No.:1) for the ISAV and REO fusion proteins. As shown in Figure 8, a significant decrease is observed only at 72 hours in cells transfected with quitosan / pDNA REO fusion protein (Flg. 8A). Under the other conditions, no significant changes were observed for any of the times measured. For the controls, the naked plasmid and the positive transfection control, llpofectamlna, were used. For the naked plasmid no significant changes were observed at any of the times measured, but a significant decrease was observed using lipofectamna with the plasmid containing the gene of the fusion proteins at 24, 48 and 72 hours post transfection, confirming the high toxicity of this reagent in transfections.

2.6 Characterization of the potential for the use of viral NPS in the CT26 model.

Animals are challenged with live tumor cells as described in section 1.8 above and after this when a tumor size of 2 mm ³ is detected, treatment with the composition of the invention comprising the NPs described above is started. In all the groups analyzed, the time of tumor onset was similar, between days 7-10 (Fig. 9). After the tumor appearance, the tumor was expected to reach a volume of 200 mm ³ and the animal was treated by injecting 100 pL of chitosan nanoparticles with the genes for the different fusion proteins (CH-REO (SEQ ID No .: 1) and CH-ISAV) with an N / P ratio = 28 using 10 pg of pDNA for the generation of the nanoparticles. Figure 10 shows a graph of tumor volume versus post-emergence days, and compares the different experimental conditions. When the effect of the treatment with chitosan alone is compared, no differences are observed in the tumor growth compared to the control, also the treatment with CH-ISAV does not present great differences compared to the control. When the analysis of the tumor growth is performed in the animals treated with CH-REO, it can be observed that there is a delay with respect to the control of chitosan and the untreated animals, this is supported by the analysis of Intersection with the x-axis (Table 3 ). On the other hand it is observed through the analysis of slopes that once the growth reaches an exponential phase the growth speed increases tumor in mice treated with CH-REO (Table 4). In addition to this, some of the animals treated with the 2 working conditions presented necrosis in the area of the tumor, which led to the decrease in tumor volume (Figure 11). Table 3. Analysis of intersections with the x axis. The values of the Intersections with the x-axis of the tumor growth curves (Flg.10) for each analyzed mouse are shown, values obtained by means of a linear regression of the curves.

Table 4. Analysis of slopes. The values of the slopes of the tumor growth curves (Flg.10) for each mouse analyzed are shown, values obtained by means of a linear regression of the curves.

Table 5. Analysis of survival of the animals. Comparison of the days of survival of the animal between the chitosan vehicle control and the treatments performed. The cut-off point was taken from the graphs of tumor growth including animals with necrosis. A Flsher Test was performed for the statistical analysis.

After the animals were sacrificed and in order to evaluate if there were changes in the lymphocyte populations at a systemic level, associated with the treatment that could account for the changes in the tumor growth, the lymphocyte populations CD4 +, CD8 +, Th17, were quantified. Th1 and Treg in spleen. Figure 12B shows the percentage of CD8 and CD4 lymphocyte populations present in the spleen. A significant decrease of the CD4 + population is observed in the treatments with CH-REO in relation to that obtained in mice without treatment. For the CD8 + population, a significant decrease was observed with respect to the control in the CH-ISAV treatment. In addition to quantifying the CD8 and CD4 lymphocyte populations, the CD4 + subpopulation present in the spleen, implicated in the antitumor response, was also analyzed by flow cytometry, which correspond to Th1, Th17 and Treg (Table 6). Table 6: Percentage of lymphocyte populations in spleen of treated animals

Figure 13 shows an analysis of the Th1 sub-population present in the spleen, in Figure 13A the representative hlstograms are observed for each of the treatments where the CD4 + TBET + population was analyzed. No significant changes were observed for any of the treatments used (p = 0.0754 - 0.34452), but a tendency to increase in the treatments with the nanoparticles could be observed with the genes of the fusion proteins.

