MX2012010920A - Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof. - Google Patents

Abstract

This invention relates to the field of therapeutics. Most specifically, the invention provides methods of generating conditionally expressing vectors for one or more immunomodulators under the control of a gene expression modulation system in the presence of activating ligand and uses for therapeutic purposes in animals. These vector may be provided to treat a variety of disorders, e.g., neoplastic disorders, through direct injection or through in vitro engineered cells, such as dendritic cells.

Description

VECTORS THAT EXPRESS CONDITIONALLY THERAPEUTIC PROTEINS, HOSPITAL CELLS THAT COMPRISE VECTORS, AND USES OF THEMSELVES Field of the Invention This invention is related to the field of gene therapy for the treatment of diseases and disorders, for example, cancer, lysosomal storage disorders, eye diseases, liver diseases, or infectious diseases. In one embodiment, the invention provides engineering manipulation of immune cells or therapeutic support cells (TSCs) to express one or more therapeutic proteins (eg, immunomodulators) and the use of cells as therapeutics. In another embodiment, the invention includes a vector, e.g., an adenovirus, for conditional expression of therapeutic proteins (e.g., immunomodulators) described herein, e.g., IL-12, TNF-alpha and method for using these vectors.

Background of the Invention Interleukin-12 (IL-12) is a member of the type I cytokine family, involved in contributing to a number of biological processes including, but not limited to, protective immune response and suppression of tumorigenesis (Abdi et al. , 2006; Adorini, 1999; Adorini, REF. : 235720 2001; Adorini et al, 2002; Adorini et al, 1996; Akhtar et al, 2004; Akiyama et al, 2000; Al-Mohanna et al, 2002; Aliberti et al, 1996; Allavena et al, 1994; There and K ar, 2004; Alzona et al, 1996; Araeraiya et al, 2006; Araujo et al, 2001; Arulanandam et al, 1999; Athie et al, 2000; Athie-Morales et al, 2004; Bertagnolli et al, 1992; Bhardwaj et al, 1996; Biedermann et al, 2006; Brunda and Gately, 1994; Buchanan et al, 1995; Romani et al, 1997; Rothe et al, 1996; Satoskar et al, 2000; Schopf et al, 1999; Thomas et al, 2000; Tsung eC al, 1997; Wolf et al, 1994; Yuminamochi et al, 2007). Growing evidence suggests that IL-12 may be a promising target for controlling human diseases (eg, cancer).

Despite the fact that IL-12 remains promising as a therapeutic agent against cancer, based on its potent supporting activity on Type 1 antitumor NK cells, CD4 + T cells and CD8 + T cells (Trinchieri, 2003), reported toxicity of recombinant IL-12 (rhIL-12) in patients (Atkins et al, 1997), together with limited sources of GMP grade rhIL-12 for clinical application, have prevented successful therapeutic approaches based on IL- 12 Thus, it seems reasonable that gene therapy approaches can represent safer, more appropriate treatment options. Of course, phase I clinical trials that implement the intra- or peri-tumor distribution of IL-12 cDNA based on recombinant virus (Sangro et al, 2004; Triozzi et al, 2005) or on plasmids (Heinzerling et al. , 2005), or autologous fibroblasts modified with the IL-12 gene (Kang et al, 2001) have been found to be safe and well tolerated.

However, the objective clinical responses in patients with melanoma or a diverse range of carcinomas receiving these gene therapies have been rare, variable, transient, and largely focused on the treatment site (Heinzerling et al, 2005; Kang et al. , 2001, Sangro et al, 2004, Triozzi et al, 2005). In cases where the resolution of the disease was partial or complete, increased frequencies of tumor infiltration lymphocytes have been noted (Heinzerling et al, 2005; Sangro et al, 2004) and elevated levels of circulating tumor-specific CD8 + T cells (Heinzerling et al. al, 2005), consistent with improved cross-priming of antigen-specific T cells in these patients.

Since cross-priming of specific T cells is best achieved by dendritic cells (DCs) that serve as a natural but regulated source of IL-12 (Berard et al, 2000), recent reports from The superior pre-clinical efficacy of therapy with the DC-based IL-12 gene has been of great interest (Satoh et al, 2002; Tatsumi et al, 2003; Yamanaka et al, 2002). For example, it has been shown that intratumoral (it) injection of DCs genetically engineered to produce IL-12p70 (via infection with recombinant adenovirus), results in dramatically improved cross-priming of a CD8 + T cell repertoire. , tumor-specific, widely reactive, in concert with tumor rejection in murine models (Tatsumi et al, 2003). Given the previous use of a recombinant adenovirus that codes for mIL-12 under a CMV-based promoter (rAd.cIL12, (Tatsumi et al, 2003)), the production of DC genetically engineered IL-12, was constitutive , since the immunological impact of this cytokine early within the tumor lesion and later within the lymphatic nodes that drain the tumors, could not be resolved with respect to the therapeutic result. Thus, there is a need for DC engineered for the conditional expression of IL-12 for the purpose of regulating the level of transgene expression and for the timing of transgene activation. The invention provides a promising therapeutic result for the use of such cells.

Many of the therapeutic proteins currently under investigation in clinical or preclinical trials do not exhibit dangerous side effects when presented in a patient prior to the expression of the nucleic acid sequence in the patient's host cell or the appropriate physiological context. However, some proteins, such as tumor necrosis factor (TNF), cause adverse effects when they are expressed out of context or normal physiological tissues (for example, they are exposed to non-target tissues). The systemic or even local administration of the protein is extremely toxic to many types of non-tumor cells, promoting anaphylaxis and cachexia. In addition, prolonged exposure to TNF-alpha can produce profoundly different cellular responses than acute stimulations. For these reasons, the safe and effective therapies of TNF-alpha against cancer have remained elusive.

In view of the problems associated with gene expression of genes through compositions of vectors containing the protein encoded by the nucleic acid sequence of interest, there is a need for improved transfer vector compositions to be used by direct injection or for use in cell-based therapies.

Lysosomal storage diseases (LSDs) represent a class of inherited genetic disorders that can currently be treated only by protein therapeutics, in the form of enzyme replacement therapy.

LSDs are a class of 49 genetically inherited disorders, characterized by a deficiency of one or more lysosomal enzymes that cause accumulation of undigested macromolecules within the lysosome. The accumulation of these residual products causes the lysosomes inside the cells to enlarge, leading to degeneration and cell damage. The accumulated damage in organs and tissues results in progressive deterioration in the physical and / or mental state, and eventually death. Typically, diagnosis is made in childhood. The severity of the individual disease is variable and is correlated to the amount of residual enzymatic activity produced by the defective gene.

The incidence of LSD is approximately 1 in 5000 people (130,000 case worldwide). The severity is variable and is correlated to the amount of residual enzymatic activity produced by the defective gene. Severely affected patients can only live until their adolescence, while less severely affected patients can survive until adulthood.

Enzyme replacement therapy is the only method available to treat LSD. The therapy consists of systemic infusion of active proteins that target lysosomes and break up the accumulation of residual molecules. Examples of proteinaceous products of LSD include Fabrazyme (Genzyme) for Fabry Disease, Elaprase (Shire) for MPSII, and Myozome (Genzyme) for Pompe Disease, and Cerezyme (Genzyme) for Gaucher Disease.

Enzymatic replacement therapy is accompanied by certain disadvantages, such as the requirement for post-transductional protein modifications, replacement enzymes exhibit short half-lives in vivo, and patients develop an immune response to replacement enzymes. Therefore, the need remains in the art for an alternative enzyme replacement therapy to treat lysosomal storage disease.

Brief Description of the Invention The invention provides a recombinant vector encoding proteins that have the functions of one or more therapeutic proteins (eg, immunomodulators), under the control of one or more promoters. In one modality, the one or more promoters are conditional. In another embodiment, the one or more promoters are constitutive. In another embodiment, the vector is an adenovirus vector encoding the proteins, extracted from a promoter that can be conditionally activated by the provision of a soluble small molecule ligand such as diacyl hydrazines (e.g., RG-115819, RG-115830 or RG-115932). This vector allows the control of the expression of immune cell proteins, TSC and direct injection of the vectors comprising therapeutic proteins (for example, immunomodulators).

In one embodiment, the invention provides a vector for conditionally expressing proteins having the functions of one or more therapeutic proteins (eg, immunomodulators) comprising a polynucleotide encoding a gene change, wherein the polynucleotide encoding a gene change comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide that encodes a or more proteins that have the function of a therapeutic protein (e.g., immunomodulator) linked to a promoter that is activated by the ligand-dependent transcription factor. In one embodiment, the therapeutic protein (e.g., immunomodulator) is selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL- 10RDN or a subunit thereof, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, CCL3 (MIP-la) , CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12, (SDF-1) ), CCL21 (6CCIN), OX40L, 4-1BBL, CD4Q, CD70, GITRL, LIGHT, b-Defensin, H GB 1, Flt3L, IFN-beta, TNF-alpha, dnFADD, TGF-alpha, PD-LlRNAi, a antisense oligonucleotide of PD-L1, TGFbRII DN, ICOS-L, S100, CD40L, p53, survivin, fusion of p53-survivin, MAGE3, PSA and PS A.

In another embodiment, the invention provides a vector for expressing proteins having the functions of one or more therapeutic proteins (eg, immunomodulators) and a protein having the function of IL-12, comprising a polynucleotide that encodes a gene change , wherein the polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, (2) a polynucleotide coding for proteins that have the functions of one or more therapeutic proteins (eg, immunomodulators), and (3) a polynucleotide that encodes a protein that has the function of IL-12; wherein the at least one polynucleotide of (2) and (3) is linked to the promoter that is activated by the ligand-dependent transcription factor.

In some embodiments, the vector of the invention conditionally expresses TNF-alpha. In certain embodiments, the vector, for example, adenoviral vector, which conditionally expresses one or more proteins having the function of a therapeutic protein (eg, immunomodulator), eg, TNF-alpha, further comprises a nucleic acid sequence that codes for a signal peptide. The signal peptide can be optimized by codon. In other embodiments, the vector further comprises the 5 'untranslated region (UTR), the 3' regulatory region, or both and improves the total yield and / or expression of the protein.

The invention further provides a method for producing a population of cells, e.g., immune cells or TSCs, which express proteins having the function of one or more therapeutic proteins (e.g., immunomodulators), by modifying (e.g., transfecting, electrophoresis, etc.) cells with a recombinant vector that conditionally expresses proteins having the functions of the one or more therapeutic proteins (e.g., immunomodulators), wherein the vector comprises a polynucleotide that encodes a gene change , wherein the polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide that codes for one or more proteins that have the function of a therapeutic protein (for example, immunomod ulator), a modulator linked to a promoter that is activated by the ligand-dependent transcription factor.

In another embodiment, the invention provides a method for producing a population of cells, e.g., immune cells or TSCs, that express proteins that have the functions of one or more therapeutic proteins (e.g., immunomodulators) and a protein that has the function of IL-12, by modifying the cells with a recombinant vector comprising a polynucleotide encoding a gene change, wherein the polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein the at least one sequence of transcription factor codes for a ligand-dependent transcription factor, (2) a polynucleotide that codes for proteins that have the functions of one or more therapeutic proteins (eg, immunomodulators), and (3) a polynucleotide that codes for a protein that has the function of IL-12; wherein the at least one polynucleotide of (2) and (3) is linked to the promoter that is activated by the ligand-dependent transcription factor.

In some embodiments, the invention provides a method for increasing the expression of a therapeutic protein (e.g., immunomodulator), e.g., TNF-alpha, mRNA expression, or protein expression comprising generating the vector that conditionally expresses a or more proteins having the function of a therapeutic protein (e.g., immunomodulatory) and one or more regulatory sequences, wherein the one or more regulatory sequences improve the expression of therapeutic proteins (e.g., immunomodulators), e.g., TNF -alpha.

The invention further provides a population of cells, e.g., immune cells or TSCs, which express proteins that function as one or more therapeutic proteins (e.g., immunomodulators), which have been modified (e.g., transfected, electrophoresed). , etc.) with a recombinant vector that conditionally expresses the proteins having the functions of the one or more therapeutic proteins (eg, immunomodulators), wherein the vector comprises a polynucleotide encoding a gene change, wherein the polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) or polynucleotide that encodes one or more proteins that have the function of a therapeutic protein (for example, immunomodulator) linked to the promoter which is activated by the ligand-dependent transcription factor.

In another embodiment, the invention provides a population of cells, e.g., immune cells or TSCs, that express proteins that have the functions of one or more therapeutic proteins (e.g., immunomodulators) and a protein that has the function of IL-12. , which has been modified with a recombinant vector comprising a polynucleotide encoding a gene change, wherein the polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein the at least one sequence of transcription factor codes for a ligand-dependent transcription factor (2) a polynucleotide that codes for proteins that have functions of the one or more therapeutic proteins (eg, immunomodulators) and (3) a polynucleotide that encodes a protein that has the function of IL-12; wherein the at least one polynucleotide (2) and (3) are linked to a promoter that is activated by the ligand-dependent transcription factor.

In another embodiment, the invention provides a composition comprising two or more populations of cells of the present invention, for example, immune cells or TSC, wherein each population of cells in the composition expresses one or more therapeutic proteins (eg, immunomodulators). ) that are different from the one or more therapeutic proteins (eg, immunomodulators) expressed in the other cell populations in the composition. In one embodiment, the composition contains two populations of cells. In another embodiment, the composition contains more than two cell populations. In another embodiment, the composition contains three cell populations. In another embodiment, the composition contains four cell populations.

In another embodiment, the invention provides an engineered in vitro cell, eg, immune cell or TSC, comprising a vector comprising a polynucleotide encoding a gene change, wherein the polynucleotide comprises (1) at least one sequence of transcription factor operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide that encodes a protein that has the function of a protein therapeutic (eg, immunomodulatory) linked to a promoter that is activated by the ligand-dependent transcription factor. In another embodiment, the invention provides an engineered in vitro cell, eg, immune cell or TSC, comprising a vector comprising a polynucleotide encoding a gene change, wherein the polynucleotide comprises (1) at least one sequence of transcription factor operably linked to a promoter, wherein the at least one sequence of transcription factor codes for a ligand-dependent transcription factor, (2) a polynucleotide that encodes a protein that has the function of a therapeutic protein (e.g., immunomodulator), and (3) a polynucleotide that encodes a protein having the function of IL-12; wherein the at least one polynucleotide of (2) and (3) is linked to a promoter that is activated by the ligand-dependent transcription factor.

In another embodiment, the invention provides a composition comprising two or more cell populations engineered in vitro, eg, immune cells or TSC, of the present invention, wherein each of the populations of cells engineered in vitro by in the composition comprises a vector comprising a polynucleotide encoding a gene change, wherein the polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein the at least one factor sequence of transcription encodes a ligand-dependent transcription factor, and (2) a polynucleotide that encodes a protein that has the function of a therapeutic protein (eg, immunomodulator) linked to a promoter that is activated by the transcription factor dependent on ligand, and where each population of cells handled in vitro by composition engineering expresses one or more therapeutic proteins (e.g., immunomodulators) that are different from the one or more therapeutic proteins (e.g., immunomodulators) expressed in the other cell populations engineered in vitro into the composition. In one embodiment, the invention provides a composition comprising two or more cell populations engineered in vitro, eg, immune cell or TSC, each of the cell populations comprising a vector comprising a polynucleotide encoding a gene change, wherein the polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, (2) a polynucleotide that encodes a protein having the function of a therapeutic protein (eg, immunomodulator), and (3) a polynucleotide that encodes a protein having the function of IL-12; wherein the at least one polynucleotide of (2) and (3) is linked to a promoter that is activated by the ligand-dependent transcription factor. In one embodiment, the composition contains two populations of cells engineered in vitro. In another embodiment, the composition contains more than two cell populations engineered in vi tro. In another embodiment, the composition contains three cell populations engineered in vitro. In another embodiment, the composition contains four populations of cells engineered in vitro by engineering.

The invention provides a pharmaceutical composition comprising a population of cells, e.g., immune cells or TSC, as described herein or a composition suitable for direct injection of expression vectors absent in a population of cells, i.e., directly injected.

In one embodiment, the polynucleotide that codes for the one or more proteins that have the functions of the immunomodulator is under the control of the gene change promoter and the polynucleotide that codes for a protein having the function of IL-12 is under the control of a constitutive promoter. In another embodiment, both the polynucleotide that codes for proteins that have the functions of therapeutic proteins (eg, immunomodulators) and the polynucleotide that codes for a protein having the function of IL-12 are both under the control of a promoter. multicistronic of gene change. In another embodiment, the polynucleotide encoding a protein having the function of the therapeutic proteins (eg, immunomodulators) is under control of the gene change promoter and the polynucleotide that codes for a protein having the function of IL-12 it is under the control of a conditional promoter that is different than the gene change promoter. In a further embodiment, the system of gene regulation for the polynucleotide that encodes the proteins that have the function of the therapeutic proteins (eg, immunomodulators) and the gene regulation system for the polynucleotide having the function of IL-12 are orthogonal In a further embodiment, the system of gene regulation for each polynucleotide that codes for each protein is orthogonal.

In one embodiment, the invention also provides a cancer treatment, such as, but not limited to, melanoma tumors, glioma tumors, renal cancer and prostate cancers, as well as the cancers listed herein in Table 1. Gene therapy with IL-12 has demonstrated antitumor efficacy in studies with animal models when applied as a recombinant cDNA vector (Faure et al, 1998; Sangro et al, 2005), but still further when applied in the context of DC geneically modified (Satoh et al, 2002, Svane et al, 1999, Tatsumi et al, 2003, Yamanaka et al, 2002). However, to date human phase 1 trials of gene therapy with IL-12 that implement plasmids or viral vectors have failed to achieve clinical, objective, durable responses in the cancer scenario (Heinzerling et al, 2005; Kang et al. , 2001, Sangro et al, 2004, Triozzi et al, 2005) gene therapy as described herein provides a promising therapeutic modality.

In one embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of: (a) administer intratumorally to tumor micro-environments, in the area surrounding the tumor, or systemically, a population of immune cells, TSCs or vectors of the invention (or a combination of these), which are engineered in vitro to express from conditionally one or more proteins that have the function of a therapeutic protein (eg, immunomodulator); Y (b) administering to the mammal a therapeutically effective amount of an activating ligand; to thereby induce the expression of a protein having the function of the therapeutic protein (eg, immunomodulator) and to treat the tumor.

In one embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of: (a) administering intratumorally to tumor micro-environments a population of immune cells or TSCs, which are engineered in vitro to conditionally express one or more proteins that function as a therapeutic protein (eg, immunomodulator); Y (b) administering to the mammal a therapeutically effective amount of an activating ligand; to thereby induce the expression of a protein having the function of therapeutic proteins (eg, immunomodulators) and to treat the tumor.

In another embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of: (a) administer intratumorally to tumor microenvironments two or more populations of immune cells or TSC, which are engineered in vitro to conditionally express one or more proteins having the function of a therapeutic protein (eg, immunomodulator), wherein each Immune cell population or TSCs express a different set of one or more therapeutic proteins (eg, immunomodulators); Y (b) administering to the mammal a therapeutically effective amount of one or more activating ligands; to thereby induce the expression of proteins that have the function of therapeutic proteins (eg, immunomodulators) and to treat the tumor.

In another embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of: (a) administering intratumorally to tumor microenvironments a population of immune cells or TSC, which are engineered in vitro to conditionally express one or more proteins that have the function of a therapeutic protein (eg, immunomodulator) and a protein that have the function of IL-12, wherein the at least one of the proteins that have the function of the therapeutic protein (eg, immunomodulator) or IL-12 is under the control of a conditional promoter that is activated by a ligand; Y (b) administering to the mammal a therapeutically effective amount of the activating ligand; to thereby induce the expression of a protein having the function of the therapeutic protein (eg, immunomodulator) and / or the protein having the function of IL-12 and to treat the tumor.

In another embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of: (a) intratumorally administering to tumor micro-environments two or more populations of immune cells or TSC, which are engineered in vitro to conditionally express one or more proteins having the function of a therapeutic protein (eg, immunomodulator) and a protein having the function of IL-12, wherein each population of immune cells or TSC expresses a different set of one or more proteins that have the function of a therapeutic protein (eg, immunomodulator), wherein the at least one of proteins that have the function of the therapeutic protein (eg, immunomodulator) or IL-12 is under the control of a conditional promoter that is activated by a ligand; Y (b) administering to the mammal a therapeutically effective amount of one or more activating ligands; to thereby induce the expression of a protein having the function of the therapeutic proteins (eg, immunomodulator) and / or the protein having the function of IL-12 and to treat the tumor.

In another embodiment, the invention provides a method for treating a disease or disorder in a mammal, comprising the steps of: (a) administering to the mammal a population of modified cells, which are modified to conditionally express one or more proteins having the function of a therapeutic protein (eg, immunomodulator); Y (b) administering to the mammal a therapeutically effective amount of an activating ligand; to thereby induce the expression of a protein having the function of the therapeutic protein (eg, immunomodulator) and to treat the disease or disorder.

In another embodiment, the invention provides a method for treating a disease or disorder in a mammal, comprising the steps of: (a) administering to the mammal two or more populations of modified cells, which are modified to conditionally express one or more proteins having the function of a therapeutic protein (eg, immunomodulator), wherein each population of modified cells expresses a set different from one or more therapeutic proteins (eg, immunomodulators); Y (b) administering to the mammal a therapeutically effective amount of one or more activating ligands; to thereby induce the expression of proteins having the function of therapeutic proteins (eg, immunomodulators) and to treat the disease or disorder.

In another embodiment, the invention provides a method for treating a disease or disorder in a mammal, comprising the steps of: (a) administering to the mammal a population of modified cells, which are modified to conditionally express one or more proteins having the function of a therapeutic protein (eg, immunomodulator) and a protein having the function of IL-12, wherein the at least one of the proteins having the function of the therapeutic protein (eg, immunomodulator) or IL-12 is under the control of a conditional promoter that is activated by a ligand; Y (b) administering to the mammal a therapeutically effective amount of the activating ligand; to thereby induce the expression of a protein having the function of the therapeutic protein (eg, immunomodulator) and / or the protein having the function of IL-12 and to treat the disease or disorder.

In another embodiment, the invention provides a method for treating a disease or disorder in a mammal, comprising the steps of: (a) administering to the mammal two or more modified cell populations, which are modified to conditionally express one or more proteins having the function of a therapeutic protein (eg, immunomodulator) and a protein having the function of IL-12, wherein each population of modified cells expresses a different set of one or more proteins having the function of a therapeutic protein (eg, immunomodulator), wherein the at least one of the proteins having the function of the therapeutic protein ( for example, immunomodulator) or IL-12 is under the control of a conditional promoter that is activated by a ligand; Y (b) administering to the mammal a therapeutically effective amount of one or more activating ligands; to thereby induce the expression of a protein having the function of therapeutic proteins (eg, immunomodulators) and / or the protein having the function of IL-12 and to treat the disease or disorder.

The invention also provides a method for determining the efficiency of therapy based on engineered cells, e.g., immune cells or TSC by measuring the level of expression or activity of IFN-gamma in a patient prior to the initiation of therapy, thereby generating mode a level of control, followed by the administration of engineered cells to express one or more proteins having the functions of a therapeutic protein (eg, immunomodulator) and optionally a protein having the function of IL-12, administering an effective amount of an activating ligand, and then measuring the level of IFN-gamma expression to generate a test level, and comparing the level of control to the test level to determine whether the therapeutic regimen is effective.