 In parallel, the Th17 sub-population present in spleen was measured (Flg. 14), where the CD4 + RORY + population was analyzed. A significant increase was observed with respect to the control of the Th17 population for all treatments performed (CH-REO p = 0.0336, CH-ISAV p = 0.0240, CH P = 0.0125).

For the Treg sub-population present in the spleen, the CD4 + CD25 + FOXP3 + population (Fig. 15) was analyzed, where no significant changes were observed for any of the treatments used (CH-REO p = 0.1718; CH-ISAV p = 0.1414).

Adclclonally, after sacrifice, the levels of CD4 + and CD8 + lymphocytes were measured in tumors (Figure 16) where a significant decrease in the CD4 + population was observed for the treatments with qultosan (p = 0.0022) and CH-REO (p = 0.0265). In the tumor CD8 + population, no significant changes were observed with respect to the control. (CH-REO p = 0.0935; CH-ISAV p = 0.1234). (Table 7) Table 7: Percentage of tumor-infiltrating populations.

In this way, nanoparticles were generated and characterized in order to be used as in vivo transfection mechanisms for their therapeutic use in tumors, expressing deleterious genes in these. The use of nanoparticles of qultosan as a vector for the release of genes was characterized by the complex coacervation technique. This process is presented as a concrete alternative to the attempts made in recent years that have proposed various gene delivery systems for gene therapy as alternatives to the viral vectors commonly used.

The qultosano has been used as a vehicle for the transference of genes in cells due to its intrinsic characteristics. This molecule is able to condense and encapsulate DNA to form complexes that are captured on the cell surface, endocycled and transported, being able to migrate to the nucleus through pathways that, to date have not yet been elucidated (Robert S Cofflin. to oncolytlc mmunotherapy: where are we now? Current Opinion in Vlrology 2015, Vol.13, pp. 93-100, DOI: 10.1016 / j.covlro.2015.06.005). Three factors have been determined that are necessary for the entry of the nanoparticles of qultosan / DNA into the cell, among which are: the ratio of amine groups of qultosan against DNA phosphates (ratio N / P), the zeta potential and size (Sun Y., Zhang S, Peng X, Gong Z, L¡X, Yuan Z, L¡Y, Zhang D, Peng Y. They prepared, characterization and transfection efficacy of chitosan nanoparticles containing the intestinal trefoil factor gene. Molecular Blology Report. 2012, Vol. 39, No. 2, pp. 945-52. DOI: 10.1007 / s11033-011 -0820-4). This is why we proceeded to the characterization of the nanoparticles generated with chitosan and pDNA-GFP. For this, the load efficiency was measured, in which levels greater than 60% of DNA encapsulation were observed in all the N / P ratios used, which indicates that the pDNA used is efficiently encapsulated by chitosan. These high load efficiencies on the part of chitosan would be explained by the presence of high positive charges given by the amino groups of its surface, this charge strongly attracts the negative charge of the pDNA promoting its encapsulation (Bateman Andrew R., Harrlngton Kevln J., Kottke Tim, Ahmed Atique, Melcher Alan A., Gough Mlchael J., Llnardakls Emmanouela, Riddle David, Dletz Alian, Lohse Chrlstine M., Strome Scott, Peterson Tim, Simarl Robert and Vile Richard G. Viral fusogenlc membrane glycoproteins klll solid tumor cells by nonapoptotic mechanics that promote cross presentation of tumor antigens by dendritic cells, Cancer Research, 2002, Vol. 62, No. 22, pp. 6566-78, http://cancerres.aacriQurnals.org/coriient/62/22/ 6566.lQng). The smaller particles have the advantage of entering the cells through endocytosis and / or pinocytosis, which increases the rate of transfection.

To study the ability to encapsulate the chitosan pDNA, the migration of NPs was evaluated by agarose gel electrophoresis. For chitosan it was observed that the ability to encapsulate DNA is dependent on the N / P ratio, at higher N / P ratios, greater stability of the nanoparticle chitosan / pDNA, which is attributable to higher amounts of chitosan, greater interaction. electrostatic with the pDNA. In addition, the evaluation of the transfection efficiency of the nanoparticles generated in the CT26 cell line corresponding to colon carcinoma was carried out, where it was observed that the NPs were able to transfect CT26 cells with all the N / P ratios used, demonstrating that NPs are capable of interacting with the plasma membrane and entering the cell.

It was thus possible to determine that quitosa nanoparticles / pDNA could be used for the gene treatment against cancer, due to its ability to efficiently transfect the CT26 tumor line. Therefore, we proceeded to evaluate the use of a composition comprising nanoparticles of qultosan using vectors containing the viral fusogenic protein gene to transfect the CT26 tumor line.

The use of viral fusion proteins alone is a different alternative in the anti-tumor treatment and in the vlroterapla. In recent decades, viruses have been used to treat cancer, thanks to the characteristics of cancer cells, which make them particularly sensitive to viral infection and replication. A key advantage of the use of viruses as an anti-cancer agent is that to use the treatment it is not necessary to know the particular genetic alterations of each type of cancer, since this strategy is based on the fact that the tumor cells are mostly susceptible to death by selective viral infection based on underlying mutations in the tumor, which can be considerably different in different tumor cells (Robert S Coffln, From vlrotherapy to oncolytlc mmunotherapy: where are we now? Current Opinion in Vlrology 2015, Vol.13, pp. 93-100, DOI: 10.1016 / j.covlro.2015.06.005).

The disadvantage of using active viruses is that they can spread in the treated patient and mutate, recovering their pathogenic potential; the malignant cells can become resistant to the vlroterapla product of its genomic instability; in addition to heterogeneous and relatively Incomplete diffusion of virus in neoplastic lesions (Pol Jonathan, Buqué Aitziber, Aranda Fernando, Bloy Norma, Cremer Isabelle, Eggermont Alexande, Erbs Phlllppe, Fuclkova Jltka, Galón Jéróme, Llmacher Jean-Marc, Prevllle Xavier, Sautés-Frldman Catherlne, Splicek Radek, Zltvogel Laurence, Kroemer Guido and Galluzzl, Lorenzo. Trlal Watch-Oncolytlc vlruses and cancer therapy, Oncolmmunology, 2016, Vol. 5, No. 2. DOI: 10.1080 / 2162402X.2015.1 1 17740).

An alternative to avoid using active viruses is the use of the proteins that carry out the toxic effect on the cell, which is why the present invention uses 2 types of fierogenic viral proteins (FMG). These proteins are capable of generating the process of cell-cell fusion, which leads to the formation of cellular agglomerates that are biologically, structurally and biochemically highly unstable, known as slnclclos. This formation of slides is carried out in 3 main stages: 1) cell-cell contact, 2) hemlfuslón, intermediate formed by the fusion of the two closest monolayers without fusion of the dlasta monolayers and 3) opening of the pore and expansion (Stephen C. Harrlson, Viral membrane fusion, Vlrology, 2015, Vol.479-480, pp. 498-507, DOI: 10.1016 / j.vlrol.2015.03.043).

The clitoxicity produced by the FMGs is observed early 24 hours post-transfection of their genes, because at this time they begin to form slnclclos producing cell death, which increases gradually with time (Bateman Andrew R., Harrlngton Kevln J., Kottke Tlm, Ahmed Atlque, Melcher Alan A., Gough Mlchael J., Llnardakls Emmanouela, Riddle David, Dletz Alian, Lohse Chrlstlne M., Strome Scott, Peterson Tlm, Robert Slmarl and Vile Richard G. Viral fusogenlc membrane glycoprotelns klll solid tumor cells by nonapoptotlc mechanlsms that promote cross presented of tumor antigens by dendritic cells, Cancer Research, 2002, Vol. 62, No. 22, pp. 6566-78.