Additionally included is method for treating a tumor, for reducing the size of a tumor, or for preventing the formation of a tumor in a mammal in need thereof, which comprises (a) administering a therapeutically effective amount of the vector that expresses so conditionally at least one therapeutic protein (e.g., immunomodulatory), e.g., IL-12, TNF-alpha, in the mammal, (b) administering to the mammal a therapeutically effective amount of one or more activating ligands, wherein the activating ligand activates the expression of the protein having the function of the therapeutic protein (eg, immunomodulator), thus inducing the expression of the protein having the function of the therapeutic protein (eg, immunomodulator) and to treat the tumor.

In one embodiment, the invention provides a method for determining the efficiency of a therapeutic regimen based on cells engineered in vitro, eg, immune cells or TSC, in a patient, comprising: (a) measuring the level of expression or the level of activity or both of interferon-gamma (IFN-gamma) in a first biological sample obtained from the patient in need thereof before the administration of cells engineered in vitro, for generate in this way a level of control; (b) administering to a patient in need thereof the cells engineered in vitro, engineered to conditionally express one or more proteins having the functions of a therapeutic protein (eg, immunomodulator) and optionally a protein having the function of IL-12; (c) administering to the patient in need thereof an amount of an activating ligand; (d) measuring the level of expression or activity level or both of IFN-gamma in a second biological sample obtained from the patient in need of the same after administration of immune cells engineered in vitro and activating the ligand, generating in this way a test level; Y (e) comparing the level of control at the IFN-gamma test level, wherein an increase in the level of test expression, activity or both of IFN-gamma relative to the control level indicates that the therapeutic regimen is effective in the patient in need of the same.

In one embodiment, the invention provides a method for treating a tumor, reducing the size of a tumor, or preventing the formation of a tumor in a mammal in need thereof, comprising: (a) administering a vector intratumorally to tumor microenvironments to conditionally express proteins having the functions of one or more therapeutic proteins (eg, immunomodulators), the vector comprising a polynucleotide having a gene change, wherein the polynucleotide comprises (1) at least one factor sequence of transcription that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide that encodes one or more proteins that have the function of a therapeutic protein (eg, immunomodulator) operably linked to a promoter that is activated by the transcription factor of ligand slope, wherein the one or more therapeutic proteins (eg, immunomodulators) are selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL -9, IL-10R DN or a subunit thereof, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, IFN-alpha 1, IFN-alpha 2, IL-15-R-alpha, CCL3 (MIP-la), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCLIO (IP-10), CXCL12 (SDF-1), CCL21 (6Cinema), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, Flt3L, IFN-beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1 RNAi, an antisense oligonucleotide of PD-L1, TGFbRII DN, ICOS-L, S100, CD40L, OX40L, p53, survivin, fusion of p53-survivin, MAGE3, PSA and PSMA, wherein the vector is not contained within a cell; and (b) administering to the mammal a therapeutically effective amount of one or more activating ligands; thereby inducing the expression of one or more proteins having the functions of the therapeutic protein (eg, immunomodulator) and to treat the tumor.

The present invention also provides a method for treating a disease in a mammal in need thereof, comprising: (a) administering to the mammal a vector for conditionally expressing proteins, the vector comprising a polynucleotide encoding a gene change wherein the polynucleotide comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and ( 2) a polynucleotide that codes for one or more proteins operably linked to a promoter that is activated by the ligand-dependent transcription factor, wherein the vector is not contained within a cell; and (b) administering to the non-human animal a therapeutically effective amount of one or more activating ligands; thus inducing the expression of the one or more protein proteins and to treat the disease.

The present invention also provides a method for treating a lysosomal storage disorder in a mammal in need thereof, comprising: (a) administering to the mammal a vector for conditionally expressing one or more proteins, the vector comprising a polynucleotide encoding for a gene change, wherein the polynucleotide comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a dependent transcription factor. of ligand, and (2) a polynucleotide encoding one or more proteins operably linked to a promoter that is activated by the ligand-dependent transcription factor, wherein the vector is not contained within a cell prior to administration in vivo.; and (b) administering to the mammal a therapeutically effective amount of one or more activating ligands; thereby inducing the expression of the one or more proteins and for treating the lysosomal storage disorder.

The present invention also provides a method for treating a liver disease in a mammal in need thereof, comprising: (a) administering to the mammal a vector for conditionally expressing proteins, the vector comprising a polynucleotide that codes for a change gene, wherein the polynucleotide comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and ( 2) a polynucleotide that encodes one or more proteins operably linked to a promoter that is activated by the ligand-dependent transcription factor, wherein the vector is not contained within a cell prior to in vivo administration; and (b) administering to the non-human animal a therapeutically effective amount of one or more activating ligands; thereby inducing the expression of the one or more proteins and to treat liver disease.

Brief Description of the Figures Figure 1 shows a plasmid map for a regulated promoter expression system, for a bicistronic transcript encoding hIL-12.

Figure 2 shows a plasmid map for a regulated promoter expression system for a bicistronic transcript encoding hIL-21 and hIL-15.

Figure 3 shows a plasmid map for a regulated promoter expression system for a bicistronic transcript encoding mIL-12.

Figure 4 shows a plasmid map for a regulated promoter expression system for a bicistronic transcript encoding mIL-21 and mIL-15.

Figure 5 shows a plasmid map for a regulated promoter expression system for hIL-21.

Figure 6 shows a plasmid map for a regulated promoter expression system for mIL-21.

Figure 7 shows a plasmid map for. a regulated promoter expression system for a tricistronic transcript encoding hIL-12 and hIL-21.

Figure 8 shows a plasmid map for a regulated promoter expression system for a tricistronic transcript encoding mIL-12 and mIL-21.

Figure 9 shows the structure of the vector rAd.RheoIL12 in which the El and E3 regions have been deleted and the components (RTS) -IL-12 of the RheoSwitch® Therapeutic System, replace the El region. The box marked "IL 12" represents the coding sequences of IL-12p40 and IL-12p35, separated by IRES.

Figure 10 shows a summary chart for translation, post-transcription and post-translation and translation processes.

Figure 11 shows modular elements with representation of production pivots.

Figure 12 shows a schematic diagram of the fundamental structure ULTRA VECTORR compatible with adenovirus with synthetic gene modulated inducible and exchange.

Figure 13 shows an adenoviral vector map (Vector 43318) for a regulated promoter expression system comprising TNFwt 5'UTR, TNFwtUV signal peptide, open reading frame (ORF) of TNFwt UV , and the 3 'regulatory region of SV40e + pA.

Figure 14 shows an adenoviral vector map (Vector 43319) for a regulated promoter expression system comprising TNFwt 5'UTR, TNFoptUV signal peptide, TNFopt UV ORF, and 3 'regulatory region of SV40e + pA.

Figure 15 shows an adenoviral vector map (Vector 43320) for a regulated promoter expression system comprising TNFwt 5'UTR, IL-2optUV signal peptide, TNFoptUV ORF, and 3 'regulatory region of SV40e + pA.

Figure 16 shows an adenoviral vector map (Vector 43321) for a regulated promoter expression system comprising 5U2 5'UTR, TNFwtUV signal peptide, TNFwtUV ORF, and 3 'regulatory region of SV40e + pA.

La Fugura 17 shows an adenoviral vector map (Vector 43322) for a regulated promoter expression system comprising 5U2 5'UTR, TNFoptUV signal peptide, TNFoptUV ORF, and 3 'regulated region of SV40e + pA.

Figure 18 shows an adenoviral vector map (Vector 43323) for a regulated promoter expression system comprising 5U2 5'UTR, IL-2optUV signal peptide, TNFoptUV ORF, and 3 'regulated region of SV40e + pA.

Figure 19 shows an adenoviral vector map (Vector 43324) for a regulated promoter expression system comprising TNFwt 5'UTR, TNFwtUV signal peptide, TNFwtUV ORF, and 3 'regulated region of hGH + pA.

Figure 20 shows an adenoviral vector map (Vector 43325) for a regulated promoter expression system comprising TNFwt 5'UTR, TNFoptUV signal peptide, TNFoptUV ORF, and 3 'regulated region of hGH + pA.

Figure 21 shows an adenoviral vector map (Vector 43326) for a regulated promoter expression system comprising TNFwt 5'UTR, IL-2optUV signal peptide, TNFoptUV ORF, and 3 'regulated region of hGH + pA.

Figure 22 shows an adenoviral vector map (Vector 43327) for a regulated promoter expression system comprising 5U2 5'UTR, TNFwtUV signal peptide, TNFwtUV ORF, and the 3-regulated region of hGH + pA.

Figure 23 shows an adenoviral vector map (Vector 43328) for a regulated promoter expression system comprising 5U2 5'UTR, TNFwtUV signal peptide, TNFwtUV ORF, and the 3 'regulated region of hGH + pA.

Figure 24 shows an adenoviral vector map (Vector 43329) for a regulated promoter expression system comprising 5U2 5'UTR, TNFwtUV signal peptide, TNFwtUV ORF, and the 3 'regulated region of hGH + pA.

Figure 25 shows an adenoviral vector map (Vector 43533) for a regulated promoter expression system comprising TNFwt5 'UTR, TNFwtUV signal peptide, TNFwtUV ORF, and TNFwt 3' UTR.

Figure 26 shows an adenoviral vector map (Vector 43534) for a regulated promoter expression system comprising TNFwt 5'UTR, full length ORF of TNFwt 3 'UTR.

Figure 27 shows a graph showing normalized levels of secreted TNF-alpha proteins after transfection of HEK293 cells with vectors with induction of PT3 (- / +) components varied by RHEOSWITCH ™ ligand.

Figure 28 shows a graph representing the normalized levels of secreted TNF-alpha protein after transfection of CHO-Kl cells with vectors with induction of PT3 (- / +) components varied by the ligand RHEOSWITCH ™.

Figure 29 shows the times of difference in TNF-alpha secretion after transfection of HEK293 cells.

Figure 30 shows the differences in protein secretion between 5U2 5'UTR and wtUV TNF-alpha 5'UTR.

Figure 31 is a line graph showing a dose response study of Ad-RTS-IL-12 in the mouse B16F0 melanoma model. Mice were treated on day 12 with a single injection of Ad-RTS-IL-12 at lee7 dose; lee8; read9; 5ee9; read them; 5eel0 viral peptides (vp)) and the ligand was distributed using food starting on day 11. The x axis shows days after inoculation of tumor cells and the y axis indicates tumor volume. The dose levels showed substantial anti-tumor effect. The percentage of tumor reduction compared to the control is indicated.

Figure 32 is a line graph showing the changes in body weight during the course of the study. The changes are shown as% body weight on the y axis.

Figures 33A and 33B are linear graphs showing the antitumor activity (Figure 33A) and safety (Figure 33B) of Ad-RTS-mIL12 in the Lewis mouse lung carcinoma (LLC) model. The Lewis lung tumor was cultured subcutaneously in immunocomponent C57b / 6 mice. When the tumor reached the desirable size, treatment was started. The animals received a single dose of AdRTS-mIL12 (lelO vp) on Day 6, 9, 13 after inoculation of tumor cells. Ad-RTS-mILl2 with activator showed marked antitumor activity in relation to the control animals. No major toxicity was perceived.

Figures 34A and 34B are linear graphs showing the efficiency (Figure 34A) and safety (Figure 34B) in a mouse melanoma model (B16F0) in which animals were treated with Ad-RTS-mIL12. The tumor s.c. on the flank of C57b / 6 mice. An individual dose of Ad-RTS-mIL12 (lelO vp) administered intratumorally (i.t.) on Day 13 after inoculation of cells (arrows). The activating ligand (50, 100, 250, 500, 750 and 1000 mg / kg) was given 24 hours before the injection of the vector until the end of the experiment. The inhibition of tumor growth is indicated as a percentage and compared to control animals. Ad-RTS-IL12 showed therapeutic benefit with wide window of activator dose. Treatment was well tolerated .

Figures 35A and 35B are linear graphs showing the efficiency (Figure 35A) and safety (Figure 35B) of Ad-RTS-mIL12 in a mouse colon cancer model (CT26Luc). Murine colon tumor was cultured subcutaneously in the flank region of Balb / C mice. Mice were treated intratumorally (i.t.) twice with Ad-RTS-mIL12 at a dose level of 10 vp / 100ul leiO at 11 and 18 after cell inoculation (arrows). Activation started 24 hours before the injection of the vector. Tumor volume and body weight were monitored. Throughout the entire experiment. The treatment with Ad-RTS-mIL12 led led to outstanding inhibition of tumor growth (100%) in relation to control animals. Notably, all animals treated with Ad-RTS-mIL12 plus activators were tumor free.

Figures 36A and 36B are linear graphs showing the efficiency (Figure 36A) and safety (Figure 36B) of Ad-RTS-mIL12 in a pancreatic cancer model (PA 02). Mice having subcutaneous PAN02 tumor were treated intratumorally (i.t.) with a single dose of Ad-RTS-mIL12 at a dose level of 10 vp / 100ulle on Days 7 and 14 (arrow) after inoculation with tumor cells. Activator feed was fed to animals one day before administration of the vector until the end of the experiment. The control group or with vector only received normal rodent food. The result suggests that the Ad-RTS-IL12 has a significant antitumor activity (97%) in relation to control animals, no major changes in body weight were found as a result of conical therapy Ad-RTS-IL12.

Figure 37 is a vector map for AD-RTS-mlFN-alpha.

Figure 38 is a vector map for AD-RTS-mTNF-alpha.

Figures 39A and 39B are linear graphs showing the efficiency (Figure 39A) and safety (Figure 39B) of Ad-RTS-mIL12 in a breast cancer model (4T1). 4T1 tumors were cultured subcutaneously (s.c.) on the flank of BALB / C mice. the mice that had tumor were randomized into four groups with 5 animals each; control without treatment, activator ligand (L) alone, Ad-RTS-mIL12 alone and Ad-RTS-mIL12 with ligand activator. An individual injection of Ad-RTS-mIL12 was given intratumorally (i.t.) at a dose level of 10?.? .// ???? PBS in three different time points (arrow). The activating ligand was given to mice through feed 24 hours before the injection of the vector until the end of the experiment. Tumor sizes (volume) and body weights (%) are shown as mean + SE. Animals without treatment (control) have rapid tumor growth. Treatment with activating ligand alone or three doses of Ad-RTS-mIL12 without ligand activator have slight inhibition of tumor growth of 22% and 35% respectively in relation to the control. Notably, treatment with Ad-RTS-IL12 plus activator ligand leads to significant inhibition of tumor growth of 82% compared to untreated control animals. No major loss of body weight was found in any treatment.

Figure 40 is a vector map for Interferon alfa-2a.

Detailed Description of Sequences Therapeutic Proteins Cytokines The polynucleotide sequences of interleukin 1 (IL-1), which are important cytokines for the inflammatory response against infection, are available from public databases as access numbers M28983 (human IL-1); M15330 (human IL1); AF201830 (IL-? D human); AF201831 (IL-? E human); AF201832 (IL- ?? human); AF201833 (human IL-ln); NM_010554 (mouse IL-α); NM 008361 (mouse IL-? ß); NM_019451 (Mouse L-168); NM_019450 (IL-lf6 of mouse); N _027163 (mouse IL-1β8); NMJ53511 (mouse IL-lf9); NM_204524 (IL-? ß chicken); NM_017019 (rat IL-α); and NM 031512 (rat ILip), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 1 (IL-1) are available from public databases as access numbers AAA59134 (IL-? Human); AAA59135 (human IL-? ß); AAF25210 (IL-? D human); AAF25211 (human IL-e); AAF25212 (IL- ?? human); AAF25213 (IL- ?? human); NP_034684 (mouse IL-α); NP_032387 (mouse IL-? ß); NP_062324 (Mouse L-? D); NP_062323 (mouse IL-lf6); NP_081439 (mouse IL-lf8); NP 705731 (mouse IL-lfP); NP_989855 (IL-? ß chicken); NP 058715 (IL-rat); and NP_ 113700 (rat IL-1β), sequences of which are incorporated by reference herein. Laurent et al, Psychiatr. Genet 7: 103 (1997) identified polymorphic mutations in the human interleukin-1 beta gene.

The polynucleotide sequences of interleukin-2 (IL-2), which belongs to a family including IL-4, IL-7, IL-9, IL-15, and IL-21, are available from public databases as numbers of access U25676 (human); NM 008366 (mouse); NM_204153 (chicken); and NM_053836 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 2 (IL-2) are available from public databases as access numbers AAA70092 (human); NP_032392 (mouse); NP_989484 (chicken); and NP_446288 (rat), sequences of which are incorporated by reference herein.

Liu et al, Appl. Biochem. Biotechnol. 133: 11 (2006) generated mutant human IL-2, and Lorberboum et al, J. Biol. Chem. 265: 16311 (1990) describe the generation of chimeric IL-2.

The polynucleotide sequences of interleukin 4 (IL-4), which is a cytokine that induces differentiation of intact helper T cells to T cells to Th2, are available from public databases as access numbers M23442 (human); NM_021283 (mouse); NM_001007079 (chicken); and NM_201270 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 4 (IL-4) are available from public databases as access numbers AAA59150 (human); NP_067258 (mouse); NP_001007080 (chicken); and NP_958427 (rat), sequences of which are incorporated by reference herein.

Kawashima et al., J. Med. Genet. 35: 502 (1998) describe the polymorphisms in the IL-4 gene, which are associated with atopic dermatitis.

Interleukin 7 (IL-7) is an important cytokine for the development of B and T cells. The polynucleotide sequences of IL-7 are available from public databases as access numbers J04156 (human); NM_008371 (mouse); NM_001037833 (chicken); and NM_013110 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 7 (IL-7) are available from public databases as access numbers AAA59156 (human); NP_032397 (mouse); NP_001032922 (chicken); and NP_037242 (rat), sequences of which are incorporated by reference herein.

Feng et al, Genetics 175: 545 (2007) have identified point mutations in IL-7 that result in functional deficiency.

Interleukin 9 (IL-9) is a cytokine produced by T cells and is a regulator of hematopoietic cells. The polynucleotide sequences of IL-9 are available from public databases as access numbers NM_000590 (human); M_008373 (mouse); N _001037825 (chicken); and M_001105747 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 9 (IL-9) are available from public databases as access numbers NP_000581 (human); NP 032399 (mouse); NP_001032914 (chicken); and NP_001099217 (rat), sequences of which are incorporated by reference herein.

IL-12 is a cytokine that can act as a growth factor for activated T and NK cells, to enhance the lytic activity of NK / lymphokine-activated killer cells, and to stimulate the production of IFN-gamma by the rest of peripheral blood mononuclear cells (PBMC). The polynucleotide sequences of IL-12 are available from public databases as access numbers NM_000882 (human IL12A); NM_002187 (human IL12B); NM_008351 (mouse IL12a); NM_008352 (mouse IL 12b); NM_213588 (chicken IL 12A); NM_213571 (chicken IL12B); NM_053390 (rat IL12a); and NM_022611 (rat IL 2b), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 12 (IL-12) are available from public databases as access numbers NP_000873 (human IL 12A); NP_002178 (human IL12B); NP_032377 (mouse IL 2a); NP_032378 (mouse IL12b); NP_998753 (chicken IL12A); NP_998736 (chicken IL12B); NP_445842 (rat IL 2a); and NP_072133 (rat IL 12b), sequences of which are incorporated by reference herein.

Interleukin 15 (IL-15) is a cytokine that regulates the activation and proliferation of T cells and natural killer cells. The polynucleotide sequences of IL-15 are available from public databases as access numbers U14407 (human); M_008357 (mouse); EU334509 (chicken); and AF015719 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 15 (IL-15) are available from public databases as access numbers AAA21551 (human); NP_032383 (mouse); ABY55312 (chicken); and AAB94536 (rat), sequences of which are incorporated by reference herein.

Interleukin 18 (IL-18), a cytokine produced by macrophages that together with interleukin 12 induce cell-mediated immunity after infection with microbial products. The polynucleotide sequences of IL-18 are available from public databases as access numbers U90434 (human), - M_008360 (mouse), -EU747333 (chicken) and AY258448 (rat), sequences of which are incorporated by reference in the present.

The amino acid sequences of interleukin 18 (IL-18) are available from public databases as access numbers AAB50010 (human); NP_032386 (mouse); ACE79188 (chicken); and AAP14669 (rat), sequences of which are incorporated by reference herein.

The polynucleotide sequences of interleukin 21 (IL-21), which is a cytokine that has potent regulatory effects on cells of the immune system, include natural killer cells and cytotoxic T cells, by induction of cell proliferation, are available from the public databases as access numbers AF254069 (human); NM_021782 (mouse); NM_001024835 (chicken); and NM_001108943 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 21 (IL-21) are available from public databases as access numbers, AAG29348 (human); NP_068554 (mouse); NP_001020006 (chicken); and NP_001102413 (rat), sequences of which are incorporated by reference herein.

Interleukin 27 (IL-27) is a cytokine that plays an important role in regulating the activity of B and T lymphocytes. The polynucleotide sequences of IL-27 are available from public databases as access numbers AY099296 (human) ); NM 145636 (mouse); and XM_344962 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interleukin 27 (IL-27) are available from public databases as access numbers AAM34498 (human); NP 663611 (mouse); and XP 344963 (rat), sequences of which are incorporated by reference herein.

The polynucleotide sequences of interferon beta 1 (IFNBl), which is a member of the group of interferon proteins that bind to specific cell surface receptors (IFNAR), and stimulates macrophages and natural killer (NK) cells to promote an antiviral response, they are available from public databases as access numbers M_002176 (human); NM_010510 (mouse); N _001024836 (chicken); and NM_019127 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interferon beta 1 (IFNBl) are available from public databases as access numbers NP_002167 (human); NP_034640 (mouse); NP_001020007 (chicken); and NP_062000 (rat), sequences of which are incorporated by reference herein.

Gamma interferon (IFN-gamma) is a soluble cytokine that is the only Type II interferon and has anti-viral, immunoregulatory and antitumor activity. The polynucleotide sequences of IFN-gamma are available from public databases as access numbers NM_000619 (human); NM_008337 (mouse); and NM_138880 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of interferon gamma (IFN-gamma) are available from public databases as access numbers NP_000610 (human); NP 032363 (mouse); and NP_620235 (rat) sequences of which are incorporated by reference herein.

The polynucleotide sequences of tumor necrosis factor (TNF-alpha), which is a multifunctional pro-inflammatory cytokine secreted predominantly by monocytes / macrophages that has effects on lipid metabolism, coagulation, insulin resistance, and endothelial function, are available from public databases as access numbers X02910 (human); NM_013693 (mouse); and BCl 07671 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of TNF-alpha are available from public databases as access numbers CAA26669 (human); NP_038721 (mouse); and AAI07672 (rat), sequences of which are incorporated by reference herein.

Human TNF-alpha (abbreviated herein as hTNF-alpha, or simply hTNF) is a human cytokine that exists as a soluble form of 17kD (sTNF-alpha) and a membrane-associated form of 26 kD (tmTNF-alpha), the biologically active form of which is composed of a trimer of 17kD molecules not covalently bound. The structure of hTNF-alpha is described, for example, in Pennica, D., et al. (1984) Nature 312: 724-729; Davis, J. , et al. (1987) Biochemistry 26: 1322-1326; and Jones, E. Y., et al. (1989) Nature 338: 225-228. TNF-alpha can bind TNF-1 receptor (TNFR-1) or TNF-2 receptor (TNFR-2) and is involved in the regulation of immune cells, in the induction of apoptosis or inflammation, or in inhibition of tumorigenesis or viral replication. Cell signaling cascades produced by the binding of TNF / TNFR are described, for example, in Wajant, H., et al. (2003) Cell Death Differ. 10 (1): 45-65 or Chen, G., et al. (2002) Science 296: 1634-5.