adhere to no theory it would seem that this death caused by the formation of problems is by not apoptotic. Necrosis seems to play an important role, since signs of mitochondrial insufficiency, ATP deficiency and generation of autophagy have been observed in cells transfected with the GALV fusion protein gene (Bateman Andrew R., Harrington Kevin J., Kottke Tim , Ahmed Atlque, Melcher Alan A., Gough Michael J., Linardakis Emmanouela, Riddle David, Dietz Alian, Lohse Christlne M., Strome Scott, Peterson Tim, Slmari Robert and Vile Richard G. Viral fusogenic membrane glycoprotelns kill solid tumor cells by That Promote cross nonapoptotlc Mechanisms of tumor antlgens presented by dendritic cells Cancer Research 2002, Vol 62, No. 22, pp 6566-78 http:..... //cancerres.aacriournals.or¾/content ^/ 62/22 / 6566.1onq ) · It has been observed that during the process of syncytium death, it releases vesicles of exosomes called syncytomasomes. These are vesicles that can be loaded with tumor-associated antigens, which can be recognized by dendritic cells, mediating communication with T cells. Furthermore, it has been seen that moribund syncytia could release toxic factors in their environment to mediate the death of unfused cells. (EH Un. Fusogenic membrane glycoproteins induces syncytia formation and death in vitro and in vivo: a potential therapy agent for lung cancer, Cancer Gene Therapy, 2009, Vol. 17, pp. 256-265 DOI: 10.1038 / cgt.2009.74 ). Once the CT26 cells were transfected by means of the chitosan NPs with the genes coding for the viral fusion proteins of REO and ISAV, their effect on cell viability was evaluated where the formation of syncytia should lead to cell death. For the cells transfected with CH-REO, a significant decrease in cell viability was observed at 72 hours, which could be attributable to the formation of syncytia. In the viability assays using CH-ISAV, no significant changes were observed at any of the times evaluated in the cells transfected with chitosan, but in the cells transfected with llpofectamna. The decrease in cell viability when using llpofectamlna can be attributed directly to the toxic effect of this compound and not by the expression of the proteins at 72 hours (Zhong YQ et al., Toxicity of cationlc liposome Upofectamlne 2000 in human pancreatic cancer Capan-2 cells, Journal of Central South Unversity of Technology. Vol. 28, No. 11, pp. 1981 -1984, DOI: 1673-4254 (2008) 11 -1981- 04). On the other hand, the fact that no significant changes in cell viability are observed in the cells transfected with CH-ISAV can be attributable to the fact that ISAV-F was evaluated in mammalian cells that does not constitute the species in which it can naturally be detected, since ISAV is a fish pathogen (Cook, D. and cois, Electrostatic Archltecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein lllustrates to Carboxyh Carboxylate pH Sensor, Journal of Blological Chemlstry, 2015, Vol. 290, No. 30 , pp. 18495-18504, DOI: 10.1074 / jbc.M1 15.644781). Another point to consider is that the tests were performed under physiological conditions at neutral pH and 370, conditions that are not optimal for ISAV-F to carry out its action, since the fusion of membranes mediated by ISAV-F is triggered in response to a pH decrease at temperatures between 10 to 160 (Cook, D. and cois, Electrostatic Archltecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein lllustrates to CarboxyhCarboxylate pH Sensor, Journal of Blological Chemlstry. 2015, Vol 290, No. 30, pp. 18495-18504, DOI: 10.1074 / jbc.M115.644781).

The kinetics of formation of syncytia by action of these fusion proteins is also relevant, since this may be greater than those of other fusogenic proteins described in literature such as the F proteins of the GALV virus (6, Dunn, Gavln et al. mmunoediting: from mmuno-surveillance to tumor scape, Nature mmunology, 2002, Vol. 3, No. 11. Pgs .: 991 -998, DOI: 10.1038 / ni1102-991) where it is shown that the formation of syncytia occurs between 24 and 72 hours post transfection and the disintegration of the syncytium occurs within 120 hours post transfection. So without adhering to any theory it would seem that, for the fusion proteins used, it must evaluate longer post-transfection times to observe changes in cell viability.

After the in vitro studies, the in vivo studies were carried out, where Balb / c mice were treated with the nanoparticles for the different viral fusion proteins. A post-challenge tumor growth monotherapy was performed, which showed a delay in tumor growth in the mice treated with CH-REO. In the tumoral growth curves a clear dispersion in the growth of the mice subjected to the same treatment is observed, this may be due to the method of preparation of the vaccine and the chemical characteristics of the qultosan. The qultosano is very little soluble in water, reason why acid must be added to him to increase its solubility due to the protonaclón of the amines groups, this solubility also is dependent of the anions that are in solution. The NPs were resuspended in PBS, it is known from literature that the phosphate and sulfate groups decrease the solubility of qultosan (Berthold A et al., Prepared and characterized by chltosan as drug carriers for prednisolone sodium phosphate as model for antiflammatory drugs. Release 1996, Vol. 39, No.1, pp. 17-25.bttp: //vwAv.sclencedlrect.com/science/artlcle/pii/0168365995001298), which would favor its precipitation and with this will diminish its ability to spread for the micro tumor, so the most optimal solution for resuspending the nanoparticles should have been found. Another point to consider is that the efficiency of encapsulation of pDNA by qultosan is not 100%, so that the amount of plasmid that binds to qultosan can vary and thereby affect the transfection of the tumor cells.

We observed a group of animals that had necrosis in the tumor after treatment, which implies a decrease in tumor size. Considering the low number of animals studied, it would be risky to propose conclusions, however, without adhering to any theory it would seem that in these animals the formation of syncytia would lead to tumor cell death through a process called necroptosls (Loves-Aaes, Tania and cois. Vaccination with Necroptotlc Cancer Cells Induces Efficlent Anti- tumor Immunity, Cell Reports, 2016, Vol.15, No. 2, pp. 274-287, DOI: 10.1016 / j.celrep.2016.03.037). The discovery of necroptosls showed that cells can execute necrosis in a programmed manner and that apoptosis is not always preferable to necrotic cell death. In addition, the immunogenic nature of the necroptosls favors its use in certain circumstances, such as helping the orientation of pathogens by the immunological system. Works by Loves et al. in 2016, they demonstrated that necroptotic CT26 cells can be immunogenic in vitro and in vivo, generating an immune response through cytotoxic CD8 + lymphocytes, promoting cross-presentation; and by means of the production of interferon and in response to tumor antigens of necrotic cells (Loves-Aaes, Tania et al., Vaccination with Necroptotlc Cancer Cells Induces Efficiency Anti- tumor Immunity, Cell Reports, 2016, Vol. 15, No 2, pp. 274-287, DOI: 10.1016 / j .celrep.2016.03.037).

Although an effect on tumor growth was obtained at least for the case of REO-F, the mechanisms responsible for this effect are not yet clear and neither do they have an important immune component. However, this effect may be due to the rapid formation of syncytia by the REO protein. The REO fusion protein corresponds to a small fusion-associated membrane protein, which retains within its structure all that it requires for the fusion process. This protein does not need others to mediate cell fusion, since the interaction of its endodomain with the cadherins mediates the initial binding to the membrane and the ectodomain with the target membrane, being able to maintain the formation of the pore. In addition, their endodominies interact with factors that act in the actin remodeling, promoting pore expansion and slncltlogeness (Boutilier J. et al., The Reovirus Fuslon-Assoclated Small Transmembrane (FAST) Protelns: Virus-Encoded Cellular Fusogens, Current Toplcs, Membranes, 2011, Vol. 68, p. 107-140 DOI: 10.1016 / B978-0-12-385891 -7.00005-2). This capacity of REO to be self-sufficient for the formation of tumors could be the key to its rapid activity and responsible for the control of tumor growth.