The TNF-alpha polypeptide, human, full-length consists of a cytoplasmic domain, a transmembrane domain, and an extracellular domain. A polypeptide sequence of 233aa was reported as a polypeptide sequence of human TNF-alpha and is designated herein as SEQ ID NO: 37, which has a cytoplasmic domain of amino acids 1-35 of SEQ ID NO: 37, a transmembrane domain of amino acids 36-56 of SEQ ID NO: 37, and an extracellular amino acid domain 57-233 of SEQ ID NO: 37. SEQ ID NO: 37 is a nucleotide sequence coding for SEQ ID NO: 35 or 36. The variants of human TNF-alpha include, but is not limited to, polypeptides with one or more of the following mutations: L105S, R108W, L112F, A160V, S162F, V167A, E222K, F63S, PSD84 -86VNR, or E183R.

Chemokines Chemokine ligand 1 (portion C) (XCL1, also known as lymphotatin) is chemotactic for CD4 + and CD8 + T cells, but not for monocytes, and induces an elevation in intracellular calcium in peripheral blood lymphocytes. The polynucleotide sequences of XCL1 are available from public databases as access numbers NM_002995 (human); NM_008510 (mouse); and NM_134361 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of XCL1 are available from public databases as access numbers NP_002986 (human); NP_032536 (mouse); and NP_599188 (rat), the sequences of which are incorporated by reference herein. U.S. Patent No. 6,022,534 describes lymphotactin and the use of either to attract cytotoxic T cells, and / or NK cells, and / or to induce the proliferation of resident cells. Methods for the isolation and use of an anti-lymphotactin antibody, and the XCL1 fusion protein are also described.

The polynucleotide sequences of chemokine CC ligand 3 (CCL3), also known as macrophage inflammatory protein 1 (MIP-1), which is a so-called monoclin (a type of cytokine produced mainly by monocytes and macrophages), which is involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes, are available from public databases as access numbers _002983 (human); _011337 (mouse); and NM_013025 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of CCL3 are available from public databases as access numbers NP_002974 (human); NP_035467 (mouse); and NP_037157 (rat), sequences of which are incorporated by reference herein.

The polynucleotide sequences of CCL5 (RANTES), which is a pro-inflammatory cytokine involved in inflammation and asthma, are available from public databases as access numbers AF043341 (human); M_013653 (mouse); and NM_031116 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of CCL5 are available from public databases as access numbers AAC03541 (human); NP_038681 (mouse); and NP_112378 (rat), sequences of which are incorporated by reference herein.

The polynucleotide sequences of chemokine CC ligand 7 (CCL7), which is a chemokine involved in the recruitment of macrophages during inflammation and cancer invasion, are available from public databases as access numbers M_006273 (human); M_013654 (mouse); and NM_001007612 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of CCL7 are available from public databases as access numbers NP_006264 (human); NP_038682 (mouse); and NP_001007613 (rat), sequences of which are incorporated by reference herein.

Chemokine ligand 9 (CXC portion) (CXCL9, also known as MIG) is a T cell chemoattractant inducible by interferon gamma. The polynucleotide sequences of CXCL9 are available from public databases as access numbers NM_002416 (human); M_108599 (mouse); and NM_145672 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of CXCL9 are available from public databases as access numbers NP_002407 (human); NP_032625 (mouse); and NP_663705 (rat), sequences of which are incorporated by reference herein.

Ligand 10 of chemokine (portion C-X-C) (CXCL10) is a small cytokine with functions in chemoattraction for cells in the immune system, adhesion of T cells to endothelial cells, antitumor activity and angiogenesis. The polynucleotide sequences of CXCL10 are available from public databases as access numbers X02530 (human); NM_021274 (mouse); and BC058444 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of chemokine ligand 10 (portion C-X-C) (CXCL10) are available from public databases as access numbers CAA26370 (human); NP_067249 (mouse); and AAH58444 (rat), sequences of which are incorporated by reference herein.

Chemokine ligand 12 (portion C-X-C) (CXCL12), also known as factor 1 derived from stromal cells (SDF-1), is a small cytokine that belongs to the family of intercrines, members of which activate leukocytes and are often induced by pro-inflammatory stimuli such as LPS, TNF or ILL. The polynucleotide sequences of CXCL12 are available from public databases as access numbers NM_000609 (human); NM_001012477 (mouse); NM_204510 (chicken); and M_001033883 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of CXCL 12 are available from public databases as access numbers NP_000600 (human); NP_001012495 (mouse); NP_989841 (chicken); and NP_001029055 (rat), sequences of which are incorporated by reference herein.

Hansson et al, Microbes and Infection 8: 841 (2006) discuss that the interaction between chemokine receptor 7 (CC portion) (CCR7) and chemokine ligand 19 (CC portion) (CCL19, also known as ??? - 3ß) is crucial for the generation of primary immune responses. The polynucleotide sequences of CCR7 are available from public databases as access numbers _001838 (human); and M_007719 (mouse), sequences of which are incorporated by reference herein.

The amino acid sequences of CCR7 are available from public databases as access numbers NP_001829 (human); and NP_031745 (mouse), sequences of which are incorporated by reference herein.

The polynucleotide sequences of CCL19 are available from public databases as access numbers M_006274 (human); and NM_011888 (mouse), sequences of which are incorporated by reference herein.

The amino acid sequences of CCL19 are available from public databases as access numbers NP_006265 (human); and NP_036018 (mouse), sequences of which are incorporated by reference herein.

The polynucleotide sequences of CC chemokine 21 ligand (CCL21), a well-established ligand for CCR7, which is necessary for CD4 + T cells but not for CD8 + T cells to reach their "resting point" of resting state and perturbations in the expression of CCL21, can alter susceptibility to autoimmunity, are available from public databases as access numbers AB002409 (human); M_011335 (mouse CCL21a); NM_0111124 (mouse CCL21b); and NM_023052 (mouse CCL21c); sequences of which are incorporated by reference herein.

The amino acid sequences of CCL21 are available from public databases as access numbers BAA21817 (human); NP_035465 (mouse CCL21a); NP_035254 (mouse CCL21b); and NP_075539 (from mouse CCL21c), sequences of which are incorporated by reference herein.

Interleukin-8 (IL-8), a chemokine, also called neutrophil activation peptide 1 or SCYB8, is a peptide derived from tissues secreted by various cell types in response to inflammatory stimuli. U.S. Patent Nos. 6,133,426 and 6,177,980 describe the amino acid and polynucleotide sequences of humanized anti-IL-8 antibodies. The polynucleotide sequence of human IL-8 is available from public databases as accession number NM_000584, sequence of which is incorporated by reference herein.

The amino acid sequence of human IL-8 is available from public databases as accession number NP_000575, sequence of which is incorporated by reference herein.

Growth factors The granulocyte-macrophage colony stimulation factor (GM-CSF) is a cytokine that functions as a growth factor for white blood cells, stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. The polynucleotide sequences of GM-CSF are available from public databases as access numbers M111734 (human); NM_009969 (mouse), · EU520303 (chicken); NM_001037660 (rat Csf2ra); and NM_133555 (rat Csf2rb), sequences of which are incorporated by reference herein.

The amino acid sequences of the granulocyte / macrophage colony stimulation factor (GM-CSF) are available from public databases as access numbers AAA52122 (human); NP_034099 (mouse); ACB11534 (chicken); NP_001032749 (rat Csf2ra); and NP_598239 (Csf2rb), sequences of which are incorporated by reference herein.

The polynucleotide sequences of the tyrosine kinase ligand related to FMS (ligand FLT3 / FLK2, Flt3L), which can function as a growth factor receptor on hematopoietic stem cells or progenitor cells or both, are available from the databases public as access numbers U04806 (human); and M_013520 (mouse), sequences of which are incorporated by reference herein.

The amino acid sequences of the FLT3 / FLK2 ligand (Flt3L) are available from public databases as access numbers AAAl 7999 (human); and NP_038548 (mouse), sequences of which are incorporated by reference herein.

The polynucleotide sequence of transforming growth factor, alpha (TGF-alpha), which is supra-regulated in some human cancers, can reversibly confer the transformed phenotype on cultured cells, is available from public databases as access numbers NM_001099691 (human); NM_031199 (mouse), - NM_001001614. (of chicken); and M_012671 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of TGF-alpha are available from public databases as access numbers NP_001093161 (human); NP_112476 (mouse); NP_001001614 (chicken); and NP_036803 (rat), sequences of which are incorporated by reference herein. Adjuvants Beta-defensins are antimicrobial peptides involved in the innate immune response against many Gram-negative and Gram-positive bacteria, fungi and viruses. The polynucleotide sequences of beta-defensins are available from public databases as access numbers X92744 (human hBD-1); AJ000152 (human hBD-2); AF217245 (human beta defensin-3); AJ314835 (human beta defensin-4); AB089180 (human hBD-5); AY122466 (human beta 106 defensin, DEFB106); AF540979 (human beta defensin 107, DEFB 107); AF529416 (human beta defensin, DEFB 108); DQ012014 (human beta defensin 110, DEFB110); DQ012015 (human beta defensin 111, DEFB111); DQ012016 (human beta defensin 112, DEFB112); DQ012017 (human defensin beta 113, DEFB113); DQ012018 (human beta defensin 114, DEFBl 14); DQ012019 (human defensin beta 115, DEFB 115); COD 12020 (human defensin beta 116, DEFBl 16); DQ012021 (human beta defensin 117, DEFBl 17); M_007843 (mouse beta 1 defensin); NM_010030 (mouse beta 2 defensin, Defb2); NM_013756 (mouse beta 3 defensin, Defb3); NM_019728 (mouse beta4 defensin, Defb4); NM_030734 (mouse beta 5 defensin, Defb5); NM_054074 (mouse beta 6 defensin, Defb6); N _139220 (mouse beta 7 defensin); NM_153108 (mouse beta 8 defensin, Defb8); _139219 (mouse beta 9 defensin, Defb9); and NM_139225 (mouse beta 10 defensin, DefblO); sequences of which are incorporated by reference herein.

The amino acid sequences of beta-defensins are available from public databases as access numbers CAA63405 (human hBD-1); CAB65126 (human hBD-2); AAF73853 (human beta defensin-3); CAC85520 (human beta defensin-4); BAC10630 (human hBD-5); AAM93908 (human beta 106 defensin, DEFB106); AA 33115 (human beta defensin 107, DEFB 107); AAQ09525 (human beta defensin, DEFB 108); AAY59750 (human beta defensin 110, DEFBl 10); AAY59751 (human beta defensin 111, DEFBl 11); AAY59752 (human beta defensin 112, DEFBl 12); AAY59753 (human beta defensin 113, DEFBl 13); AAY59754 (human beta defensin 114, DEFBl 14) AAY59755 (human defensin beta 115, DEFB115); AAY59756 (human defensin beta 116, DEFB116); AAY59757 (human beta defensin 117, DEFB117); NP 031869 (mouse beta 1 defensin); NP_034160 (mouse beta 2 defensin, Defb2); NP_038784 (mouse beta 3 defensin, Defb3); NP_062702 (mouse beta4 defensin, Defb4); NP_109659 (mouse beta 5 defensin, Defb5), · NP_473415 (mouse beta 6 defensin, Defb6); NP_631966 (defensin beta 7 mouse mouse, Defb7); NP_694748 (mouse beta 8 defensin, Defb8); NP_631965 (mouse beta 9 defensin, Defb9); and NP_631971 (mouse beta 10 defensin, DefblO), sequences of which are incorporated by reference herein. See also U.S. Patent No. 5,242,902 for additional human and rat peptide sequences.

The proteins in Table 1 of the high mobility group (HMGBl) are non-histone chromosomal proteins that function as cytokines, mediating local and systemic responses to necrotic cell death and cancer, invasion by pathogens, trauma and sepsis. The polynucleotide sequences of the HMGB1 proteins are available from public databases as access numbers NM_002128 (human); M_010439 (mouse); NM_204902 (chicken); and NM_012963 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of table 1 of the high mobility group (HMGBl) are available from public databases as access numbers NP 002119 (human); NP_034569 (mouse); NP_990233 (chicken); and NP_037095 (rat), sequences of which are incorporated by reference herein.

S100 phagocytic proteins are mediators of inflammatory responses and recruit inflammatory cells to sites of tissue damage, and are members of the molecules of the molecular pattern associated with damage (DAMP) that are important for innate immunity. See Foell et al, J. Leukocyte Biol. 81: 1 (2006). The polynucleotide sequences of S100 proteins are available from public databases as access numbers BCO 14392 (S100 Al human); BC002829 (human S100 A2); BC012893 (human S100 A3); BC016300 (human S100 A4); Z 18954 (human S100D); BC001431 (human S100 A6); BC 034687 (human S100 A7); BC005928 (human S100 A8); BC047681 (human S100 A9); BC015973 (human S100 A10); D38583 (human clagizzarin); NM_011309 (SlOOal of mouse); NM_009115 (SlOOb of mouse); NM_013650 (mouse S100a8); NM_009114 (mouse S100a9); NM_011310 (mouse S100a3); NM_011311 (mouse S100a4); and NM_011312 (mouse S100a5), sequences of which are incorporated by reference herein.

The amino acid sequences of S100 proteins are available from public databases as access numbers AAH14392 (S100 Al human); AAH02829 (human S100 A2); AAH12893 (human S100 A3); AAH16300 (human S100 A4); CAA79479 (human S100D); AAHO 1431 (human S100 A6); AAH34687 (human S100 A7); AAH05928 (human S100 A8); AAH47681 (human S100 A9); AAH15973 (human S100 A10); BAA07597 (human clagizzarin); NP_035439 (SlOOal of mouse); NP_033141 (SlOOb of mouse); NP_038678 (mouse S100a8); NP_033140 (mouse S100a9); NP_035440 (mouse S100a3); NP_035441 (mouse S100a4); and NP_035442 (mouse S100a5), sequences of which are incorporated by reference herein.

Manana, a plant polysaccharide, which is a sugar polymer, is useful for the generation of an immune response. U.S. Patent No. 5,807,559, describes the immunogenic conjugates of the morning meal which may be useful for generating immunity of T cells against carbohydrate cultures associated with the tumor, or against carbohydrate structures expressed on infectious agents and / or infected host cells U.S. Patent No. 5,773,425 describes the use of the morning meal to alleviate symptoms and / or cure viral diseases and to improve the immune response.

Calmette-Guerin bacilli (BCG), live attenuated Mycobacterium species, are used as vaccines to prevent severe and fatal tuberculosis. U.S. Patent No. 7,393,541 describes the generation of an adjuvanted vaccine to produce a T cell-mediated immune response in vivo to a mycobacterium in a mammalian subject. See also Hubbard and Collins, Infect. Immun. 59 (2): 570. U.S. Patent No. 5,292,513 discloses a method for priming macrophages in vivo in patients in need of enhanced bactericidal and antiviral activity, with heat-killed BCG. The complete genome sequence of BCG is available from the public databases as access number NC_008769 (M bovis BCG str Pasteur 1173P2, full genome).

Bacterial lipopolysaccharides (LPS) are endotoxins that induce a strong immune response after infection with Gram-negative bacteria. U.S. Patent No. 4,148,877 describes the fractionation of LPS from bacterial culture and the use of the fraction as a drug to induce resistance to bacterial infection. U.S. Patent No. 5,292,513 discloses a method for priming macrophages in vivo, in patients in need of enhanced bactericidal and anti-viral activity, with LPS.

Co-stimulatory molecules (Positive) Ligand 0X 0 (0X4OL) belongs to member 4 of the tumor necrosis factor (ligand) superfamily (Tnfsf4), is expressed on dendritic cells and promotes Th2 cell differentiation. The polynucleotide sequences of the OX40 ligand are available from public databases as access numbers X79929 (human); U12763 (mouse); and AF037067 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of ligand 0X40 (0X4OL) are available from public databases as access numbers CAA56284 (human); AAA21871 (mouse); and AAC67236 (rat), sequences of which are incorporated by reference herein.

The ligand 4-1BB (4-1BBL) belongs to member 9 of the tumor necrosis factor (ligand) superfamily (Tnfsf9), which is a transmembrane type 2 protein and is expressed on activated T lymphocytes. The polynucleotide sequences of 4-IBBL are available from public databases as access numbers NM 003811 (human); N _009404 (mouse); and AY332409 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of the ligand 4-IBB (4-IBBL) are available from the public databases as access numbers NP_003802 (human); NP_033430 (mouse); and AAQ01228 (rat), sequences of which are incorporated by reference herein.

The CD40 protein belongs to member 5 of the turaoral necrosis factor superfamily, it is essential in the mediation of a wide variety of immune and inflammatory responses, including the change of T cell-dependent immunoglobulin class, the development of memory B cells , and the formation of the germinal center. The polynucleotide sequences of CD40 proteins are available from public databases as access numbers X60592 (human); NM_170701 (mouse); NM_204665 (chicken); and NM_134360 (rat), sequences of which are incorporated by reference herein.

The amino acid sequences of CD40 proteins are available from public databases as access numbers CAA43045 (human); NP_733802 (mouse); NP_989996 (chicken), - and NP_599187 (rat), sequences of which are incorporated by reference herein.

CD40L (ligand CD40, or CD154) is expressed primarily on activated T cells and is a member of the TNF molecule superfamily. It binds to CD40 in cells that present the antigen. CD40L plays the role of a co-stimulatory molecule and induces activation in cells that present the antigen in association with stimulation of the T cell receptor by MHC molecules in the cells that present the antigen. CD40L has three binding partners: CD40, 5β1 integrin and aIIbP3. The CD40L sequences are available from public databases as access numbers MN_000074 and MP_000065 (human and MN_011616 and NP_035746 (mouse).

The protein related to the glucocorticoid-induced tumor necrosis factor receptor (GITR) family can cause effective tumor immunity via T-cell stimulation. Administration of the anti-GITR monoclonal antibody (mAb) can elicit potent tumor-specific immunity , and eradicate established tumors without promoting a patent autoimmune disease. See Ko et al, J. Exp. Med 7: 885 (2005). U.S. Patent No. 6,503,184 Bl describes an anti-GITR antibody.

The polynucleotide sequences of the GITR ligand (GITRL) are available from public databases as access numbers AY358868 (human); and AY359852 (mouse), sequences of which are incorporated by reference herein.

The amino acid sequences of the GITR ligand (GITRL) are available from public databases as access numbers AAQ89227 (human); and AAQ55265 (mouse), sequences of which are incorporated by reference herein.

The binding ligand of the herpes virus entry mediator (HVEM) (HSVgD), also referred to as p30, or LIGHT is a member of the TNF family involved in the co-stimulation of T cells. it has two receptors, the mediator of the herpes virus entry (HVEM) and the lymphotoxin-β receptor (LT-R). Being a ligand for HVEM, HSVgD activates T cells by acting as a co-stimulatory factor for T cells that results in T cell proliferation and cytokine secretion. See U.S. Patent No. 7,118,742 for the polynucleotide and amino acid sequences of LIGHT. U.S. Patent 5,654,174 discloses a variant gD protein with deletion of the carboxyl-terminal residues.

CD70 is a cytokine that binds to CD27. This plays a role in the activation of T cells. It induces the proliferation of co-stimulated T cells and intensifies the generation of cytolytic T cells. The polynucleotide sequences of CD70 are available from public databases as access numbers NM_001252 (human); N _011617 (mouse); and M_001106878 (rat), sequences of which are incorporated by reference herein.

The CD70 amino acid sequences are available from public databases as access numbers NP_001243 (human); NP 035747 (mouse); and NP_001100348 (rat), sequences of which are incorporated by reference herein.

ICOS-L is a ligand for the ICOS receptor on the specific cell surface of T cells and acts as a co-stimulatory signal for T cell proliferation and cytokine secretion. ICOS-L also induces B cell proliferation and differentiation in plasma cells. ICOS-L could play an important role in mediating local tissue responses to inflammatory conditions, thus modulating the secondary immune response by co-stimulating the function of memory T cells. The polynucleotide sequences of ICOS-L are available from public databases as access numbers NM_015259 (human); and N _015790 (mouse), sequences of which are incorporated by reference herein.

The amino acid sequences of ICOS-L are available from public databases as access numbers NP_056074 (human); and NP_056605 (mouse), sequences of which are incorporated by reference herein.

PD-Ll protein (also known as CD274) is expressed in activated monocytes, T and B cells. PD-Ll is supra-monozyme after treatment with IFN-gamma, and in dendritic cells and keratinocytes after treatment with IFN-gamma, together with other activators. The polynucleotide sequences of PD-Ll proteins are available from public databases as access numbers NM_014143 (human) and NM_021893 (mouse), sequences of which are incorporated by reference herein.

The amino acid sequences of PD-Ll proteins are available from public databases as access numbers NP_054862 (human); and NP_068693 (mouse), sequences of which are incorporated by reference herein.

Co-stimulatory molecule (negative) Compound 4 associated with cytotoxic T lymphocytes (CTLA4) is a member of the immunoglobulin superfamily and is a co-stimulatory molecule expressed in activated T cells. U.S. Patent Nos. 7,034,121 and 6,984,720 describe methods of preparation and use of antibodies against CTLA4. U.S. Patent 6,984,720 also describes the amino acid sequences of the heavy and light chain of the anti-CTLA4 antibody.

The PD-1 molecules are members of the immunoglobulin gene superfamily, which binds to the ligand PD-1 (PD-Ll). The binding of a PD-1 receptor on a T cell by PD-Ll transmits a co-stimulatory signal to the cell, which prevents cells from progressing through the cell cycle, and increases the proliferation of T cells. an interaction between PD-Ll and the receptor on the T cell with an anti-PD-Ll antibody, results in the sub-regulation of the immune response, termed as immune cell energy. U.S. Patent No. 7,029,674 describes the methods for the preparation and the sequence of the anti-PD-Ll antibody.

PD-L2 is primarily known as a ligand for PD-1 (or the human homolog PDCD1). However, it has been reported that PD-12 is involved in the co-stimulatory signal, essential for the proliferation of T lymphocytes and the production of IFN-gamma in an independent manner of PDCD1. The interaction with PDCD1 inhibits the proliferation of T cells by blocking the progression of the cell site and the production of cytokine. Yamazaki et al, J "of Immunol., 169: 5538 (2002) and Ansari et al, J. Exp. Med. 198: 63 (2003) describe the preparation of anti-PD-L2 monoclonal antibodies.

Supresores Immunitarros Contrarios (Inhibitors of Tolerance) Beta transforming growth factor (TGF-β) is a multifunctional protein that regulates cell proliferation and differentiation, by interacting with one of the two transmembrane serine / threonine kinase receptors, type I and type II. See Chen et al, Science 28: 1335 (1993). The type II TGF receptor (TGFR2) phosphorylates and activates type I receptors which are autophosphorylated, then bind to and activate the SMAD transcriptional regulators. Lynch MA et al, Cancer Res. 58: 4227 (1998) describes mutations in the gene of the type II receptor of transforming growth factor-ß (TGFBR2) that are associated with human ovarian carcinomas. Brand et al, J. Biol. Chem. 255: 11500-11503 (1993) describes that the suppression of the predicted cytoplasmic domain of serine / teronin-kinase (nucleotides 1172-2036 of TGFPR2 cDNA H2-3FF, available from the bases of public data such as accession number M85079 and the amino acid sequences available as accession number AAA61164) impairs the expressions of the genes dependent on the three TGF-β (1, 2 and 3). TGF-ß is produced in most human tumors and inhibits specific cellular immunity or tumor antigen. Foster et al, J. Immunother. 31: 500 (2008) describes that the expression of? T? The dominant negative 2, in cytotoxic T lymphocytes, can lead to resistance to the inhibitory effects of TGF-β.

TGF acts synergistically with TGF in the induction c of transformation. It also acts as a negative autocrine growth factor. Deregulation of TGFP activation and signaling may result in apoptosis. Ziyadeh et al, Proc. Nati Acad. Sci. 97: 8015 (2000) discloses that administration of the anti-TGF antibody can prevent renal failure and glomerulosclerosis and db / db mice, a model of type II diabetes that develops evident nephropathy. Methods of generation and use of monoclonal antibodies to TGF are described in the United States Patent 10 No. 6,419,928. Barcellos -Hoff et al., Axn J. Pathol. 147: 5 (1995) also describes a method for the generation of the antibody for TGF. The nucleotide and amino acid sequences for the TGF fusion protein constructs are described in U.S. Pat. No. 15 6,756,215.