The composition comprising NPs with the genes coding for the fusion protein ISAV, showed no significant effects on tumor growth, without adhering to any theory, this could be due to the need for post-translational modifications for the protein to be active, specific pH requirements for its action, since ISAV that acts optimally at low pH (Cook D. Jonathan, Soto- Montoya Hazel, Korpela Markus, Lee E. Jeffrey, Electrostatlc Architecture of the Infectlous Salmon Anemia Virus (ISAV) Core Fusion Proteln lllustrates a CarboxyhCarboxylate pH Sensor, Journal of Biologcal Chemistry, 2015, Vol. 290, No. 30, pp. 18495-18504, DOI: 10.1074 / jbc.M115.644781), or it could also be, the need for accessory proteins for carry out your action.

When evaluating the effect of the treatments on the lymphocyte populations in spleen, a decrease of CD4 + lymphocytes was observed, without changes in the CD8 + T population. Several studies have shown the importance of CD4 + lymphocyte subpopulations in the antitumour response, which is why they were analyzed. Treg lymphocytes are CD4 + CD25 OXP3 + cells and their main characteristics are their ability to actively inhibit CD8 + lymphocytes, dendritic cells, Natural Killer (NK) cells and B cells, promoting tumor growth (Sundstrom, Patrik and cois, Regulatory T cells from colon cancer patiens inhibit effector T cell migratlon through an adenosine-dependent mechanism, Cancer Immunology Research, 2016, Vol.4, No. 3, pp.183-93, DOI: 10.1158 / 2326-6066.CIR-15-0050) . In addition, regulatory T cells have been shown to promote the growth of human colon cancer (CC). This type of cancer is characterized by having an Inflammatory response that would be modulated by Treg as important actors in the inhibition of T cells that would activate antitumour immunity, presenting high levels of Treg lymphocytes in tumor (Sundstrom, Patrik and cois, Regulatory T cells from colon cancer patiens inhibit effector T cell mlgratlon through an adenosine-dependent mechanicsm Cancer Immunology Research 2016, Vol.4, No. 3, pp.183-93 DOI: 10.1158 / 2326-6066 CIR-15-0050 ). Despite this background, the results obtained do not show significant changes for the Treg populations in any of the treatments used.

On the other hand, the Th1 population has been associated with a potent anti-tumor response in various types of cancer. When this population was analyzed in the spleen, although no significant difference was obtained between the treated and untreated mice, a tendency to increase this population was observed in the treated mice.

Th17 lymphocytes play a complex and controversial role in tumor immunity, since they can act by favoring tumor development or an anti-tumor response depending on the malignancy and temporality of tumor growth (Bailey, Stefanle et al., Th17 cells n cancer: the ultímate dentlty crisis Frontlers n Immunology 2014, Vol 5, Artlcle 276. DOI 10.3389 / flmmu.2014.00276). In the evaluation of this subpopulation in spleen, a significant increase of Th17 was obtained in all the treatments used. In the literature it has been shown that Th17 cells are negatively correlated with the presence of Treg cells and positively with Immune effector cells, including Th1, these observations are supported by data from studies in human and mouse model. Considering the action of Th1 and Th17, its increase in the treated mice could explain it obtained in the tumoral growth curves, because these two subpopulations promote a antitumor response from the Immune system (Balley, Stefanle et al., Th17 cells cancer: the last crisis, Frontlers Immunology, 2014, Vol. 5, Artcle 276. DOI: 10.3389 / flmmu.2014.00276). It may be an additional contribution to determine exactly what happens with the decrease in the population of CD4 + lymphocytes in both spleen and tumor since it is unknown which CD4 + lymphocyte sub-population is the one that is decreasing. However, for this it would be necessary to carry out the analysis of a greater number of CD4 + sub-populations to relate them to the effect of the treatments tested.

But the results set forth in the present application show that the composition of the invention comprising nanoparticles of natural origin can be used as vectors for the encapsulation of genes whose proteins are capable of controlling tumor development, creating an immune response against this or both, by direct treatment of the tumor. The expression of the REO protein in the tumor caused a delay in tumor growth, which would be associated with the death of the tumor by the generation of syncytia, rather than the induction of an immune response by the T lymphocytes evaluated. The present composition also comprises nanoparticles that can optionally encapsulate genes whose proteins are able to control tumor development and create an immune response against it, by direct treatment of the tumor, where the gene has a sequence comprising an identity of at least 50% , preferably at least 60%, and more preferably at least 70%, still more preferably at least 80%, and more preferably at least 90% of the nucleotides (ie, sample sequence identity) shown in SEQ ID NO: 1; or wherein the protein has a sequence comprising an identity of at least 50%, preferably at least 60%, and more preferably at least 70%, even more preferably at least 80%, and more preferably at least 90% of the amino acids (ie, sequence identity sample) shown in SEQ ID NO: 2.