IL-10 is a cytokine produced by activated Th2 cells, B cells, keratinocytes, monocytes, and macrophages. IL-10 inhibits the synthesis of a number of cytokines, including IFN-gamma, IL-2, IL-3, TNF and GM-CSF 2Q produced by activated macrophages and by helper T cells. IL-10 is useful in promoting the development and differentiation of activated human B cells, inhibiting Thl responses to prevent rejection of transplantation and autoimmune disorders mediated by , c cells T. O'Farrell eü al., EMBO J. 17: 1006 (1998); Kanbayashi et al, Cell Immunol. 171: 153 (1996); Fukushima et al, Br. J. Ophthal ol. 90: 1535 (2006); and van Lent et al, Ann. Rheum. Dis. 66: 334 (2007) describe the preparation of anti-IL10 antibodies. U.S. Patent No. 7,326,567 describes the polynucleotide sequence of the antibody for IL-10. U.S. Patent No. 5,837,232 describes a method for treating an autoimmune disorder mediated by B cells, with anti-IL-10 antibodies.

The suppressor of cytokine signaling families (SOCS) is part of a classical negative feedback system, which regulates the transduction of cytokine signals. Alexander et al. Cell 98: 597 (1999) describes that the suppressor of cytokine signaling 1 (S0CS1) is a critical inhibitor of interferon-gamma signaling, and prevents the potentially fatal neonatal actions of this cytokine. Hilton et al, Proc. Nati Acad. Sci. USA 95: 114 (1999) discusses that SOCS1 is involved in the negative regulation of cytokines that signal through the JAK / STAT3 pathway. Ohya et al J. Biol. Chem. 272: 27178 (1997) discloses that SOCS proteins appear to be a major regulator of signaling by interleukin 6 (IL-6) and leukemia inhibitory factor (LIF). U.S. Patent No. 6,534,277 describes a method for the preparation and use of the anti-SOCSl antibody, wherein a nucleic acid sequence encoding the SOCS1 antibody is introduced into the cells, such that the antibody is expressed by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. U.S. Patent Nos. 6,323,317 and 7,049,418 also describe anti-SOCSl antibodies.

TGF-a is a mitogenic polypeptide that is capable of binding to the EGF receptor, and of acting synergistically with TGF-β to promote cell proliferation independent of anchoring, in soft agar. Ellis et al, N. Engl. J. Med. 317: 15% (1987) describes that TGF-a plays a role in certain paraneoplastic manifestations of melanoma. U.S. Patent No. 4,742,003 and Xian et al, The J. of Histochem. & Cytochem. 47: 949 (1999) describe the methods of preparation of anti-GF- antibodies.

Both the tumor necrosis factor receptor (TNFR1) and Fas contain the cytoplasmic protein associated with Fas, with death domain (FADD), which is essential for signaling induced by Fas and TNF, for programmed cell death (apoptosis) and oligomerization of the receptor. A mammalian protein designated FADD that has the ability to bind to the cytoplasmic region or the Fas receptor domain that inhibits FAS-mediated apoptosis has been identified. The polynucleotide sequence of FADD is available from public databases as accession number U24231, and the amino acid sequence as accession number AAA86517, which are incorporated by reference herein. The FADD fragment or the nucleic acid encoding it is a dominant negative inhibitor of functionally intact native FADD, which is described in U.S. Patent No. 6,562,797 Bl. p53 (also known as protein 53 or tumor protein 53), is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is included in the prevention of cancer. The amino acid and polynucleotide sequences for p53 are available as access numbers NM_00546 and NP_000537 (human) and NM_01 1640 and NP_035770 (mouse).

Survivin is a member of the family inhibitor of apoptosis. The survivin protein functions to inhibit the activation of caspase, thereby leading to the negative denial of apoptosis or programmed cell death. This has been shown by the breaking of the survivin induction pathways that lead to the increase in apoptosis and to the decrease in tumor growth. The survivin protein is highly expressed in most human tumors and fetal tissue, but it is completely absent in terminally differentiated cells. Therefore, this fact makes survivin an ideal target for cancer therapy since cancer cells are targeted as long as normal cells are left alone. The expression of survivin is also highly regulated by the cell cycle and is only expressed in the G2-M phase. It is known that survivin is located in the mitotic spindle by interaction with tubulin during mitosis and may play a contributory role in the regulation of mitosis. The regulation of survivin appears to be linked to the p53 protein. The amino acid and polynucleotide sequences for p53 are available as access numbers M_001012270 and NP_001012270 (human) and NM_001012273 and NP_001012273 (mouse).

The amino acid sequence of melanoma-associated antigen 3 (MAGE3) is found as accession number P43357-1 (UniParc).

Prostate-specific antigen (PSA) is a protein produced by the cells of the prostate gland. PSA is present in small amounts in the serum of man with a healthy prostate, but it is frequently elevated in the presence of prostate cancer and in other prostate disorders.

The prostate-specific membrane antigen (PSMA) is a type 2 integral membrane glycoprotein found in prostate tissue and in a few other tissues. It is a possible therapeutic target for prostate cancer.

SEQ ID No.: 1 is a polynucleotide sequence of a construct that codes for mIL-12 and m-IL21.

SEQ ID No. 2 is a polynucleotide sequence of a construct that codes for hIL-12 and hIL-21.

SEQ ID NO: 3 is a polynucleotide sequence of a construct that codes for mIL-21 and mIL-15.

SEQ ID No. 4 is a polynucleotide sequence of a construct that codes for mIL-12.

SEQ ID No. 5 is a polynucleotide sequence of a construct that codes for hIL-21 and hIL-15.

SEQ ID No .: 6 is a polynucleotide sequence of a construct that codes for hIL-21.

SEQ ID NO: 7 is a polynucleotide sequence of a construct that codes for mIL-21.

SEQ ID No. : 8 is a polynucleotide sequence of a construct that codes for hIL-21.

SEQ ID NO: 9 is a polynucleotide sequence that codes for mIL-21.

SEQ ID No .: 10 is an amino acid sequence of mIL-21.

SEQ ID NO: 11 is a polynucleotide sequence that codes for mIL-15.

SEQ ID NO: 12 is an amino acid sequence of mIL-15.

SEQ ID No .: 13 is a polynucleotide sequence that codes for mp40 of mIL-12.

SEQ ID NO: 14 is the amino acid sequence of mp40 of mIL-12.

SEQ ID No .: 15 is a polynucleotide sequence that codes for mp35 of mIL-12.

SEQ ID NO: 16 is the amino acid sequence of mp35 of mIL-12.

SEQ ID NO: 17 is a polynucleotide sequence that codes for hIL-21.

SEQ ID NO: 18 is the amino acid sequence of hIL-21.

SEQ ID No.: 19 is a polynucleotide sequence that codes for hIL-15.

SEQ ID NO: 20 is the amino acid sequence of hIL-15.

SEQ ID NO: 21 is a polynucleotide sequence that codes for hIL-12 p40.

SEQ ID NO: 22 is the amino acid sequence of p40 of hIL-12.

SEQ ID NO: 23 is a polynucleotide sequence that codes for p35 of hIL-12.

SEQ ID NO: 24 is the amino acid sequence of p35 of hIL-12.

SEQ ID No.: 25 is a nucleotide acid sequence of an ecdysone response element, found in Drosophila.

SEQ ID NO: 26 is a nucleotide acid sequence of an element of the ecdysone response found in Drosophila melanogaster.

SEQ ID NO: 27 is a nucleotide acid sequence of an element of the ecdysone response found in Drosophila melanogaster.

SEQ ID No.: 28 is a restriction site of a domestic endonuclease (HE) enzyme (I-Scel) SEQ ID NO: 29 is a DNA sequence of the adenoviral vector comprising the coding sequence of human IL-12: Ad-RTS-hIL-12 (SPl-RheoIL-12).

SEQ ID NO: 30 is a 5'UTR wild type nucleic acid sequence of human TNF.

SEQ ID NO: 31 is a nucleic acid sequence of 5U2 5'UTR.

SEQ ID NO: 32 is a nucleic acid sequence optimized by codon coding for the IL-2 signal peptide.

SEQ ID NO: 33 is a wild-type nucleic acid sequence that codes for the human TNF-alpha signal peptide.

SEQ ID NO: 34 is a nucleotide sequence optimized by codon coding for the human TNF-alpha signal peptide.

SEQ ID NO: 35 is a wild-type nucleic acid sequence that encodes human TNF-alpha pair.

SEQ ID NO: 36 is a nucleic acid sequence optimized by codon coding for human TNF-alpha.

SEQ ID NO: 37 is an amino acid sequence of human TNF-alpha.

SEQ ID NO: 38 is a nucleic acid sequence of the 3 'regulatory region comprising a nucleotide sequence encoding an SV40 polyadenylation signal.

SEQ ID NO: 39 is a nucleic acid sequence of the 3 'regulatory region comprising a nucleotide sequence encoding a human growth hormone polyadenylation signal.

SEQ ID NO: 40 is a nucleic acid sequence comprising 3 'UTR of wild-type human TNF-alpha.

SEQ ID NO: 41 is an imitant nucleic acid sequence of 31 UTR AtoC of human TNF-alpha.

SEQ ID NO: 42 is a human GAST 3 'UTR nucleic acid sequence.

SEQ ID NO: 43 is a nucleic acid sequence of the synthetic 3 'regulatory sequence.

SEQ ID NO: 44 is a human 5 'UTR GAPDH nucleic acid sequence.

SEQ ID NO: 45 is a wild type nucleic acid sequence of insulin SP.

SEQ ID NO: 46 is a wild type nucleic acid sequence encoding the human FGF-19 signal peptide.

SEQ ID NO: 47 is a nucleic acid sequence of Vector 43318.

SEQ ID NO: 48 is a nucleic acid sequence of Vector 43319.

SEQ ID NO: 49 is a nucleic acid sequence of Vector 43320.

SEQ ID NO: 50 is a nucleic acid sequence of Vector 43321.

SEQ ID NO: 51 is a nucleic acid sequence of Vector 43322.

SEQ ID NO: 52 is a nucleic acid sequence of Vector 43323.

SEQ ID NO: 53 is a nucleic acid sequence of Vector 43324.

SEQ ID NO: 54 is a nucleic acid sequence of Vector 43325.

SEQ ID NO: 55 is a nucleic acid sequence of Vector 43326.

SEQ ID NO: 56 is a nucleic acid sequence of Vector 43327.

SEQ ID NO: 57 is a nucleic acid sequence of Vector 43328.

SEQ ID NO: 58 is a nucleic acid sequence of Vector 43329.

SEQ ID NO: 59 is a nucleic acid sequence of Vector 43533.

SEQ ID NO: 60 is a nucleic acid sequence of Vector 43534.

SEQ ID NO: 61 is a nucleic acid sequence of Vector WN2823 (Ad-RTS-hIL-12).

SEQ ID NO: 62 is a nucleic acid sequence of Vector WN2539 (Ad-RTS-mlL-12).

Detailed description of the invention Definitions Unless defined otherwise, all terms of the art, notations, and other scientific terms or scientific terminology used herein, are intended to have the meanings commonly understood by those of ordinary experience in the art. which this invention belongs to. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for easy reference and understanding, and the inclusion of such definitions herein should not necessarily be considered as a substantial difference over what is in general understood in the technique. Commonly understood definitions of molecular biology terms and / or methods and / or protocols thereof can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York , 1991; Lewin, Genes V, Oxford University Press: New York, 1994; Sambrook et al, Molecular Cloning, A Laboratory Manual (3d ed 2001) and Ausubel et al, Current Protocols in Molecular Biology (1994). As appropriate, procedures that involve the use of commercially available kits and / or reagents are generally carried out in accordance with the guide and / or the protocols and / or manufacturer's parameters, unless otherwise indicated. another way.

The term "isolated" for the purposes of the invention, designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or in an animal is not isolated, however, the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present is considered "isolated".

The term "purified", as applied to biological materials, does not require that the material be present in a form that shows absolute purity, excluding the presence of other compounds. This is rather a relative definition.

"Nucleic acid", "nucleic acid molecule", "oligonucleotide", "nucleotide" and "polynucleotide" are used interchangeably and refer to the polymeric form of the phosphate ester of the ribonucleosides (adenosine, guanosine, uridine or cytidine, "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, either in the form of a single strand, or in a double stranded helix. Double-stranded helices of DNA-DNA, DNA-RNA and RNA-RNA are possible. The term nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule; and do not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (eg, restriction fragments), plasmids, supercoiled DNA and chromosomes. In the discussion of the structure of the particular double-stranded DNA molecules, the sequences can be described herein, according to the normal convention of only giving the sequence in the 5 'to 3' direction along the non-transcribed strand of DNA (for example, the strand that has a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. The DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.

The term "fragment," as applied to polynucleotide sequences, refers to a nucleotide sequence of reduced length, relative to the reference nucleic acid and comprising, on the common portion, a nucleotide sequence identical to the reference nucleic acid . Such a nucleic acid fragment according to the invention can be, where appropriate, included in a larger polynucleotide of which this is a constituent. Such fragments comprise, or alternatively consist of, oligonucleotides in the length range of at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000, or more consecutive nucleotides of a nucleic acid according to the invention.

As used herein, an "isolated nucleic acid fragment" refers to an RNA or DNA polymer that is single-stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a DNA polymer can be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

A "gene" refers to a polynucleotide comprising nucleotides that encode a functional molecule, including functional molecules produced by transcription only (eg, a species of bioactive RNA) or by transcription and translation (eg, a polypeptide). The term "gene" encompasses the cDNA and the nucleic acids of genomic DNA. "Gene" also refers to a fragment of nucleic acid that expresses a specific RNA, protein or polypeptide, including the regulatory sequences that precede (sequences of non-coding 5 ') and that follow (sequences of non-coding 3') the sequence of coding. "Native gene" refers to a gene as it is found in nature, with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and / or coding sequences that are not found together in nature. In consecuense, a chimeric gene can comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a different way from that found in nature. A chimeric gene may comprise the coding sequences derived from different sources and / or regulatory sequences derived from different sources. "Endogenous gene" refers to a native gene in its natural position in the genome of an organism. A "foreign" or "heterologous" gene refers to a gene not normally found in the host organism, but is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. For example, the gene for interleukin-12 (IL-12) codes for the IL-12 protein. IL-12 is a heterodimer of a 35-kD subunit (p35) and a 40-kD subunit (p40) linked through a disulfide bond to make fully functional IL-12p70. The IL-12 gene encodes the p35 and p40 subunits.

"Heterologous DNA" refers to DNA not naturally located in the cell, or at a chromosomal site in the cell. The heterologous DNA may include a gene foreign to the cell.

The term "genome" includes chromosomal, as well as mitochondrial, chloroplast and viral DNA or RNA.

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single-stranded form of the nucleic acid molecule can be annealed to the other nucleic acid molecule under the appropriate conditions of temperature and ionic strength in solution. Hybridization and washing conditions are well known and exemplified in Sambrook et al. in Molecular Cloning:? Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 in this one). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

Demanding conditions can be adjusted to select moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. For the preliminary selection for the homologous nucleic acids, the conditions of low existence hybridization, corresponding to a Tm of 55 ° C, for example, 5X SSC, 0.1% SDS, 0.25% milk, and without formamide, can be used. or 30% formamide, 5X SSC, 0.5% SDS. Hybridization conditions of moderate stringency correspond to a Tm, higher, for example, 40% formamide, with 5X or 6X SSC.

The high stringency hybridization conditions correspond to the highest Tm, for example, 50% formamide, 5X or 6X SSC.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, disparities between bases are possible. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing with one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the invention also includes isolated fragments of nucleic acid that are complementary to the complete sequences, as described or used herein, as well as those of substantially similar nucleic acid sequences.

In one embodiment of the invention, the polynucleotides are detected by employing hybridization conditions comprising a hybridization step at Tn, of 55 ° C, and using conditions as described above. In other embodiments, the Tm is 60 ° C, 63 ° C, or 65 ° C.

The post-hybridization washes also determine the stringency conditions. One set of conditions uses a series of washes beginning with 6X SSC, 0.5% SDS at room temperature for 15 minutes (min), then repeated with 2X SSC, 0.5% SDS at 45 ° C for 30 min, and then repeated with 0.2X SSC, 0.5% SDS at 50 ° C for 30 min. A preferred group of demanding conditions uses higher temperatures in which the washings are identical to those previously mentioned, except for the temperature of the two final 30 min washes in 0.2X SSC, 0.5% SDS which is increased to 60 ° C . Another preferred group of highly demanding conditions uses two final washes in 0. IX SSC, 0.1% SDS at 65 ° C.

The appropriate stringency for the hybridization of nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for the hybrids of the nucleic acids that have those sequences. The relative stability (corresponding to the highest Tm) of the nucleic acid hybridizations, decreases in the following order: RNA: RNA, AD: AR, AD: AD. For hybrids more than 100 nucleotides in length, the equations for calculating Tm have been derived (see Sambrook et al, supra, 9.50-0.51). For hybridization with shorter nucleic acids, eg, oligonucleotides, the position of the disparities becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra, 11.7-11.8).

In one embodiment of the invention, the polynucleotides are detected by employing hybridization conditions comprising a hybridization step in at least 500 mM and at least 37 ° C, and a washing step in 2X SSPE at a temperature of minus 63 ° C. In yet another embodiment, the hybridization conditions comprise less than 200 mM salt and minus 37 ° C for the hybridization step. In a further embodiment, the hybridization conditions comprise 2X SSPE and 63 ° C for the hybridization and washing steps.

In another embodiment, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably, a minimum length for a hybridizable nucleic acid is at least 15 nucleotides; for example, at least about 20 nucleotides; for example, at least 30 nucleotides. In addition, the person skilled in the art will recognize that the temperature and concentration of the salt of the washing solution can be adjusted as necessary according to factors such as the length of the probe.

The term "probe" refers to a single-stranded nucleic acid molecule that can base pairing with a complementary single-stranded target nucleic acid to form a double-stranded molecule.

As used herein, the term "oligonucleotide" refers to a short nucleic acid that is hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule. Oligonucleotides can be labeled, for example, with 32 P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. A labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. Oligonucleotides (one or both of which can be labeled) can be used as PCR primers, either for full-length cloning or a fragment of a nucleic acid, for DNA sequencing or for detecting the presence of an acid nucleic. An oligonucleotide can also be used to form a triple helix with a DNA molecule. In general, the oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with phosphoester analogues of non-natural origin, such as thioester bonds, etc.

A "primer" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double-stranded nucleic acid region that can serve as a starting point for DNA synthesis under suitable conditions. Such primers can be used in a polymerase chain reaction or for DNA sequencing.

The "polymerase chain reaction" is abbreviated as PCR and refers to an in vitro method to enzymatically amplify specific nucleic acid sequences. PCR involves a repetitive series of temperature cycles with each cycle comprising three steps: the denaturation of the template nucleic acid to separate the strands from the target molecule, annealing a single-stranded PCR oligonucleotide primer to the nucleic acid template, and the extension of the primer (s) annealed by DNA polymerase. PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the initial pool of nucleic acids.

"Reverse transcription polymerase chain reaction" is abbreviated RT-PCR and refers to an in vitro method to enzymatically produce a target cDNA molecule or molecules from one molecule or several RNA molecules, followed by enzymatic amplification of a specific sequence or sequences of nucleic acid or sequences within the target cDNA molecule or molecules, as described above. RT-PCR also provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the initial pool of nucleic acids.

A "coding sequence" of DNA refers to a double-stranded DNA sequence that codes for a polypeptide and can be transcribed and translated into a polypeptide in a cell in vitro or in vivo, when placed under the control of regulatory sequences adequate. The "suitable regulatory sequences" refer to the nucleotide sequences located in the 5 'direction (5' non-coding sequences), within, or in the 3 'direction (3' non-coding sequences) of a coding sequence, and it influences the transcription, the processing and the stability of the RNA, or the translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and trunk-loop structures. The boundaries of the coding sequences are determined by a start codon at the 5 'end (amino) and a translation stop codon at the 3' end (carboxyl). A coding sequence can include, but is not limited to, prokaryotic cDNA sequences from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and the transcription termination sequence will usually be located 3 'to the coding sequence.

"Open reading frame" is abbreviated ORF and refers to a frequency length of nucleic acid, either DNA, cDNA or RNA, comprising a start translation signal or start codon, such as an ATG or AUG, and a stop codon, and can potentially be translated into a polypeptide sequence.

The term "head to head" is used herein to describe the orientation of two polynucleotide sequences one relative to the other. Two polynucleotides are placed in a head-to-head orientation when the 5 'end of the coding strand of a polynucleotide is adjacent to the 5' end of the coding strand of the other polynucleotide, whereby the transcription direction of each polynucleotide proceeds away of the 5 'end of the other polynucleotides. The term "head to head" can be abbreviated (5 ') - a- (5') and can also be indicated by the symbols («-?) Or (3 '< -5 * 5'? 3 ').

The term "tail-to-tail" is used herein to describe the orientation of two polynucleotide sequences one relative to the other. Two polynucleotides are placed in the tail-to-tail orientation when the end 31 of the coding strand of a polynucleotide is adjacent to the end 31 of the coding strand of the other polynucleotide, whereby the transcription direction of each polynucleotide proceeds towards the other polynucleotide. The term "tail-to-tail" can be abbreviated (3 ') - a- (3!) And can also be indicated by the symbols (< -?) Or (5'? 3 '3'? 5 ') · The term "head-to-tail" is used herein to describe the orientation of two polynucleotide sequences one relative to the other. Two polynucleotides are placed in a head-to-tail orientation when the 5 'end of the coding strand of a polynucleotide is adjacent to the end 31 of the coding strand of the other polynucleotide, whereby the transcription direction of each polynucleotide is in the same direction as that of the other polynucleotide. The term "head-to-tail" can be abbreviated (5 ') -a- (31) and can also be indicated by the symbols (??) Or (5I? 315 '? 3 ' ) .

The term 3 'refers to a nucleotide sequence that is located 31 to a reference nucleotide sequence. In particular, nucleotide sequences with 3 'direction are generally referred to following the initial point of transcription. For example, the start codon of the translation of a gene is located 3 'from the transcription start site.

The term 5 'refers to a nucleotide sequence that is located 51 to a reference nucleotide sequence. In particular, nucleotide sequences with a 5'-direction generally refer to sequences that are located on the 5'-side of a coding sequence or the initial point of transcription. For example, most of the promoters are located at address 51 of the transcription start site.

The terms "restriction endonuclease" and "restriction enzyme" are used interchangeably and refer to an enzyme that binds to and cuts within a specific nucleotide sequence within the double-stranded DNA.

"Homologous recombination" refers to the insertion of a foreign DNA sequence into another DNA molecule, for example, the insertion of a vector into a chromosome. Preferably, the vector is directed to a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficient long regions of homology to the chromosome sequences, to allow complementary binding and incorporation of the vector into the chromosome. . Longer regions of homology, and higher degrees of sequential similarity, may increase the efficiency of homologous recombination.