 The present invention also comprises fragments and derivatives of the nucleotide sequences shown in SEQ ID No .: 1 or of the amino acid sequences shown in SEQ ID No: 2. Also, the present invention comprises functional equivalents of the above sequences.

 For the purposes of the present invention, a "fragment" of a nucleotide sequence is defined as a contiguous sequence of about at least 6, preferably at least 8, more preferably at least 10 nucleotides, and even more preferably at least 15 nucleotides that correspond to a region of the specific nucleotide sequence.

Claims

A pharmaceutical composition for intratumoral injection characterized in that it comprises chitosan nanoparticles that encapsulate DNA from a protein capable of controlling tumor development, creating an immune response against the tumor or both; a fragment or derivative of said DNA, with the same function.

2. The pharmaceutical composition of claim 1 characterized in that said protein is a viral fusogenlca protein.

3. The pharmaceutical composition of claim 2 characterized in that said viral fusogenlca protein is a fusogenic membrane glycoprotein.

4. The pharmaceutical composition of claim 3 characterized in that said membrane fusogenlca glycoprotein is the fusion protein of Reovlrus Aviar (REO) sequence SEQ ID No.2 or a protein with a sequence comprising at least 50% identity, preferably at least 60%, and more preferably at least 70%, still more preferably at least 80%, and more preferably at least 90% identity with the sequence of the fusion protein of Avian Reovlrus.

5. The pharmaceutical composition of claim 4 characterized in that said membrane fusogenlca glycoprotein is the Reovlrus Aviar (REO) fusion protein of sequence SEQ ID No.:2.

6. The pharmaceutical composition of claim 1 characterized in that said

DNA has sequence SEQ ID No.:1 or a sequence comprising an identity of at least 50%, preferably at least 60%, and more preferably at least 70%, still more preferably at least 80%, and more preferably at least 90 % identity with said sequence SEQ ID NO: 1.

7. The pharmaceutical composition of claim 6 characterized in that said DNA has sequence SEQ ID No.:1.

The pharmaceutical composition of claim 1 characterized in that said DNA of the Avian Reovirus fusion protein (REO) is inserted into an expression vector.

9. The pharmaceutical composition of claim 8 characterized in that said vector is selected from the expression vector plRES2.

10. The pharmaceutical composition of claim 1 characterized in that said nanoparticles have a radius of average size between 25-56 nm.

11. The pharmaceutical composition of claim 1 characterized in that said

DNA has a ratio of amine group of chitosando (N) to phosphate of DNA (P), ratio N / P equal to 28.

12. Use of the pharmaceutical composition of any of claims 1 to 11, characterized in that it serves to prepare a medicament useful in the treatment of cancer and tumors.

13. The use according to claim 12 characterized in that said tumors are selected from tumors that are in the early stages of development.

14. The use according to claim 13, characterized in that said tumor is selected from melanoma.

15. The use according to claim 12, characterized in that said cancer is selected from carcinoma.

16. The use according to claim 15, characterized in that said carcinoma is selected from colon carcinoma.

17. Treatment method for cancer or tumors characterized in that it comprises intratumorally administering a pharmaceutical composition comprising chitosan nanoparticles that encapsulates the DNA of the Reovlrus Avian fusion protein (REO), and thereby, cause cell death and stimulate the immune system .

18. The method of claim 17, characterized in that it comprises administering said pharmaceutical composition in early stages of tumor development.

19. The method of claim 17, characterized in that it comprises administering said pharmaceutical composition in the tumor zone.