Various methods known in the art can be used to propagate a polynucleotide according to the invention. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As described herein, expression vectors that can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast virus; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

A "vector" refers to any vehicle for the cloning and / or transfer of a nucleic acid within a host cell. A vector can be a replicon to which another DNA segment can be linked to thereby give rise to the replication of the linked segment. A "replicon" refers to any genetic element (eg, plasmid, phage, cosmid, chromosome, virus) that functions as an autologous unit of DNA replication in vivo, for example, capable of replication under its own control. The term "vector" includes viral and non-viral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors known in the art can be used to manipulate nucleic acids, to incorporate response elements and promoters into genes, etc. Possible vectors include, for example, plasmids or modified viruses including, for example, bacteriophages, such as lambda derivatives or plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector. Yet another example of vectors that are useful in the invention is the UltraVector ™ Production System (Intrexon Corp., Blacksburg, VA) as described in WO 2007/038276. For example, the insertion of the DNA fragments corresponding to the response elements and the promoters into a suitable vector can be achieved by ligating the appropriate DNA fragments within a chosen vector having complementary cohesive ends. Alternatively, the ends of the DNA molecules can be enzymatically modified or any site can be produced by ligation of nucleotide sequences (linkers) within the DNA ends. Such vectors can be manipulated to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome. Such markers allow the identification and / or selection of host cells that incorporate and express the proteins encoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in a wide variety of applications of gene distribution in cells, as well as live animal subjects. Viral vectors that can be used include, but are not limited to, retroviruses, adeno-associated viruses, smallpox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes and biopolymers. In addition to a nucleic acid, a vector can also comprise one or more regulatory regions and / or selectable markers useful in the selection, measurement and monitoring of nucleic acid transfer results (transfer to which tissues, duration of expression, etc. .).

The term "plasmid" refers to an extrachromosomal element that often possesses a gene that is not part of the cell's central metabolism, and are usually in the form of circular, double-stranded DNA molecules. Such elements can be autonomous replication sequences, genomic integration sequences, phage or nucleotide sequences, linear, circular, or supercoiled of a single-stranded or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been linked or recombined into a single construct that is capable of introducing a promoter fragment and the DNA sequence for a selected gene product, together with the appropriate 3 'untranslated sequence, into a cell.

A "cloning vector" refers to a "replicon", which is a unit length of a nucleic acid, preferably a DNA, which replicates sequentially and which comprises an origin of replication, such as a plasmid, phage, or cosmid , to which another nucleic acid segment can be linked, thus giving rise to the replication of the linked segment. Cloning vectors may be capable of replication in one cell type and expression in another ("shuttle vector"). The cloning vectors may comprise one or more sequences that can be used for the selection of cells comprising the vector and / or one or more multiple cloning sites for the insertion of the sequences of interest.

The term "expression vector" refers to a vector, plasmid, or vehicle designed to make possible the expression of an inserted nucleic acid sequence after transformation within the host. The cloned gene, for example, the inserted nucleic acid sequence, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like. The initiation control regions or promoters, which are useful for placing the expression of a nucleic acid in the desired host cell, are numerous and familiar to those skilled in the art. Virtually any promoter capable of promoting the expression of these genes can be used in an expression vector, including, but not limited to, viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, promoters, constituents, promoters specific to tissues, promoters related to pathogenesis or disease, specific promoters of development, inducible promoters, promoters regulated by light; CYC1, HIS3, GAII, GAL4, GALIO, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichia); the promoters of β-lactamase, lac, ara, tet, trp, IPL, IPR, 11, tac, and trc (useful for expression in Escherichia coli); 35S cauliflower mosaic virus, specific ovarian, specific to pollen, specific to seeds, regulated by light. CMV 35S minimum, cassava vein mosaic virus (CsVMV), chlorophyll a / b binding protein, ribose 1, 5-bisphosphate-carboxylase, shoot-specific, root-specific, chitinase, stress-inducible, virus bacilliforme of tungro root, super-plant promoter, potato leucine-aminopeptidase, nitrate-reductase, mannopine-synthase, nopalin-synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells); animal and mammalian promoters known in the art to include, but not be limited to, the SV40e promoter early region (SV40e), the promoter contained in the 3 'long terminal repeat (LTR) of the SV40e virus. Rous sarcoma (RSV), E1A promoters or adenovirus major adenovirus (MLP) delayed promoter (Ad) genes, the cytomegalovirus (CMV) thin promoter, the promoter of thymidine kinase (TK) of herpes simplex virus (HSV), an IE1 vaculovirus promoter, an elongation factor 1 alpha promoter (EF1), a phosphoglycerate kinase promoter (PGK) , a ubiquitin promoter (Ubc), an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and similar), the promoters of the intermediates (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of DR, CFTR or factor VIII, and the like), promoters related to pathogenesis or disease, and promoters showing specificity of tissue and have been used in transgenic animals, such as the control region of the elastase I gene that is activated in pancreatic acinar cells; the control region of the insulin gene, active in pancreatic beta cells, the control region of the active immunoglobulin gene, in lymphoid cells, the control region of the mouse mammary tumor virus active in testicular, breast, lymphoid cells and mast cells, the albumin gene, the active Apo AI and Apo AII control regions in the liver, the control region of the active alpha-fetoprotein gene in the liver, the control region of the antitrypsin alpha 1-antitrypsin gene in liver, the control region of the active beta-globin gene in myeloid cells, the control region of the active myelin basic protein gene in oligodendrocyte cells in the brain, the control region of the light chain 2 gene of active myosin in skeletal muscle, and the control region of the gonadotropin-releasing hormone gene, active in the hypothalamus, the pyruvate-kinase promoter, the villin promoter, the promoter or of the fatty acid binding protein, the alpha-actin promoter of smooth muscle cell and the like. In addition, these expression sequences can be modified by the addition of enhancer or regulatory sequences and the like.

Vectors can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, lipofection (lysosome fusion), of a gene gun, or a DNA vector or transporter (see for example, Wu et al, J. Biol. Chem. 267: 963 (1992), Wu et al, J. Biol. Chem. 263: 14621 (1988). ), and Hartmut et al, Canadian Patent Application No. 2, 012, 311).

A polynucleotide according to the invention can also be introduced in vivo by lipofection. In the last decade, there has been an increasing use of liposomes for the encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and hazards encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al, Proc. Nati Acai. USA 84: 7413 (1987) Mackey et al, Proc. Nati.

Acad. Sci. USA 55: 8027 (1988); and Ulmer et al, Science 259: 1745 (1993)). The use of cationic lipids can promote the encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner et al, Science 337: 387 (1989)). Particularly useful lipid compounds and compositions for the transfer of nucleic acids are described in W095 / 18853, W096 / 17823 and U.S. 5,459,127. The use of lipofection can introduce exogenous genes into specific organs in vivo, which has certain practical advantages. The molecular direction of liposomes to specific cells represents an area of benefit. It is clear that the direction of transfection to particular cell types, could be particularly preferred in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain, the lipids can be chemically coupled to other molecules for the purpose of direction to the objective (Mackey et al., 1988, supra). Targeted peptides, for example, hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules, could be chemically coupled to liposomes.

Other molecules are also useful for facilitating the transfection of a nucleic acid in vivo, such as a cationic oligopétide (e.g., 095/21931), peptides derived from DNA binding proteins (e.g., WO96 / 25508), or a polymer cationic (for example, W095 / 21931).

It is also possible to introduce an in vivo vector as a naked DNA plasmid (see U.S. Patent Nos. 5,693,622, 5,589,466 and 5,580,859). Methods of distribution of DNA mediated by receptor can also be used (Curiel et al., Hum Gene 3: 147 (1992) and Wu et al, J. Biol. Chem. 262: 4429 (1987)) .

The term "transfection" refers to the absorption of AR or exogenous or heterologous DNA by a cell. A cell has been "transfected" by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced into the cell. A cell has been "transformed" by exogenous or heterologous RNA or DNA when the RNA or DNA effects a phenotypic change. The transformation of RNA or DNA can be integrated (covalently linked) into the chromosomal DNA that makes up the genome of the cell.

"Transformation" refers to the transfer of a nucleic acid fragment within the genome of a host organism, resulting in genetically stable inheritance. Host organisms that contain transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

In addition, the recombinant vector comprising a polynucleotide according to the invention can include one or more origins for replication in cellular hosts in which its amplification or its expression is sought, in selectable markers or markers.

The term "selectable marker" refers to an identification factor, usually a gene for resistance to antibiotics or chemicals, that is capable of being selected based on the effect of the marker gene, for example, resistance to an antibiotic, resistance to a herbicide, colorimetric labels, enzymes, fluorescent labels, and the like, wherein the effect is used to trace the inheritance of a nucleic acid of interest and / or to identify a cell or organism that has inherited the nucleic acid of interest. Examples of known and used in the art selectable marker genes include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, for example, anthocyanin regulatory genes, isopentanyl transferase gene, and the like.

The term "reporter gene" refers to a nucleic acid encoding an identification factor that is capable of being identified based on the effect of the reporter gene, where the effect is used to trace the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and / or to measure the induction of gene expression or transcription. Examples of known and used in the art reporter genes include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), beta-galactosidase (LacZ), beta-glucuronidase (Gus), and the like. Selectable marker genes can also be considered indicator genes.

The "promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3 'to a promoter sequence. The promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters". Promoters that cause a gene to be expressed at a specific stage of cell development or differentiation are commonly referred to as "developmental specific promoters" or "cell-differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light or the like that induces the promoter is commonly referred to as "inducible promoter "or" adjustable promoter ". It is further recognized that in most cases the exact boundaries of the regulatory sequences have not been fully defined, the different lengths of DNA fragments may have identical promoter activity.

In any of the vectors of the present invention, the vector optionally comprises a promoter described herein. In one embodiment, the promoter is a promoter listed in Table 1 herein.

In any of the vectors of the present invention, the vector optionally comprises a tissue-specific promoter. In one embodiment, the tissue-specific promoter is a tissue-specific promoter described herein. In yet another embodiment, the tissue-specific promoter is a tissue-specific promoter listed in Table 2 herein.

The promoter sequence is typically limited at its 3 'end by the transcription initiation site and extends in the 5' direction (5 'direction) to include the minimum number of bases or elements necessary to initiate transcription at detectable levels above. of the antecedent. Within the promoter sequence is found a transcription initiation site (conveniently defined for example, by mapping with the nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of the RNA polymerase.

"Therapeutic change promoter" ("TSP") refers to a promoter that controls the expression of a gene change component. The gene changes and their various components are described in detail elsewhere in the present. In certain modalities, a TSP is constitutive, for example, continuously active. A constitutive TSP may be either ubiquitous constitutive (eg, it generally functions without the need for additional factors or regulators, in any tissue or cell) or tissue-specific or constitutive cell (eg, in general in functions without the need for additional factors or regulators in a specific tissue type or cell type). In certain embodiments, a TSP of the invention is activated under conditions associated with a disease, disorder or condition. In certain embodiments of the invention where two or more TSPs are involved, the promoters may be a combination of constitutive and activatable promoters. As used herein, an "activated promoter under conditions associated with a disease, disorder or condition" includes, without limitation, disease-specific promoters, promoters that respond to particular physiological, developmental, differentiating or pathological conditions, promoters. which respond to specific biological molecules and promoters specific for a particular tissue or cell type, associated with the disease, disorder or condition, for example, tumor tissue or malignant cells. The TSPs may comprise the sequence of promoters of natural origin, modified sequences derived from promoters of natural origin, or synthetic sequences (for example, insertion of a response element within a minimal promoter sequence to alter the responsiveness of the promoter).

A coding sequence is "under the control" of transcriptional and translational control sequences in a cell, when RNA-polymerse transcribes the coding sequence within the mRNA, which is then trans-spliced RNA (if the coding sequence contains introns ) and translated into the protein encoded by the coding sequence.

"Transcriptional and translational control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, which provide for the expression of a coding sequence in a host cell. In eukaryotic cells, the polyadenylation signals are control sequences.

The term "response element" refers to one or more cis-acting DNA elements, which confer responsiveness to a promoter, mediated through the interaction with the DNA-binding domains, of a transcription factor . This DNA element can be either palindromic (perfect or imperfect) in its sequence or composed of sequence portions or mediated sites separated by a variable number of nucleotides. The media sites can be similar or identical and accommodated either as direct or inverted repeats or as a single site medium or multimers of adjacent media sites in tandem. The response element may comprise a minimal promoter isolated from different organisms, depending on the nature of the cell or organism into which the response element is incorporated. The DNA binding domain of the transcription factor binds, in the presence or absence of a ligand, to the DNA sequences of a response element to initiate or suppress the transcription of or genes in the 3 'direction under the regulation of this element of response. Examples of DNA sequences for natural ecdysone receptor response elements include: RRGG / TTCANTGAC / ACYY (SEQ ID No .: 25) (see Cherbas et al., Genes Dev. 5: 120 (1991)); AGGTCAN (n) AGGTCA, where N (n) can be one or more spacer nucleotides (SEQ ID NO: 26) (see D'Avino et al, Mol Cell Endocrinol 775: 1 (1995)); and GGGTTGAATGAATTT (SEQ ID No: 27) (see Antoniewski et al, Mol Cell Biol. 14: 4465 (1994)).

The term "operably linked" refers to the association of the nucleic acid sequences on a single nucleic acid fragment, so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence, when it is capable of affecting the expression of that coding sequence (eg, that the coding sequence is under the transcriptional control of the promoter). The coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term "expression" as used herein, refers to the transcription and stable accumulation of sense mRNA or antisense RNA derived from a nucleic acid or polynucleotide. The expression can also refer to the translation of the mRNA into a protein or polypeptide.

The terms "cassette", "expression cassette" and "gene expression cassette" refers to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination. The DNA segment comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure the insertion of the cassette into the appropriate reading structure for transcription and translation. "Transformation caste" refers to a specific vector comprising a polynucleotide that encodes a polypeptide of interest that has elements in addition to the polynucleotide that facilitate the transformation of a particular host cell. The cassettes, expression cassettes, gene expression cassettes and transformation cassettes of the invention may also comprise elements that allow improved expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

For the purposes of this invention, the term "gene change" refers to the combination of a response element associated with a promoter, and a system based on transcription factor, dependent on the ligand, which, in the presence of one or more ligands, modulate the expression of a gene within which the response element and the promoter are incorporated. The term "a polynucleotide encoding a gene change" refers to the combination of a response element associated with a promoter, and a polynucleotide that codes for a system based on the ligand-dependent transcription factor which, in the presence of one or more ligands, modulates the expression of a gene within which the response element and the promoter are incorporated.

The therapeutic change promoters of the invention may be any promoter that is useful to treat, ameliorate or prevent a specific disease, disorder or condition. Examples include, without limitation promoters of genes that show increased expression only during a disease, disorder or specific condition, and promoters of genes that show increased expression under specific cellular conditions (eg, proliferation, apoptosis, change in pH, oxidation state, oxygen level). In some embodiments where the gene change comprises more than one sequence of the transcription factor, the specificity of the therapeutic methods can be increased by combining a specific promoter of the disease or condition with a specific promoter of the tissue or cell, to limit the tissues in which the therapeutic product is expressed. Thus, tissue-specific or cell-type promoters are encompassed within the definition of therapeutic change promoter.

As an example of disease-specific promoters, promoters useful in treating cancer include promoters of oncogenes. Examples of oncogene classes include, but are not limited to, growth factors, growth factor receptors, protein kinases, programmed cell death regulators, and transcription factors. Specific examples of oncogenes include, but are not limited to, sis, erb B, erb B-2, ras, abl, myc and bel-2 and TERT. Examples of other cancer-related genes include tumor-associated antigen genes, and other genes that are overexpressed in neoplastic cells (eg, MAGE-1, carcinoembryonic antigen, tyrosinase, prostate-specific antigen, prostate-specific membrane antigen, p53). , MUC-1, UC-2, MUC-4, HER-2 / neu, T / Tn, MART-1, gplOO, GM2, Tn, sTn, and Thompson-Friedenreich antigen (TF)).

Examples of promoter sequences and other regulatory elements (e.g., enhancers) that are known in the art, and are useful as therapeutic change promoters in the present invention are described in the references listed in Tables 1 and 2, along with the specificity of disease / disorder (Table 1) or tissue (Table 2) associated with each promoter. The promoter sequences described in these references are incorporated herein by reference, in their entirety.

Table 1 Table 2 Other genes that show changes in the level of expression during specific disorders or diseases, and can therefore provide promoters that are useful in the present invention include, without limitation, the genes (together with the associated disorder / disease) listed in Table 3 Table 3 Once a gene with an expression pattern that is modulated during a disease, disorder or condition is identified, the gene promoter that can be used in the gene switching of the invention. The sequence of many genes including the promoter region is known in the art and is known in public databases, for example, GenBank. In this way, once an appropriate gene is identified, the promoter sequence can be easily identified and obtained. Yet another aspect of the present invention is directed to the identification of suitable genes whose promoter can be isolated and placed within a gene change. The identity of the gene, therefore, may not be critical to the specific embodiments of the present invention, with the proviso that the promoter can be isolated and used in scenarios or subsequent environments. The present invention thus includes the use of promoters from genes that have not yet been identified. Once the appropriate genes are identified, it is a matter of routine skill or experimentation to determine the genetic sequences necessary for the promoter function. Of course, there are several commercial protocols to assist in the determination of the promoter region of the genes of interest. As an example, Ding et al. recently elucidated the promoter sequence of the new Sprouty4 gene (Am. J. Physiol, Lung Cell, Mol Physiol 287: L52 (2004), which are incorporated by reference) by progressively suppressing the 5'-flanking sequence of the human Sprouty4 gene. In summary, once the transcription initiation site was determined, the PCR fragments were generated using the common PCR primers to clone the segments of the 5'-flanking segment in a unidirectional manner. The generated segments were cloned into a luciferase indicator vector and the luciferase activity was measured to determine the promoter region of the human Sprouty4 gene.

Yet another example of a protocol for acquiring and validating gene promoters includes the following steps: (1) acquiring samples of diseased and non-diseased cells / tissues from a similar / equal tissue type; (2) isolate the RNA or total mRNA from the samples; (3) perform the differential microarray analysis of the sick and non-diseased RNA; (4) identify candidate transcripts specific for the disease; (5) identify the genomic sequences associated with the specific transcripts of the disease; (6) acquiring or synthesizing the DNA sequence upstream and downstream from the predicted transcription start site of the disease-specific transcript; (7) design and produce the promoter indicator vectors using different lengths of DNA from step 6; and (8) testing the promoter indicator vectors in diseased and non-diseased cells / tissues, as well as in unrelated cells / tissues.

The source of the promoter that is inserted into the gene change can be natural or synthetic, and the source of the promoter should not limit the scope of the invention described herein. In other words, the promoter can be directly cloned from the cells, or the promoter may have been previously cloned from a different source, or the promoter may have been synthesized.

Gene Change Systems The gene change can be any gene change that regulates the expression of the gene by addition or elimination of a specific ligand. In one embodiment, gene change is one in which the level of gene expression is dependent on the level of ligand that is present. Examples of ligand-dependent transcription factor complexes, which can be used in the gene changes of the invention include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoid, estrogen, progestin, retinoid , ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect of the invention, the gene change is a gene change based on EcR. Examples of such systems include, without limitation, the systems described in U.S. Patent Nos. 6,258,603, 7,045,315, U.S. Patent Applications Published Nos. 2006/0014711, 2007/0161086, and the International Published Application. No. Or 01/70816. Examples of chimeric ecdysone receptor systems are described in U.S. Patent No. 7,091,038, U.S. Patent Applications Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and 2006 / 0100416, and published International Applications Nos. WO 01/70816, WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO 02/29075, and WO 2005/108617, each of the which are incorporated by reference in their entirety. An example of a system regulated by the non-steroidal ecdysone agonist is the RheoSwitch'1 'Mammalian Inducible Expression System (New England Biolabs, Ipswich, MA). In yet another aspect of the invention, the gene change is based on the heterodimerization of the FK506 binding protein (FKBP) with the rapamycin-associated protein FKBP (FRAP) and is regulated through rapamycin or its non-immunosuppressive analogues. . Examples of such systems, include without limitation, the ARGENTMR Transcriptional Technology (ARIAD Pharmaceuticals, Cambridge, MA) and the systems described in U.S. Patent Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595.

In one embodiment, the gene change comprises a sequence of the single transcription factor, which codes for a complex of the transcription factor dependent on the ligand under the control of a therapeutic change promoter. The sequence of the transcription factor can encode a complex of the ligand-dependent transcription factor, which is a complex of the transcription factor dependent on the ligand, of natural or artificial origin. An artificial transcription factor is one in which the natural sequence of the transcription factor has been altered, for example, by mutation of the sequence or by the combination of domains from different transcription factors. In one embodiment, the transcription factor comprises a ligand binding domain of the nuclear receptor of Group H. In one embodiment, the ligand binding domain of the nuclear receptor of Group H is derived from an ecdysone receptor, a ubiquitous receptor ( UR), an orphan receptor 1 (OR-1), a nuclear steroid hormone receptor 1 (NER-I), an interaction protein 15 with the retinoid receptor X (RIP-15), a hepatic β-receptor X (LXR) ), a protein similar to the steroid hormone receptor (RLD-I), a hepatic receptor X (LXR), a hepatic receptor X (LXROÍ), a farnesoid X receptor (FXR), a protein 14 interaction with the receptor (RIP-14), or a farnesol receptor (FIRR-1). In another embodiment, the LBd of the nuclear receptor of group H is derived from an ecdysone receptor.

A. Change of Gene Based on Ecdysone The EcR and the other nuclear receptors of Group H are members of the nuclear receptor superfamily where all members are generally characterized by the presence of an amino-terminal transactivation domain (AD, also referred to interchangeably as "TA" or "TD"), optionally fused to a heterodimerization partner (HP) to form a co-activation protein (CAP), a domain DNA linkage (DBD); and an LBD fused to the DBD via a hinge region to form a ligand-dependent transcription factor (LTF). As used herein, the term "DNA binding domain" comprises a minimal polypeptide sequence of a DNA binding protein, up to the full length of a DNA binding protein, as long as the DNA binding domain. work to associate with a particular response element. Members of the nuclear receptor superfamily are characterized by the presence of four or five domains: A / B, C, D, E, and in some F members (see U.S. Patent 4,981,784 and Evans, Science 240: 889 (1988)). The domain "A / B" corresponds to the transactivation domain. "C" corresponds to the DNA binding domain. "D" corresponds to the hinge region, and "E" corresponds to the ligand binding domain. Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD corresponding to "F".

The following polypeptide sequence was reported as a polypeptide sequence of the Ecdysone receptor (Ecdysteroid receptor) (20-hydroxy-ecdysone receptor) (20E receptor) (EcRH) (member 1 of group H of subfamily 1 of nuclear receptors) and has the access number P34021 in Genbank.

Ecdysone Receptor (878aa) from Drosophila melanogaster (fruit fly) (SEQ ID No. 5) 1 mkrrwsnngg fmrlpeesss evtsssnglv lpsgvnmsps sldshdycdq dlwlcgnesg 61 fggsnghgl sqqqqqsvall amhgcsstlp aqttiiping nangnggstn gqyvpgatnl 121 galangmlng gfngmqqqiq nghglinstt pstpttplhl qqnlggaggg giggmgilhh. 181 angtpnglig wgggggvgl gvggggvggl gmqhtprsds vnsissgrdd lspssslngy 241 sanescdakk skkgpa rvq eelclvcgdr asgyhynalt cegckgffrr svtksavycc 301 kfgracemdm ymrrkcqecr lkkclavgmr pecwpenqc amkrrekkaq kekdkmttsp 361 ssqhggngsl asgggqdfvk keildlmtce ppqhatipll pdeilakcqa rnipsltynq 421 laviykliwy qdgyeqpsee dlrrimsqpd enesqtdvsf rhiteitilt vqlivefakg 481 lpaftkipqe dqitllkacs sevmmlrmar rydhssdsif fannrsytrd sykmagmadn 541 iedllhfcrq mfsmkvdnve yalltaivif sdrpglekaq Iveaiqsyyi dtlriyilnr 601 hcgdsmslvf yakllsilte lrtlgnqnae mcfslklknr klpkfleeiw dvhaippsvq 661 erleraermr shlqitqeen idcdsastsa asvggaitag aaaaaqhqpq pqpqpqpssl 721 tqndsqhqtq pqlqpqlppq lqgqlqpqlq pqlqtqlqpq iqpqpqllpv sapvpasvta 781 pgslsavsts seymggsaai gpitpattss itaavtasst tsavpmgngv gvgvgvggnv 841 smyanaqtam almgvalhsh qeqliggvav ksehstta The DBD is characterized by the presence of two zinc fingers of cysteine between which are two amino acid portions, Table P and Table D, which contain specificity for the response elements. These domains can be either native, modified or Chimeras from different protein domains of the heterologous receptor. The EcR, as a subset of the nuclear receptor family, also has fewer well-defined regions responsible for the heterodimerization properties. Because nuclear receptor domains are modular in nature, LBD, DBD, and AD can be exchanged.

In yet another embodiment, the transcription factor comprises an AD, a DBD that recognizes a response element associated with the therapeutic protein or the therapeutic polynucleotide whose expression is to be modulated; and a LBD of the Group H nuclear receptor. In certain embodiments, the LBD of the Group H nuclear receptor comprises a substitution mutation.

In another embodiment, the gene change comprises a first sequence of the transcription factor, for example, a CAP, under the control of a first therapeutic change promoter (TSP-I) and a second sequence of the transcription factor, for example, a LTF, under the control of a second therapeutic change promoter (TSP-2), where the proteins encoding the first sequence of the transcription factor and the second sequence of the transcription factor interact to form a protein complex (LDTFC, its acronym in English), for example, a gene change based on "double change" or "two hybrid". The first and second TSPs can be the same or different. In this modality, the presence of two different TSPs in the gene change that are required for the expression of the therapeutic molecule, intensifies the specificity of the therapeutic method (see Figure 2). Figure 2 also demonstrates the ability to modify therapeutic gene change to treat any disease, disorder or condition simply by inserting the appropriate TSPs.

In a further embodiment, the first and second transcription factor sequence, eg, a CAP or an LTF, is under the control of a simple therapeutic change promoter (eg, TSP-1 in Figure 1). The activation of this promoter will generate CAP and LTF with a simple open reading structure. This can be achieved with the use of a transcriptional linker such as an IRES (internal ribosomal entry site). In this embodiment, both portions of the ligand-dependent transcription factor complex are synthesized after the activation of TSP-1. TSP-1 can be a constitutive or only activated promoter under conditions associated with the disease, disorder or condition.

In a further embodiment, a sequence of the transcription factor, e.g., an LTF, is under the control of a therapeutic-only promoter activated under conditions associated with the disease, disorder or condition (e.g., TSP-2 or TSP-3 in Figure 4) and the other sequence of the transcription factor, e.g., CAP, is under the control of a constitutive therapeutic change promoter (e.g., TSP-I in Figure 4). In this embodiment, a portion of the ligand-dependent transcription factor complex is constitutively present, while the second portion will only be synthesized under conditions associated with the disease, disorder or condition.

In another embodiment, a sequence of the transcription factor, e.g., CAP, is under the control of a first TSP (e.g., TSP-1 in Figure 3) and two or more second sequence of the different transcription factor, e.g. , LTF-1 and LTF-2 are under the control of different TSPs (for example, TSP-2 and TSP-3 in Figure 3). In this embodiment, each of the LTFs may have a different DBD that recognizes a different sequence of the promoter regulated by the factor (eg, a DBD-A binds to a response element associated with the promoter regulated by factor 1 ( FRP-1) and DBD-B bind to a response element associated with the promoter 2 regulated by the factor (FRP-2) Each of the promoters regulated by the factor can be operably linked to a different therapeutic gene. this way, multiple treatments can be provided simultaneously.

In one embodiment, the first sequence of the transcription factor codes for a polypeptide comprising an AD, a DBD that recognizes a response element associated with the sequence of the therapeutic product whose expression is to be modulated; and a LBD of the Group H nuclear receptor, and the second sequence of the transcription factor codes for a transcription factor comprising a LBD of the nuclear receptor selected from a vertebrate X retinoid (RXR) receptor, an invertebrate RXR, a ultrapyracle protein (USP), or a chimeric nuclear receptor comprising at least two different polypeptide fragments of the ligand binding domain, from the different nuclear receptor, selected from a vertebrate RXR, an invertebrate RXR, and a USP (see WO 01 / 70816 A2 and United States Patent US 2004/0096942 Al). The ligand binding domain of the "partner" nuclear receptor may further comprise a truncation mutation, a deletion mutation, a substitution mutation, or other modification.

In another embodiment, the gene change comprises a first sequence of the transcription factor that codes for a first polypeptide comprising a LBD nuclear receptor and a DBD that recognizes a response element associated with the sequence of the therapeutic product whose expression is to be modulated, and a second sequence of the transcription factor encoding a second polypeptide comprising an AD and an LBD of the nuclear receptor, wherein one of the LDLs of the nuclear receptor is a LBD of the nuclear receptor of the H group. In a preferred embodiment, the The first polypeptide is substantially free of an AD and the second polypeptide is substantially free of a DBD. For purposes of the invention, "substantially free" means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.

In still another aspect of the invention, the first sequence of the transcription factor codes for a protein comprising a heterodimerization partner and an AD (a "CAP") and the second sequence of the transcription factor codes for a protein comprising a DBD and an LBD (an "LTF").

When only one LDL of the nuclear receptor is a LBD of Group H, the other LBD of the nuclear receptor can be from any other nuclear receptor that forms a dimer with the LBD of Group H. For example, when the LBD of the nuclear receptor of Group H is an EcR LBD, the other "partner" of the LBD of the nuclear receptor can be from an EcR, a vertebrate RXR, an invertebrate RXR, an ultraspiral protein (USP), or a chimeric nuclear receptor comprising at least two different polypeptide fragments of LBD from the nuclear receptor, selected from a vertebrate RXR, an invertebrate RXR, or a USP (see O 01/70816 A2, International Patent Application No. PCT / US02 / 05235 and U.S. Patent No. US 2004/0096942 A1, incorporated by reference in its entirety). The "partner" nuclear receptor binding domain may further comprise a truncation mutation, a deletion mutation, a substitution mutation, or other modification.

In one embodiment, the vertebrate RXR LBD is derived from a human Homo sapiens, from mouse Mus musculus, from rat Rattus norvegicus, from chicken Gallus gallus, from pig Sus scrofa domestica, from frog xenopus laevis, from zebrafish Danio rerio, of tunicate Polyandrocarpa misakiensis, or of medusa Tripedalia cysophora RXR.

In one embodiment, the ligand binding domain Invertebrate RXR is from a locusta migratory locusta migratory polypeptide ("LmUSP"), a 1 RXR homolog of the ixodid tick Amblyomma americanum ("AmaRXRl"), a RXR homolog of the ixodid tick Amblyomma americanum ("AmaRXR2") , an RXR homologue of the fiddler crab Celuca pugilator ("CpRXR"), a RXR homolog of the Tenebrio molitor beetle ("TmRXR"), an XRX homologue of the honey bee Apis mellifera ("AmRXR"), an RXR homologue of the aphid Myzus persicae ("MpRXR"), or a RXR homolog not of dipteran / no lepidoptera.

In one embodiment, (the RXR LBD comprises at least two polypeptide fragments selected from a vertebrate species RXR polypeptide fragment, an invertebrate species RXR polypeptide fragment, or a polypeptide fragment of the invertebrate species RXR homologue, no dipteran / no lipidopter A binding domain to the chimeric RXR ligand for use in the present invention may comprise at least two polypeptide fragments of RXR of different species, or when the species is the same, the two or more polypeptide fragments may be from two or more different isoforms of the RXR polypeptide fragment of the species These LBDs of chimeric RXR are described, for example, in WO 2002/066614.

In one embodiment, the chimeric RXR ligand binding domain comprises at least one vertex species RXR polypeptide fragment and a invertebrate species RXR polypeptide fragment.

In one embodiment, the chimeric RXR ligand binding domain comprises at least one vertex species RXR polypeptide fragment and a polypeptide fragment of the RXR homolog of invertebrate species not of dipterid / non-lepidopteran.

The ligand, when combined with the LBD of the nuclear receptor (s), which in turn are linked to the response element of an FRP associated with a sequence of the therapeutic product, provides external temporal regulation of the expression of the therapeutic product sequence. . The binding mechanism or the order in which the various components of this invention are linked to one another, ie, for example, the ligand to LBD, DBD to the response element, AD to the promoter, etc., is not critical .

In a specific example, the binding of the ligand to the LBD of a nuclear receptor of Group H and its LBD partner of the nuclear receptor, makes possible the expression of the sequence of the therapeutic product. This mechanism does not exclude the potential for ligand binding to the Group H nuclear receptor (GHNR) or its partner, and the resulting formation of active homodimer complexes (eg, GHNR + GHNR or partner + partner). ). Preferably, one or more of the receptor domains is varied producing a hybrid gene change. Typically, one or more of the three domains, DBD, LBD, and AD, can be chosen from a source different from the source of the other two domains, so that hybrid genes and resulting hybrid proteins are optimized in the cell or host organism chosen for the transactivation activity, the complementary link of the ligand, and the recognition of a specific response element. In addition, the response element itself can be modified or substituted with the response element for other domains of the DNA binding protein, such as the GAL-yeast protein (see Sadowski et al, Nature 555: 563 (1988)) or the LexA protein of Escherichia coli (see Brent et al., Cell 43: 729 (1985)), or the specific synthetic response elements for targeted interactions with proteins designed, modified, and selected for such specific interactions (see, for example, Kim et al, Proc. Nati, Acad. Sci. USA, 94: 3616 (1997)) to accommodate hybrid receptors. Another advantage of the two hybrid systems is that they allow the choice of a promoter used to boost the expression of the gene according to a desired final result. Such double control can be particularly important in the areas of gene therapy, especially when cytotoxic proteins are produced, because the synchronization of expression as well as the cells in which the expression occurs can be controlled. When genes, operably linked to a suitable promoter, are introduced into the cells of the subject, the expression of the exogenous genes is controlled by the presence of the system of this invention. The promoters may be constitutively or unutterably regulated, or they may be tissue-specific (i.e., expressed only in a particular type of cells) or specific to certain stages of organism development.

The DNA binding domain to the first hybrid protein binds, presence or absence of a ligand, to the DNA sequence of a response element to initiate or suppress the transcription of the gene (s) with 3 'direction under the regulation of this element of response.

Functional LDTFC, for example, an EcR complex, may also include one or more additional proteins such as immunophilins. Additional members of the nuclear receptor family of proteins, known as transcriptional factors (such as DHR38 or betaFTZ-1), may also be dependent or independent partners of the ligand for EcR, USP, and / or RXR. In addition, other co-factors may be required such as proteins generally known as co-activators (also referred to as adapters or mediators). These proteins do not specifically bind in sequence to DNA and are not involved in basal transcription. These can exert their effect on the activation of transcription through various mechanisms, including the stimulation of the DNA linkage of the activators, by affecting the structure of the chromatin, or by mediating activator-complex or initiation interactions. Examples of such co-activators include RIP 40, TIF1, RAP46 / Bag-1, ARA70, SRC-1 / NCoA-1, TIF2 / GRIP / NCOA-2, ACTR / AIBl / RAC3 / pCIP as well as the binding protein to the promiscuous B co-activator response element, CBP / p300 (for review see Glass et al, Curr Opin Cell Cell 9: 222 (1997)). Also, protein cofactors generally known as co-repressors (also known as repressors, silencers, or silencing mediators) may be required to effectively inhibit transcriptional activation in the absence of the ligand.These co-repressors may interact with the EcR unbound to silence the activity in the response element.The current evidence suggests that ligand binding changes the receptor formation, which results in the release of the co-repressor and the recruitment of the co-activators described above, with which suppresses its silencing activity.Examples of co-repressors include N-CoR and SMRT (for review, see Horwitz et al., Mol Endocrinol., 10: 1167 (1996).) These co-factors can be either endogenous within the cell or organism, or they can be added exogenously, as transgenes to be expressed in a regulated or unregulated manner.

B. Change of Rapamycin-based Gene The present invention further provides a gene switching system that uses the F 506 binding protein as the ligand-dependent transcription factor complex, and rapamycin as the ligand. In one embodiment, the construct that codes for gene change comprises: (a) a first polynucleotide that codes for a first chimeric protein that binds to rapamycin or an analogue thereof, and that comprises at least one domain of the FK506 binding protein (FKBP) and at least one protein domain heterologous to this, wherein the FKBP domain comprises a peptide sequence selected from: (1) a FKBP of natural origin (2) a variant of a FKBP of natural origin in which up to 10 amino acid residues have been deleted, inserted or replaced with substitute amino acids, and (3) an FKBP that encodes a DNA sequence that hybridizes selectively to a DNA sequence encoding an FKBP of (1) 6 (2); (b) a second polynucleotide encoding a second chimeric protein that forms a complex with (a) rapamycin or a rapamycin analog (b) the first chimeric protein, and comprising at least one FKBP binding domain: rapamycin ( FRB) and at least one protein domain heterologous thereto, wherein the FRB domain comprises a peptide sequence selected from: (4) a natural FRB domain, (5) a variant of a FRB domain of natural origin, in which up to 10 amino acid residues have been deleted, inserted, or replaced with substitute amino acids, and (6) an FRB domain encoded by a DNA sequence that hybridizes selectively to a DNa sequence encoding an FRB of (4) or (5).

In this system of gene switching, each of the first polynucleotide and the second polynucleotide are under the control of one or more therapeutic change promoters, as described elsewhere herein. In addition, in certain embodiments, at least one protein domain heterologous to the FKBP and / or FRB domains in the first and second chimeric proteins may be one or more "action" or "effector" domains. Effector domains can be selected from a wide variety of protein domains including DNA binding domains, transcription activation domains, cell localization domains and signaling domains (e.g., domains that are capable of clustering or multimerization , to trigger cell growth, proliferation, differentiation, apoptosis, and gene transcription, etc.).

In certain embodiments, a fusion protein contains at least one DNA binding domain (e.g., a DNA binding domain of GAL4 or ZFHD1) and another fusion protein contains at least one transcription activation domain (e.g. , an activation domain upon activation of VP 16 or p65). The association bound by the ligand of the fusion proteins represents the formation of a complex of the transcription factor and leads to the initiation of the transcription of a target gene linked to a DNA sequence recognized by, (for example, capable of binding to) the DNA binding domain on one of the fusion proteins. Information regarding the gene expression system has been how the ligand is described in US Patents Nos. 6,187,757 Bl, 6,649,595 Bl, 6,509, 152 Bl, 6,479,653 Bl, and 6, 117,680 Bl.

In other embodiments, the present invention provides a gene exchange system comprising polynucleotides that encode two fusion proteins which autoaggregate in the presence of a ligand, wherein (a) the first fusion protein comprises a conditional aggregation domain which binds to a selected ligand and a transcriptional activation domain, and (b) the second fusion protein comprising a conditional aggregation domain that binds to a selected ligand and a DNA binding domain, and (c) ) in the absence of a ligand, the cells express a gene operably linked to the regulatory DNA to which the DNA enlácela domain binds. Modified cells comprising the gene change system are expanded in the presence of a ligand in an amount sufficient for repression of the gene. The elimination of the ligand induces the expression of the encoded protein that causes cell death. The nucleic acids encoding the two fusion proteins are under the control of at least one conditional promoter. The gene expression system using the conditional aggregation domains is described in U.S. Publication No. 2002/0048792.

C. Prokaryotic Operator-Based Gene Shift System In one embodiment, the present invention provides the gene switching system consisting of (a) a first polynucleotide that encodes a transactivator fusion protein, comprising a eukaryotic tetracycline repressor ("tet") and a protein binding domain. euciotic transcriptional activator; and (b) a second polynucleotide encoding a therapeutic protein or therapeutic polypeptide, wherein the second polynucleotide is operably linked to a minimal promoter and at least one tet operator sequence. The first polynucleotide that codes for a transactivator fusion protein can comprise the therapeutic change promoter as described elsewhere herein. The expression of the lethal protein is supra-regulated in the absence of tetracycline, (see, for example, Gossen et al. (1992) Proc. Nati. Acad. Sci. 89: 5547-5551; .Gossen et al. (1993) TIBS 18: 471-475; Furth et al. (1994) Proc. Nati. Acad. Sci. 91: 9302-9306; and Shockett et al. (1995) Proc. Nati. Acad. Sci. 92: 6522-6526) . The Teto expression system is described in U.S. Patent No. 5,464,758 Bl.

In yet another embodiment, the gene change system comprises the lactose repressor-operator systems ("Lac") of the bacterium Escherichia coli. The gene switching system of the present invention may also comprise (a) a first polynucleotide that encodes a transactivator fusion protein comprising a prokaryotic lac I repressor and a eukaryotic transcriptional activator protein domain; and (b) a second polynucleotide that encodes a therapeutic protein or therapeutic polypeptide, wherein the second polynucleotide is operably linked to a therapeutic change promoter. In the Lac system, a lac operon is inactivated in the absence of lactose, or synthetic analogues such as isopropyl-β-D-thiogalactoside. .

Additional gene change systems include those described in the following documents: US7, 091, 038; WO2004078924; EP1266015; US20010044151; US20020110861; US20020119521; US20040033600; US20040197861; US20040235097; US20060020146; US20040049437; US20040096942; US20050228016; US20050266457; US20060100416; WO2001 / 70816; WO2002 / 29075; WO2002 / 066612; WO2002 / 066613; WO2002 / 066614; WO2002 / 066615; O2005 / 108617; US 6,258,603; US20050209283; US20050228016; US20060020146; EP0965644; US 7,304,162; US 7,304,161; MX234742; KR10-0563143; AU765306; AU2002-248500; and AU2002-306550.

D. Combination of Gene Change Systems The present invention provides nucleic acid compositions, modified cells, and bioreactors comprising two or more gene exchange systems comprising different ligand-dependent transcription factor complexes that are activated by an effective amount of one or more ligands, wherein two or more gene exchange systems comprise a first gene change and a second gene change, which selectively induce the expression of one or more therapeutic polypeptides or therapeutic polypeptides, after binding to one or more ligands. Within the scope of the present invention, there are any numbers of and / or combinations of gene change systems.

In one embodiment, the present invention provides a nucleic acid composition comprising: to. a first system of gene change comprising: i. a first expression cassette of the gene comprising a polynucleotide encoding a first hybrid polypeptide including: 1. a transactivation domain, which activates a promoter regulated by the factor, operably associated with polynucleotide encoding a therapeutic polynucleotide or therapeutic polynucleotide; Y 2. a heterodimeric partner domain, ii. a second cassette of expression of the gene comprising a polynucleotide encoding a second hybridization polypeptide comprising: 1. a DNA binding domain, which recognizes a promoter regulated by the gene, operably associated with a polynucleotide encoding a therapeutic therapeutic polypeptide or polynucleotide; Y 2. a ligand binding domain; Y iii. a third cassette of expression of the gene comprising a polynucleotide encoding a polypeptide or a therapeutic polynucleotide comprising: 1. a promoter regulated by the factor, which is activated by the transactivation domain of the second hybrid polypeptide; Y 2. a polynucleotide encoding a therapeutic polypeptide or a therapeutic polynucleotide, and b. a second gene expression system comprising: i. a first expression cassette comprising a polynucleotide encoding a hybrid polypeptide, which comprises: 1. a transactivation binding domain, which activates, a promoter regulated by the factor, operably associated with a polynucleotide encoding a therapeutic polypeptide or a therapeutic polynucleotide; Y 2. a heterodimer partner domain, ii. a second an expression cassette comprising a polynucleotide encoding a second hybrid polypeptide, which comprises: 1. a DNA binding domain, which rearranges a promoter regulated by the factor, operably associated with a polynucleotide encoding a therapeutic polypeptide or a therapeutic polynucleotide; Y 2. a ligand binding domain; Y iii. a third cassette of expression of the gene comprising a polynucleotide encoding a polypeptide or a therapeutic polynucleotide comprising: 1. a promoter regulated by a factor, which is activated by the transactivation domain of the second hybrid polypeptide; Y, 2. a polynucleotide that encodes a therapeutic polypeptide or a therapeutic polynucleotide.

The multiple inducible gene expression systems provide for the expression of a given therapeutic polynucleotide or therapeutic polypeptide, under conditions associated with different diseases, disorders or conditions, or the expression of multiple therapeutic polypeptides or polynucleotides either under the same conditions associated therewith. disease, disorder or condition, or under different conditions associated with different diseases, disorders or conditions.

In certain embodiments, the combination of two or more gene change systems can be (1) a gene expression system based on the ecdysone receptor, double-shifted, and (2) a gene change based on the ecdysone receptor of change simple. In other embodiments, the combination may be (1) a gene change based on the single or double ecdysone receptor, and (2) a gene change based on rapamycin. Alternatively, the combination of the gene switching systems may be two or identical, rapamycin-based gene switching systems, described above. Any possible combinations of gene change systems are within the scope of the invention. Examples of dual-exchange ecdysone systems can be found for example in O 2001/29075 and US 2002/0110861 Ligands As used herein the term "ligand" as applied to gene changes based on LDTFC, for example, gene changes based on the EcD complex, describes small and soluble molecules that are capable of activating a gene change to stimulate the expression of a polypeptide encoded therein. The ligand for a complex 154 of the ligand-dependent transcription factor of the invention binds to the protein complex comprising one or more ligand binding domains, the heterodimer partner domain, the DNA binding domain, and the transactivation domain. The choice of ligand to activate the complex of the ligand-dependent transcription factor depends on the type of gene change used.

Examples of ligands include, without limitation, an ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid,?,? '- diacylhydrazines such as those described in U.S. Patent Nos. 6,013, 836; 5,117", 057; 5,530,028; and 5,378,726 and US Requests Nos. 2005/0209283 and 2006/0020146; oxadiazolines as described in U.S. Published Application No. 2004/0171651; dibenzoylalkyl-cyanohydrazines such as those described in European Application No. 461,809; N-alkyl-N, N '-diaroylhydrazines such as those described in Patent No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those described in European Application No. 234,994; aroyl-N-alkyl-N'-arylhydrazines such as those described in Patent No. 4,985,461, amidoketones such as those described in U.S. Published Application No. 2004/0049037, each of which is incorporated by reference into present, and other materials and the like including 3, 5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpAGE, oxysterols, 22 (R) -hydroxycholesterol, 24 (S) - hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, famesol, bile acids, 1,1-bisphosphonate esters, juvenile hormone III, and Similar. Examples of diacylhydrazine ligands useful in the present invention include N- (1-ethyl-2, 2-dimethyl-propyl) -N '- (2-methyl-3-methoxy-benzoyl) -hydrazide) acid (3 , 5-dimethyl-benzoic acid), RG-115932 N- (1-tert-butyl-butyl) -N '- (2-ethyl-3-methoxy-benzoyl) -hydrazide) of (R) -3,5- dimethyl-benzoic, and RG-115830 N- (1-tert-butyl-butyl) - '- (2-ethyl-3-methoxy-benzoyl) -hydrazide) of (3,5-dimethyl-benzoic acid). See, for example, U.S. patent application Serial No. 12 / 155,111, and PCT application No. PCT / US2008 / 006757, which are incorporated by reference herein in their entirety.

For example, a ligand for gene change based on the ecdysone receptor can be selected from any suitable ligands. The ecdysone or ecdison analogues of natural origin (eg, 20-hydroxyecdysone, muristerone A, ponasterone A, ponasterone B, ponasterone C, 26-iodoponasterone A, inokosterone or 26-mesilinokosterone) and the non-steroidal inducers can be used as a ligand for the gene change of the present invention. Patent No. 6,379,945 Bl, describes an insect steroid receptor isolated from Heliothis virescens ("HEcR") which is capable of acting as a gene change that responds to certain steroidal and non-steroidal inducers. Non-steroidal inducers have a distinct advantage over steroids, in this and many other systems that respond to steroid and non-steroidal inducers, for a number of reasons including, for example: lower manufacturing cost, metabolic stability, lack of insects, plants or mammals, and environmental acceptability. U.S. Patent No. 6,379,945 Bl describes the utility of two dibenzoylhydrazines, 1,2-dibenzoyl-1-tert-butyl-hydrazine and tebufenozide (N- (4-ethylbenzoyl) -N 1 - (3,5-dimethylbenzoyl) ) - '-tert-butyl-hydrazine) as ligands for a gene change based on ecdysone. Also included in the present invention as a ligand are other dibenzoylhydrazines, such as those described in U.S. Patent No. 5,117,057 Bl. The use of tebufenozide as a chemical ligand for the ecdysone receptor of Drosophila melanogaster is also described in U.S. Patent No. 6,147,282. Additional non-limiting examples of ecdysone ligand are 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-0- acetylharpide, a 1,2-diacylhydrazine, an N'-substituted-N, '-disubstituted hydrazine, a dibenzoylalkyl-cyanohydrazine, an N-substituted-N-alkyl-N, -diaroyl-hydrazine, an N-substituted-N- acyl-N-alkyl-carbonylhydrazine or an N-aroyl-N '-alkyl-N' -aryl idrazine, (See U.S. Patent No. 6,723,531).

In one embodiment, the ligand for a gene exchange system based on ecdysone is a diacylhydrazine ligand or a chiral diacylhydrazine ligand. The ligand used in the gene exchange system can be the compounds of formula I.

Formula I where A is alkoxy, arylalkyloxy or aryloxy; B is optionally substituted aryl or optionally substituted heteroaryl; Y R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocycle, optionally substituted aryl, or optionally substituted heteroaryl; or the salts, hydrates, crystalline forms or amorphous forms thereof, all pharmaceutically acceptable.

In yet another embodiment, the ligand may be enantiomerically enriched compounds of Formula II Formula II where A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substituted aryl, or optionally substituted heteroaryl; B is optionally substituted aryl or optionally substituted heteroaryl; Y R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocycle, optionally substituted aryl, or optionally substituted heteroaryl; with the proviso that Rl is not equal to R2; wherein the absolute configuration at the asymmetric carbon atom possessing Rl and R2 is predominantly S; or the salts, hydrates, crystalline forms or amorphous forms thereof, all pharmaceutically acceptable.

In certain embodiments, the ligand can be enantiomerically enriched compounds of Formula III Formula III where A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substituted aryl, or optionally substituted heteroaryl; B is optionally substituted aryl or optionally substituted heteroaryl; Y R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocycle, optionally substituted aryl, or optionally substituted heteroaryl; with the proviso that Rl is not equal to R2; wherein the absolute configuration in the asymmetric carbon atom possessing R1 and R2 is predominantly salts, hydrates, crystalline forms or amorphous forms thereof, all pharmaceutically acceptable.

In one embodiment, a ligand may be N- (1-tert-butyl-butyl) -N 1 - (2-ethyl-3-methoxy-benzoyl) -hydrazide of (R) -3,5-dimethyl-benzoic acid , which has an enantiomeric excess of at least 95% or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.

The diacylhydrazine ligands of Formula I and the chiral diacylhydrazine ligands of Formula II or III, when used with an ecdysone-based gene switching system, provide the means for external temporal regulation of the expression of a therapeutic polypeptide or of a therapeutic polynucleotide of the present invention. See United States application No. 12 / 155,111, filed May 29, 2008, which is incorporated by reference herein.

The ligands used in the present invention can form salts. The term "salt or salts" as used herein, denotes the acid and / or basic salts formed with organic and / or inorganic acids and bases. In addition, when a compound of Formula I, II or III contains a basic portion and an acid portion, amphoters ("internal salts") can be formed and are included within the term "salt or salts" as used herein. Pharmaceutically acceptable salts (for example, non-toxic, pharmaceutically acceptable salts are used, although other salts are also useful, for example, in isolation or purification steps which can be used during the preparation.) The salts of the compounds of Formulas I , II or III can be formed, for example, by reacting a compound with an amount of base acid, such as an equivalent amount, in a medium such as one in which the salt is precipitated or in an aqueous medium followed by lyophilization Ligands containing a basic portion can form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, , camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glycoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), iodides, 2-hydroxyethane sulfonates, lactates, maleates (formed with maleic acid), methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulphates (such as formed with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.

Ligands containing an acidic portion can form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts, such as sodium, lithium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases (e.g., organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N, N-bis (dehydroabiethyl) ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t-butylamines, and salts with amino acids such as arginine, lysine , and similar.

Non-limiting examples of the ligands for the inducible gene expression system using the FK506 binding domain are FK506, Cyclosporin A, or Rapamycin. FK506, rapamycin, and its analogs are described in U.S. Patent Nos. 6,649,595 B2 and 6,187,757. See also United States Patents Nos. 7,276,498 and 7, 273, 874.

The ligands described herein may be administered alone or as part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition is in the form of solutions, suspensions, tablets, capsules, ointments, elixirs or injectable compositions.

In one embodiment, the vector and methods of the present invention can be used to express a polynucleotide that encodes a protein that includes, but is not limited to, a cyclin, an immunomodulator, a coagulation factor, an antibody or a fragment. of an antibody, a tumor necrosis factor receptor (TNFR), such as Ertanercept, an erythroproetin, an anti-trypsin alpha-1, an interferon (IFN), interferon-alpha, interferon-beta, interferon-gamma, interferon- beta-la, interferon-beta-1b, factor VII, Facor VIII, Factor IX, antithrombin III, a pretein of hepatitis B virus, a hormone, for example, a growth hormone (GH), human growth hormone (hGH), parathyroid hormone (PH), thyroid stimulating hormone (TSH), GCSF or fragment thereof, GM-CSF or a fragment thereof.

In one embodiment, the polynucleotide encoding an antibody codes for a monoclonal antibody.

In another embodiment, the vector and methods of the present invention can be used to express nucleic acid as a vaccine. The present invention also provides a vaccine composition comprising a vector or expression system of the present invention. In another embodiment, the vaccine composition comprises an adjuvant.

The term "ecdysone receptor-based", with respect to a gene change, refers to a gene change comprising at least a functional part of a ligand binding domain of the ecdysone receptor, of natural or synthetic origin, and the which regulates the expression of the gene in response to a ligand that binds to the ligand binding domain of the ecdysone receptor. Examples of systems that respond to ecdysone are described in U.S. Patent Nos. 7,091,038 and 6,258,603. In one embodiment, the system is the RheoSwitch® therapeutic system (RTS), which contains two fusion proteins, the domains of a mutagenized ecdysone receptor (EcR) fused to a Gal4 DNA binding domain and the EF domain of a chimeric RXR fused to a VP16 transcription activation domain, expressed under a constitutive promoter as illustrated in Figure 1.

The terms "modulate" and "modulate" mean that they induce, reduce or inhibit the expression of the nucleic acid or gene, resulting in the respective induction, reduction or inhibition of the production of protein or polypeptide.

The polynucleotides or vectors according to the invention may further comprise at least one promoter suitable for driving the expression of a gene in a host cell.

Enhancers that can be used in the embodiments of the invention include, but are not limited to: an SV40 enhancer, a cytomegalovirus enhancer (CMV), an elongation factor 1 enhancer (EF1), yeast enhancers, enhancer viral genes and the like.

The control regions of the termination, for example, the terminator or polyadenylation sequences, can also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is more preferred if it is included. In one embodiment of the invention, the termination control region may be comprised of or be derived from a synthetic sequence, synthetic polyadenylation signal, a late SV40 polyadenylation signal, a SV40 polyadenylation signal, a polyadenylation signal from the bovine growth hormone (BGH), viral terminator sequences, or the like.

The terms "3 'non-coding sequences" or "untranslated region 31 (UTR)" refer to DNA sequences located downstream (31) of a coding sequence, and may comprise polyadenylation recognition sequences [poly. (A)] and other sequences that encode regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of stretches of polyadenylic acid towards the 3 'end of the mRNA precursor.

"Regulatory region" refers to a nucleic acid sequence that regulates the expression of a second nucleic acid sequence. A regulatory region may include sequences that are naturally responsible for the expression of a particular nucleic acid (a homologous region) or may include sequences of a different origin that are responsible for the expression of different proteins or even synthetic proteins (a heterologous region). In particular, the sequences may be sequences of prokaryotic, eukaryotic or viral genes or derived sequences that stimulate or repress the transcription of a gene in a specific or non-specific manner, and in an inducible or non-inducible manner. Regulatory regions include origins of replication, splice sites to the AR, promoters, enhancers, transcription termination sequences, and signal sequence that direct the polypeptide to the secretory pathways of the target cell.

A regulatory region from a "heterologous source" refers to a regulatory region that is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are the regulatory regions from different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences that do not occur in nature but are designed by a person who has ordinary experience in the field. technique.

"RNA transcript" refers to the product resulting from transcription catalyzed by the RNA polymerase of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as a primary transcript and can be an RNA sequence derived from the post-transcriptional processing of the primary transcript, and is referred to as mature RNA. "Messenger RNA (mRNA)" refers to RNA that is without introns and that can be translated into protein by the cell. "CDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. RNA "in sense" refers to the RNA transcript that includes the mRNA and thus can be translated into protein by the cell. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene. The complementarity of an antisense RNA can be with any part of the transcript of the specific gene, for example, in the 5 'non-coding sequence, the non-coding sequence 31, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozimal RNA, or other RNA that is not yet translated but has an effect on cellular processes.

"Polypeptide", "peptide" and "protein" are used interchangeably and refer to a polymeric compound comprised of covalently linked amino acid residues.

An "isolated polypeptide", "isolated peptide" or "isolated protein" refers to a peptide or protein that is substantially free of those compounds that are normally associated with it in its natural state (eg, other proteins or polypeptides, nucleic acids , carbohydrates, lipids). "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities that do not interfere with biological activity, and which may be, for example, due to incomplete purification, the addition of stabilizers, or the composition in a pharmaceutically acceptable preparation.

A "substitution mutant polypeptide" or "substitution mutant" will mean a mutant polypeptide comprising a substitution of at least one wild type or naturally occurring amino acid with a different amino acid relative to the wild-type or naturally occurring polypeptide. A substitution mutant polypeptide may comprise only a wild-type or naturally-occurring amino acid substitution, and may be referred to as a "point mutant" or single "point mutant" polypeptide. Alternatively, a substitution mutant polypeptide may comprise a substitution of two or more wild type or naturally occurring amino acids with two or more amino acids relative to the wild-type or naturally occurring polypeptide. According to the invention. According to the invention, a ligand binding domain polypeptide of the nuclear group H receptor, comprising a substitution mutation, comprises a substitution of at least one wild type or naturally occurring amino acid, with a different amino acid in relation to to the ligand binding domain polypeptide, of the group H nuclear receptor, wild type or of natural origin.

When the substitution mutant polypeptide comprises a substitution of two or more wild type or naturally occurring amino acids, this substitution may comprise either an equivalent number of wild type or naturally occurring amino acids deleted for substitution, eg, two amino acids wild-type or of natural origin replaced with two amino acids not of wild origin or of non-natural origin, or a non-equivalent number of wild-type amino acids deleted for substitution, for example, two wild-type amino acids substituted with one amino acid of type wild type (a substitution mutation + deletion), or two wild type amino acids replaced with three non-wild type amino acids (a substitution + insertion mutation).

Substitution mutants can be described using an abbreviated nomenclature system to indicate the amino acid residue and the number replaced within the sequence of the reference polypeptide and the new substituted amino acid residue. For example, a substitution mutant in which the twentieth (20th) amino acid residue of a polypeptide is substituted, can be abbreviated as "x20z", where "x" is the amino acid to be replaced, "20" is the position of the amino acid residue or the number within the polypeptide, and "z" is the new substituted amino acid. Therefore, a substitution abbreviation interchangeably abbreviated, "E20A" or "Glu20Ala" indicates that the mutant comprises an alanine residue (commonly abbreviated in the art as "A" or "Ala") in place of glutamic acid (commonly abbreviated in the art as "E" or "Glu") at position 20 of the polypeptide.

A substitution mutation can be performed by any technique for mutagenesis, known in the art, including but not limited to site-directed mutagenesis in vitro (Hutchinson et al, J. Biol. Chem. 253: 6551 (1978); Zoller et al. al, DNA 3: 479 (1984), Oliphant et al, Gene 44: 177 (1986), Hutchinson et al, Proc. Nati Acad. ScL USA 83: 710 (1986)), the use of ® linkers (Pharmacia) , restriction endonuclease digestion / deletion or substitution of fragments, PCR-mediated / oligonucleotide-directed mutagenesis, and the like. PCR-based techniques are preferred for site-directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA," in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chap. 6, pp. 61-70).

The term "fragment", as applied to a polypeptide, refers to a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence . Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part. Such fragments of a polypeptide according to the invention can have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 or more amino acids.

A "variant" of a polypeptide or protein refers to any analogue, fragment, derivative, or mutant that is derived from a polypeptide or protein, and which retains at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants can be allelic variants characterized by differences in the nucleotide sequence of the structural gene coding for the protein, or they can involve differential splicing or post-translational modification. The person skilled in the art can produce variants that have single or multiple substitutions, deletions, additions or replacements of amino acids. These variants may include, among others: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) ) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin. The techniques for obtaining these variants, including genetic techniques (deletions, omissions, mutations, etc.), chemical and enzymatic, are known to people who have ordinary experience in the field. In one embodiment, a variant polypeptide comprises at least about 14 amino acids.

The term "homology" refers to the percent identity between two polynucleotide portions or two portions of polypeptide. The correspondence between the sequence from one portion to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by alignment of the sequence information and the use of readily available computer programs. Alternatively, homology can be determined by hybridization of the polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with one or more specific single-stranded nucleases, and size determination of the digested fragments.

As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins possessing a "common evolutionary origin", including proteins from superfamilies (for example, the superfamily of immunoglobulins), and homologous proteins from different species (e.g., light chain of .myosin etc.) (Reeck et al, Cell 50: 667 (1987)). Such proteins (and their coding genes) have sequential homology, as reflected by their high degree of sequential similarity. However, in common usage and in application, the term "homologous", when modified with an adverb such as "highly", may refer to the similarity of the sequence and not to a common evolutionary origin.

Accordingly, the term "sequential similarity" in all its grammatical forms refers to the degree of identity or correspondence between the nucleic acid or amino acid sequences of the proteins, which may or may not share a common evolutionary origin (see Reeck et al. , Cell 50: 667 (1987)). In one embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 50% (eg, at least about 75%, 90%, or 95%) of the nucleotides agree on the defined length of the DNA sequence. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in the sequence data banks, or in a Southern hybridization experiment, for example, under stringent conditions as defined for that particular system. The definition of the appropriate hybridization conditions is within the skill of the art (see for example, Sambrook et al, 1989, supra).

As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases result in the substitution of one or more amino acids, but do not affect the functional properties of the encoded protein by the DNA sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate the alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid fragments of the invention, such as the deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Each of the proposed modifications is well within the routine experience in the art, as is the determination of the biological activity retention of the coded products.

In addition, the skilled person recognizes that the substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under strict conditions (0.1X SSC, 0.1% SDS, 65 ° C and washed with 2X SSC, 0.1% SDS followed by 0.1 X SSC, 0.1% SDS), with the sequences exemplified herein. The substantially similar nucleic acid fragments of the invention are those fragments of nucleic acid whose DNA sequences are at least about 70%, 80%, 90% or 95% identical to the DNA sequence of the nucleic acid fragments reported in the I presented.

Two amino acid sequences are "substantially homologous" or "substantially similar" when more than about 40% of the amino acids are identical, or more than 60% are similar (functionally identical). Preferably, similar or homologous sequences are identified by alignment using, for example, the GCG program (Genetics Computer Group, Manual Program for the GCG package, Version 7, Madison, Wisconsin).

The term "corresponding to" is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. The alignment of the nucleic acid or amino acid sequence may include spaces. In this way, the term "corresponding to" refers to the sequence similarity and not to the numbering of amino acid residues or nucleotide bases.

A "substantial portion" of an amino acid or nucleotide sequence comprises sufficient of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by a person skilled in the art, or by automated computerized sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al, J. Mol Biol. 215: 403 (1993)); available at ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. In addition, with respect to nucleotide sequences, oligonucleotide probes specific for the gene comprising 20-30 contiguous nucleotides can be used in sequence dependent methods, gene identification (eg, Southern hybridization) and isolation ( for example, in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 to 15 bases can be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises sufficient of the sequence to identify and / or specifically isolate a nucleic acid fragment comprising the sequence.

The term "percent identity", as is known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparison of the sequences. In the art, "identity" also means the degree of sequential kinship between the polypeptide or polynucleotide sequences, as the case may be, as determined by the agreement between stretches of such sequences. The "identity" and "similarity" can be easily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). The preferred methods for determining identity are designed to give the best match between the tested sequences. Methods for determining identity and similarity are codified in publicly available computer programs. Sequential alignments and percentage identity calculations can be performed using sequence analysis software such as the Megalign program from the LASERGENE bioinformatics computation program (DNASTAR Inc., Madison, WI). Multiple alignments of the sequences can be performed using the Clustal method of alignment (Higgins et al, CABIOS 5: 151 (1989)), with the default parameters (PENALTY FOR EMPTY SPACE = 10, PENALTY FOR LENGTH OF EMPTY SPACE = 10 ). The default parameters for paired alignments using the Clustal method can be selected: KTUPLE 1, EMPTY SPACE PENALTY = 3, WINDOW = 5 and SAVED DIAGONALS - 5.

The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. The "sequence analysis software" can be commercially available or independently developed. Typical sequence analysis software includes, but is not limited to, the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J Mol. Biol. 215: 403 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA). Within the context of this application, it will be understood that where the sequential analysis software is used for the analysis, the results of the analysis will be based on the "default values" of the referred program, unless otherwise specified. As used herein, "default values" shall be understood as any group of values or parameters that are originally loaded with the software when it is first initialized.

"Chemically synthesized", related to a DNA sequence, means that the component nucleotides can be assembled in viii. Manual chemical synthesis of DNA can be achieved using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression, based on the optimization of the nucleotide sequence to reflect the deviation of the host cell codon. The person skilled in the art appreciates the probability of successful gene expression if the use of the codon is diverted towards those codons favored by the host. The determination of the preferred codons can be based on a monitoring of the genes derived from the host cell, where the sequence information is available.

As used herein, it is said that two or more individually operable gene regulation systems are "orthogonal" when; a) the modulation of each of the systems given by their respective ligand, at a chosen concentration, results in a measurable change in the magnitude of the gene expression of that system, and b) the change is statistically and significantly different from the change in the expression of all other systems simultaneously operable in the cell, tissue, or organism, notwithstanding the simultaneity or sequentiality of the effective modulation. Preferably, the modulation of each individually operable gene regulation system effects a change in gene expression at least two times greater than all other systems operable in the cell, tissue or organism, eg, 5 times, 10 times, 100 times, or 500 times higher. Ideally, the modulation of each of the systems given by their respective ligand, at a chosen concentration, results in a measurable change in the magnitude of the gene expression of that system, and no measurable change in the expression of all the others systems operable in the cell, tissue or organism. In such cases, it is said that the system of multiple inducible gene regulation is "completely orthogonal". Useful orthogonal ligands and gene expression systems based on orthogonal receptors are described in US 2002/0110861 Al.

The term "exogenous gene" means a foreign gene for the subject, i.e., a gene that is introduced into the subject through a transformation process, a non-mutated version of an endogenous mutated gene or a mutated version of a non-gene. endogenous mutant The transformation method is not critical to this invention, and can be any method suitable for the subject known to those skilled in the art. The exogenous genes can be either natural or synthetic genes that are introduced into the subject in the form of DNA or RNA, which can function through a DNA intermediate such as by reverse transcriptase. Such genes may be introduced into the target cells, directly introduced into a subject, or indirectly introduced by the transfer of transformed cells within the subject.

The term "therapeutic product" refers to a therapeutic polypeptide or therapeutic polynucleotide, which imparts a beneficial function to the host cell in which such a product is expressed. Therapeutic polypeptides can include, without limitation, polypeptides as small as three amino acids in length, single or multiple chain proteins, and fusion proteins. Therapeutic polynucleotides may include, without limitation, antisense oligonucleotides, small interfering RNAs, ribozymes, and external RNA leader sequences. The therapeutic product may comprise a sequence of natural origin, a synthetic sequence or a combination of natural and synthetic sequences.

The term "ligand-dependent transcription factor complex" or "LDTFC" refers to a transcription factor comprising one or more protein subunits, whose complex can regulate the expression of the gene driven by a "promoter regulated by the factor". "as defined herein. An LDTFC model is an "ecdysone receptor complex" which generally refers to a heterodimeric protein complex that has at least two members of the nuclear receptor family, in ecdysone receptor ("EcR") and ultraspiral protein ( "USP") (see Yao et al, Nature 366: 476 (1993)); Yao et al, Cell 71:63 (1992)). A functional LDTFC such as an EcR complex may also include one or more additional proteins such as immunophilins. Additional members of the family of nuclear receptor proteins, known as transcriptional factors (such as DHR38, betaFTZ-1 or other insect homologs), can also be dependent or independent partners of the ligand, for EcR and / or USP. An LDTFC such as an EcR complex can also be a heterodimer of the EcR protein and the vertebrate homologue of the ultraespiracle protein, the retinoic acid X receptor protein ("RXR") or a USP and RXR chimera. The terms "LDTFC" and "EcR complex" also encompass the homodimeric complexes of the EcR or USP protein, as well as the simple polypeptides or trimers, tetramers or other multimers that serve the same function.

An LDTFC such as an EcR complex can be activated by an active ecdysteroid or non-steroid ligand, linked to one of the complex proteins, including EcR, but not excluding other proteins of the complex. An LDTFC such as an EcR complex includes proteins that are members of the nuclear receptor superfamily, wherein all members are characterized by the presence of one or more polypeptide subunits that comprise an amino-terminal transactivation domain ("AD," " TD, "or" TA, "used interchangeably herein), a DNA binding domain (" DBD "), and a ligand binding domain (" LBD "). The AD can be present as a fusion with a "heterodimerization partner" or "HP". A fusion protein comprising an AD and HP of the invention is referred to herein as a "coactivation protein" or "CAP." The DBD and the LBD can be expressed as a fusion protein, herein referred to as a "ligand-inducible transcription factor (" LTF "). The fusion partners can be separated by linker, eg, a hinge region. Some members of the LTF family may also have another transactivation domain on the carboxyl-terminal side of the LBD.The DBD is characterized by the presence of two zinc fingers of cysteine, among which are two amino acid portions, the P-box and Table D, which confer specificity for the ecdysone response elements, These domains can be either native, modified or chimeras from different domains of heterologous receptor proteins.

The DNA sequences constituting the exogenous gene, the response element, and the LDTFC, for example, the EcR complex, can be incorporated into prokaryotic cells, archaebacteria, such as Escherichia coli, Bacillus subtilis, and other enterobacteria, or eukaryotic cells such as plant or animal cells. However, because many of the proteins expressed by the gene are incorrectly processed in bacteria, eukaryotic cells are preferred. The cells can be in the form of single cells or multicellular organisms. The nucleotide sequences for the exogenous gene, the response element, and the receptor complex, can also be incorporated as RNA molecules, preferably in the form of functional viral RNAs, such as the tobacco mosaic virus. Of eukaryotic cells, vertebrate cells are preferred because they naturally lack the molecules that confer responses to the ligands of this invention, for EcR. As a result, they are "substantially insensitive" to the ligands of this invention. Thus, the ligands useful in this invention will have negligible physiological effects or other effects on the transformed cells, or the entire organism. Therefore, the cells can develop and express the desired product, substantially unaffected by the presence of the ligand itself.

The term "ecdysone receptor complex" generally refers to a heterodimeric protein complex having at least two members of the nuclear receptor family, the ecdysone receptor ("EcR") and the ultrapyracle ("USP") proteins. (see Yao et al, Nature 366: 476 (1993)); Yao et al., Cell 71:63 (1992)). The functional EcR complex may also include one or more additional proteins such as immunofalynae. Additional members of the family of nuclear protein receptors, known as transcriptional factors (such as DHR38, betaFTZ-1 or other insect homologs), may also be ligand-dependent or independent partners for EcR and / or USP. The EcR complex can also be a heterodimer of the EcR protein and the vertebrate homologue of the ultraspiral protein, the retinoic acid X receptor protein ("RXR") or a USP and RXR chimera. The term EcR complex also encompasses the homodimeric complexes of the EcR or USP protein.

An EcR complex can be activated by an ecdysteroid or non-steroid active ligand, bound to one of the complex proteins, including EcR, but not excluding other proteins of the complex. As used herein, the term "ligand", as applied to gene changes based on EcR, describes small and soluble molecules that have the ability to activate a gene change to stimulate the expression of a polypeptide encoded therein. Examples of ligands include, without limitation, an ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N, N 1 -diacylhydrazines. such as those described in U.S. Patent Nos. 6,013,836; 5,117,057; 5,530,028; and 5,378,726 and Requests of the United States Nos. 2005/0209283 and 2006/0020146; oxadiazolines as described in United States Published Application No. 2004/0171651; dibenzoylalkyl-cyanohydrazines such as those described in European Application No. 461,809; N-alkyl-N, N '-diaroylhydrazines such as those described in Patent No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those described in European Application No. 234,994; N-aroyl-N-alkyl-1-arylhydrazines such as those described in Patent No. 4,985,461; amidoketones such as those described in U.S. Published Application No. 2004/0049037; and other materials and the like including 3, 5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpigide, oxysterols, 22 (R) -hydroxycholesterol, 24 (S) -hydroxycholesterol, - epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, famesol, bile acids, 1,1-bisphosphonate esters, juvenile hormone III, and the like. Examples of diacylhydrazine ligands useful in the present invention include N- (1-ethyl-2, 2-dimethyl-propyl) -N '- (2-methyl-3-methoxy-benzoyl) -hydrazide) acid (3 , 5-dimethyl-benzoic acid), RG-115932 N- (1-tert-butyl-butyl) -N| - (2-ethyl-3-methoxy-benzoyl) -hydrazide) of (R) -3,5- dimethyl-benzoic, and RG-115830 N- (1-tert-butyl-butyl) -N1 - (2-ethyl-3-methoxy-benzoyl) -hydrazide) of (3,5-dimethyl-benzoic acid). See, for example, United States patent application Serial No. 12 / 155,111, filed May 29, 2008, and PCT / US2008 / 006757 filed May 29, 2008, for additional diacylhydrazines which are useful in practice of the invention.

The EcR complex includes proteins that are members of the nuclear receptor superfamily where all the members are characterized by the presence of an amino-terminal transactivation domain ("TA"), a DNA binding domain ("DBD"), and a ligand binding domain ("LBD") separated by a hinge region. Some members of the LTF family may also have another transactivation domain on the carboxyl-terminal side of the LBD. The DBD is characterized by the presence of two zinc fingers of cysteine, among which are two amino acid portions, the P table and the D table, which confer specificity for the ecdysone response elements. These domains may be either native, modified or chimeras from different domains of heterologous receptor proteins.

The DNA sequences constituting the exogenous gene, the response element, and the EcR complex, can be incorporated into prokaryotic cells, archaebacteria, such as Escherichia coli, Bacillus subtilis, and other enterobacteria, or eukaryotic cells such as plant cells or animals. However, because many of the proteins expressed by the gene are incorrectly processed in bacteria, eukaryotic cells are preferred. The cells can be in the form of single cells or multicellular organisms. The nucleotide sequences for the exogenous gene, the response element, and the receptor complex, can also be incorporated as RNA molecules, preferably in the form of functional viral RNAs, such as the tobacco mosaic virus. Of eukaryotic cells, vertebrate cells are preferred because they naturally lack the molecules that confer responses to the ligands of this invention, for EcR. As a result, they are "substantially insensitive" to the ligands of this invention. Thus, the ligands useful in this invention will have negligible physiological effects or other effects on the transformed cells, or the entire organism. Therefore, the cells can develop and express the desired product, substantially unaffected by the presence of the ligand itself.

The EcR ligands when used with the EcR complex which in turn are linked to the response element linked to an exogenous gene (eg, IL-12), provide the means for the external temporal regulation of the expression of the exogenous gene. The order in which the various components bind to each other, i.e., the ligand to the receptor complex and the receptor complex to the response element, is not critical. Typically, the modulation of exogenous gene expression is in response to the binding of the EcR complex to a specific or regulatory control DNA element. The EcR protein, like other members of the nuclear receptor family, possesses at least three domains, a transactivation domain, a DNA binding domain, and a ligand binding domain. This receptor, as a subset of the nuclear receptor family, also has fewer well-defined regions responsible for the heterodimerization properties. The binding of the ligand to the ligand binding domain of the EcR protein, after heterodimerization with the USP or RXR protein, makes it possible for the DNA binding domains of the heterodimeric proteins to bind to the response element in an activated form, thus resulting in the expression or deletion of the exogenous gene. This mechanism does not exclude the potential for ligand binding to either EcR or USP, and the resultant formation of active homodimeric complexes (eg, EcR + EcR or USP + USP). In one embodiment, one or more of the receptor domains can be varied producing a chimeric gene change. Typically, one or more of the three domains can be chosen from a source different from the source of the other domains, so that the chimeric receptor is optimized in the chosen host cell or in the organism chosen for transactivation activity, the link complementary to the ligand and the recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domain, such as the GAL-4 protein of yeast (see Sadowski et al, Nature 335: 563 (1988) or the LexA protein of E. coli (see Brent et al, Cell 43: 729 (1985)) to accommodate chimeric EcR complexes Another further advantage of chimeric systems is that they allow the choice of a promoter used to boost the exogenous gene According to a desired end result, such double control can be particularly important in gene therapy areas, especially when cytotoxic proteins are produced, because the synchronization of the expression, as well as the cells in which the expression occurs, can be controlled When exogenous genes, operatively linked to a suitable promoter, are introduced into the cells of the subject, the expression of the exogenous genes is controlled by the presence a of the ligand of this invention. The promoters can be constitutively or inducibly regulated or they can be tissue-specific (i.e., expressed only in a particular type of cells) or specific for certain stages of organism development.

In certain modalities, the promoter of therapeutic change described in the methods is constitutive. In certain embodiments, the therapeutic change promoter is activated under conditions associated with a disease, disorder or condition, for example, the promoter is activated in response to a disease, in response to a particular physiological, developmental, differentiating or pathological condition. , and / or in response to one or more specific biological molecules; and / or the promoter is activated in a particular tissue or in particular cell types. In certain embodiments, the disease, disorder or condition responds to the therapeutic polypeptide or polynucleotide. For example, in certain non-limiting embodiments, the polynucleotide or therapeutic polypeptide is useful for treating, prevent, improve, reduce symptoms, prevent progression, or cure the disease, disorder or condition, but you do not need to achieve one or all of these things. In certain embodiments, the first and second polynucleotides are introduced to allow expression of the ligand-dependent transcription factor complex, under conditions associated with a disease, disorder or condition. In one embodiment, the therapeutic methods are carried out such that the therapeutic polypeptide or therapeutic polynucleotide is expressed and disseminated through the subject at a level sufficient to treat, ameliorate, or prevent said disease, disorder or condition. As used herein, "disseminated" means that the polypeptide is expressed and released from the modified cell, sufficiently to have an effect or activity on the subject. The dissemination can be systemic, local or anything between them. For example, the therapeutic polypeptide or the therapeutic polynucleotide can be systemically disseminated through the bloodstream or the lymphatic system. Alternatively, the therapeutic polypeptide or the therapeutic polynucleotide can be locally disseminated in a tissue or organ to be treated.

Numerous genomic nucleic acid and cDNA sequences that code for a variety of polypeptides such as transcription factors and reporter proteins are well known in the art. Here those skilled in the art have access to information on nucleic acid sequences for virtually all known genes, and can either obtain the nucleic acid molecule directly from a public depository, the institution that published the sequence, or employ methods routine to prepare the molecule. See for example the description of sequence access numbers, below.

The gene change can be any system of gene change that regulates the expression of the gene by the addition or elimination of a specific ligand. In another embodiment, the gene change is one in which the level of expression of the gene is dependent on the level at which the ligand is present. Examples of ligand-dependent transcription factors, which can be used in the gene changes of the invention include, without limitation, members of the superfamily of nuclear receptors activated by their respective ligands (eg, glucocorticoid, estrogen, progestin, retinoid , ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect of the invention, the gene change is a gene change based on EcR. Examples of such systems include, without limitation, the systems described in U.S. Patent Nos. 6,258,603, 7,045,315, U.S. Published Patent Applications Nos. 2006/0014711, 2007/0161086, and the International Published Application. No. WO 01/70816. Examples of ecdysone, chimeric receptor systems are described in U.S. Patent No. 7,091,038, U.S. Patent Publications Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and 2006/0100416, and International Published Applications Nos. WO 01/70816, WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO 02/29075, and WO 2005/108617. An example of a system regulated by non-steroidal ecdysone agonist is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs, Ipswich, MA).

In one embodiment, a polynucleotide encoding gene change comprises a sequence of the single transcription factor, which codes for a transcription factor dependent on the ligand, under the control of a promoter. The sequence of the transcription factor can encode a transcription factor dependent on the ligand, which is of natural origin or an artificial transcription factor. An artificial transcription factor is one in which the natural sequence of the transcription factor has been altered, for example, by mutation of the sequence or by combining the domains from different transcription factors. In one embodiment, the transcription factor comprises a ligand binding domain of the nuclear group H receptor (LBD). In one embodiment, the LBD of the Group H nuclear receptor is from an EcR, a ubiquitous receptor, an orphan receptor 1, an NER-I, a nuclear steroid hormone receptor, an interaction protein 15 with the retinoid X receptor , a liver X receptor, a protein similar to the steroid hormone receptor, a hepatic receptor X, a hepatic OI receptor X, a farnesoid X receptor, a protein 14 interacting with the receptor, or a farnesol receptor. In another modality, the LBD of the nuclear receptor of Group H is from an ecdysone receptor.

The EcR and the other nuclear receptors of Group H are members of the nuclear receptor superfamily, where all the members are generally characterized by the presence of an amino-terminal transactivation domain (TD), a DNA binding domain ( DBD), and a separate LBD of DBD by a hinge region. As used in this, the term "DNA binding domain" comprises a minimal polypeptide sequence of a DNA binding protein, up to the full length of a DNA binding protein, as long as the DNA binding domain functions to associate with an element of particular response. Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: A / B, C, D, E, and in some F members (see US 4,981,784 and Evans, Science 240: 889 (1988)) . The "A / B" domain corresponds to the trasactivation domain, "C" corresponds to the DNA binding domain, "D" corresponds to the hinge region, and "E" corresponds to the ligand binding domain. Some members of the family may also have another transactivation domain on the carboxyl-terminal side of the LBD corresponding to "F".

The DBD is characterized by the presence of two zinc fingers of cysteine between which are two amino acid portions, the P table and the D table, which can confer specificity for response elements. These domains can be either native, modified or chimeras of different domains of the heterologous receptor proteins. The EcR, as a subgroup of the nuclear receptor family, also has fewer well-defined regions responsible for the heterodimerization properties. Because the nuclear receptor domains are modular in nature the LBD, DBD, and TD can be exchanged.

In another embodiment, the transcription factor comprises a TD, a DBD that recognizes a response element associated with the exogenous gene whose expression is to be modulated; an LDL of the Group H nuclear receptor. In certain embodiments, the LBD of the Group H nuclear receptor comprises a substitution mutation.

In another embodiment, a polynucleotide encoding gene change comprises a first sequence of the transcription factor under the control of a first promoter, and a second sequence of the transcription factor under the control of a second promoter, wherein the proteins encoded by the first sequence of the transcription factor and the second sequence of the transcription factor interact to form a protein complex that functions as a ligand-dependent transcription factor, eg, a gene change based on "double change" or "two hybrids". The first and the second promoters can be the same or different.

In certain embodiments, the polynucleotide encoding a gene change comprises a first sequence of the transcription factor and a second sequence of the transcription factor under the control of a promoter, wherein the proteins encoded by the first sequence of the transcription factor and the The second sequence of the transcription factor interacts to form a protein complex that functions as a ligand-dependent transcription factor, eg, a "simple gene change". The first sequence of the transcription factor and the second sequence of the transcription factor can be connected by an internal ribosomal entry site (IRES, for its acronym in English). The IRES can be an EMCV IRES.

In one embodiment, the first sequence of the transcription factor codes for a polypeptide comprising a TD, a DBD that recognizes a response element associated with the exogenous gene, whose expression is to be modulated; and LBD of the Group H nuclear receptor, and the second sequence of the transcription factor codes for a transcription factor comprising a LBD of the nuclear receptor, selected from a RXR LBD, vertebrate, an RXR LBD, invertebrate, a LBD of ultrapyracle protein, and a chimeric LBD comprising two polypeptide fragments, wherein the first polypeptide fragment is derived from an RBP LBD, from vertebrate, an RXR LBD from invertebrate, or a LBD from protein ultraspiral, and the second polypeptide fragment is from RXR of different vertebrate LBD, RXR of invertebrate LBD, or LBD of protein ultraespiráculo.

In another embodiment, the gene change comprises a first transcription factor sequence encoding a first polypeptide comprising a LBD of nuclear receptor and a DBD that recognizes a response element associated with the exogenous gene, whose expression is to be modulated, and a second sequence of the transcription factor encoding a second polypeptide comprising a TD, and a LBD of the nuclear receptor, wherein one of the LBDs of the nuclear receptor is a LBD of the nuclear receptor of Group H. In a preferred embodiment, the first polypeptide is substantially free of a TD, and the second polypeptide is substantially free of a DBD. For purposes of the invention, "substantially free" means that the protein in question does not contain a sufficient sequence of the domain in question, to provide activation or binding activity.

In another aspect of the invention, the first sequence of the transcription factor codes for a protein comprising a heterodimer partner and a TD, and the second sequence of the transcription factor codes for a protein comprising a DBD and an LBD.

When only one LDL of the nuclear receptor is a LBD of Group H, the other LBD of the nuclear receptor can be from any other nuclear receptor that forms a dimer with the LBD of Group H. For example, when the LBD of the nuclear receptor of the Group H is an EcR LBD, the other "partner" of the LBD of the nuclear receptor can be from an EcR, a vertebrate RXR, an invertebrate RXR, an ultraespiráculo protein (USP), or a chimeric nuclear receptor that comprises at least two different polypeptide fragments of the nuclear receptor LBD selected from a vertebrate RXR, an invertebrate RXR, and a USP (see WO 01/70816 A2, International Patent Application No.

PCT / US02 / 05235 and US 2004/0096942 Al). The ligand binding domain of the "partner" nuclear receptor may further comprise a truncation mutation, a deletion mutation, a substitution mutation, or another modification.

In one embodiment, the vertebrate RXR LBD is derived from human Homo sapiens, mouse Mus musculus, rat Rattus norvegicus, chicken Gallus gallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafish Danio rerio, tunicate Polyandrocarpa misakiensis, or medusa Tripedalia cysophora RXR.

In one embodiment, the binding domain to the invertebrate ligand RXR from a Locusta migratory lobster ultraespiracle polypeptide ("LmUSP"), a RXR homologue of the ixodide tick A blyomma americanum ("AmaRXRl"), a homologue 2 of RXR of the ixodide tick Amblyomma americanu ("AmaRXR2"), an RXR homologue of the fiddler crab Celuca pugilator ("CpRXR"), a RXR homolog of the Tenebrio molitor beetle ("TmRXR11), an RXR homolog of the honey bee Apis mellifera ("AmRXR"), an RXR homolog of the aphid Myzus persicae ("M RXR"), or a non-dipteran / non-lepidopteran RXR homologue.

In one embodiment, chimeric RXR LBD comprises at least two polypeptide fragments selected from a polypeptide fragment of RXR of vertebrate species, a polypeptide fragment of invertebrate species RXR and a homologous polypeptide fragment of RXR from non-dipteran / non-lepidopteran invertebrate species. A chimeric RXR ligand binding domain for use in the invention may comprise RXR polypeptide fragments from at least two different species, or when the species is the same, the two or more polypeptide fragments may be from two or more different isoforms of the RXR polypeptide fragment of the species.

In one embodiment, the chimeric RXR ligand binding domain comprises at least one vertex species RXR polypeptide fragment and an invertebrate species RXR polypeptide fragment.

In yet another embodiment, the chimeric RXR ligand binding domain comprises at least one RXR polypeptide fragment of vertebrate species and a homologous polypeptide fragment of RXR of non-dipteran / non-lepidopteran invertebrate species.

The ligand, when combined with the LBD of the nuclear receptor (s), which in turn are linked to the response element linked to the exogenous gene, provides external temporal regulation of the expression of the exogenous gene. The binding mechanism or the order in which the various components of this invention are linked to one another, ie, for example, the ligand to the LBD, DBD to the response element, TD to the promoter, etc., is not critical .

In a specific example, the binding of the ligand to the LBD of a nuclear receptor of Group H and its LBD partner of the nuclear receptor, makes possible the expression of the exogenous gene. This mechanism does not exclude the potential for ligand binding to the Group H nuclear receptor (GHNR) or its partner, and the resulting formation of the active homodimeric complexes (e.g., GHNR + GHNR, or partner + partner). Preferably, one or more of the receptor domains are varied producing a hybrid gene change. Typically, one or more of the three domains, DBD, LBD, and TD, can be chosen from a source different from the source of the other domains, so that hybrid genes and resulting hybrid proteins are optimized in the cell or organism host chosen for the transactivation activity, the complementary link of the ligand, and the recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein of yeast (see Sadowski et al, Nature 335: 563 (1988)) or the protein LexA of Escherichia coli (see Brent et al, Cell 43: 729 (1985)), or specific synthetic response elements for targeted interactions with proteins designed, modified and selected for such specific interactions (see, for example, Kim et al. al., Proc. Nati. Acad.

Sci. USA, 94: 3616 (1997)) to accommodate hybrid receptors.

The functional EcR complex may also include one or more additional proteins such as immunophilins. Additional members of the nuclear receptor protein family, known as transcriptional factors (such as DHR38 or betaFTZ-1), may also be dependent or independent ligand partners for EcR, USP, and / or RXR. In addition, other cofactors may be required, such as proteins generally known as coactivators (also referred to as adapters or mediators). These proteins do not specifically bind from sequence to DNA, and are not involved in basal transcription. These can exert their effect on the activation of transcription through various mechanisms, including the stimulation of the linkage to the DNA of the activators, by affecting the structure of the chromatin, or by means of activator-initiation complex interactions. Examples of such coactivators include RIP 140, TIF1, RAP46 / Bag-1, ARA70, SRC-l / NCoA-1, TIF2 / GRIP / NCOA-2, ACTR / AIB1 / RAC3 / pCIP as well as the element binding protein. Response B of the promiscuous C coactivator, CBP / p300 (for review see Glass et al, Curr Opin Cell Biol 9: 222 (1997)). Also, protein cofactors generally known as co-repressors (also known as repressors, silencers or silencing mediators), may be required to effectively inhibit transcriptional activation in the absence of the ligand. These co-repressors can interact with unbound EcR to silence the activity in the response element. Current evidence suggests that the binding of the ligand changes the conformation of the receptor, which results in the release of the co-repressor and the recruitment of the coactivators described above, thereby suppressing its silencing activity. Examples of co-repressors include N-CoR and SMRT (for review, see Horwitz et al., Mol Endocrinol, 70: 1167 (1996)). These cofactors can be either endogenous within the cell or organism, or they can be added exogenously as transgenes to be expressed in a regulated or unregulated manner.

The exogenous gene is operably linked to a promoter comprising at least one response element that is recognized by the DBD or the ligand-dependent transcription factor, encoded by the gene change. In one embodiment, the promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of the response element. The promoters comprising the desired response elements can be naturally occurring promoters or artificial promoters created using techniques that are well known in the art, for example one or more response elements operably linked to a minimal promoter.

A gene encoding an immunomodulator, for example, IL-12, TNF-alpha, signal peptides or any of the transcription factors herein can also be optimized by codon. In one embodiment, a coding region of an immunomodulator, for example, IL-12, TNF-alpha, a signal peptide, or a transcription factor is codon optimized for expression in human. As appreciated by one skilled in the art, several nucleic acid coding regions will code for the same polypeptide due to the redundancy of the genetic code. Deviations in the nucleotide sequence comprising the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence encoding the b gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which code for amino acids (the remaining three codons encode for signals terminating translation). The "genetic code" shows that codons code for which amino acids are reproduced in the present as Table 4. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are encoded by four triplets, serine and arginine by six, while tryptophan and methionine are encoded by only one triplet. This degeneracy allows the composition of DNA bases to vary over a wide variety without altering the amino acid sequence of the polypeptides encoded by the DNA.

Table 4: The Normal Genetic Code It will be appreciated that any polynucleotide that codes for a polypeptide encoding for polypeptide in accordance with the invention falls within the scope of this invention, notwithstanding the codons used.

Many organisms exhibit a deviation for the use of particular codons to encode for the insertion of a particular amino acid into a growing polypeptide chain. The codon preference or codon deviation, differences in codon usage between organisms, is facilitated by the degeneracy of the genetic code, and is well documented among many organisms. Codon deviation frequently correlates with translation efficiency of messenger RNA (mRNA), which in turn is believed to be dependent, inter alia, on the properties of the codons that are translated and the availability of particular transfer RNA molecules. (TRNA) The predominance of selected tRNAs in a cell in general is a reflection of the codons most frequently used in peptide synthesis. Therefore, genes can be adapted for optimal gene expression in a given organism based on codon optimization.

The polynucleotides are prepared by incorporating preferred codons for use in the genes of a given species in the DNA sequence.

Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are easily available, for example, in the "Codon Usage Datábase" available at http: // www. kazusa or. jp / codon / (accessed May 30, 2006), and these tables can be adapted in many ways. See Nakamura, Y., et al, "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucí. Acids Res. 28: 292 (2000). The codon usage tables for humans calculated from GenBank Relase 151.0, is reproduced below as Table 5 (from http://www.kazusa.or.jp/codon/supra). These tables use nomenclature of AR m, and in this way instead of thymidine (T) that is in the DNA, the tables use uracil (U) that is in the RNA. The tables have been adapted so that the frequencies for each amino acid are calculated, instead for all 64 codons.

Table 5: Table of Use of Codon for Human Genes (Homo sapiens) By using these or similar tables, one skilled in the art can apply the frequencies to any polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region that codes for the polypeptide, but uses optimal codons for a given species.

Several options are available to synthesize codon-optimized coding regions designed by any of the methods described above, using normal and routine molecular biological manipulations well known to those skilled in the art.

In one embodiment, the coding region encoding the immunomodulator, eg, TNF-alpha, in the vector of the invention is optimized by codon. In another embodiment, the coding region is optimized by codon for expression in human. In a particular embodiment, TNF-alpha in the invention is encoded by a nucleic acid sequence optimized by codon.

To introduce in vivo or ex vivo the polynucleotides within the cells, a vector can be used. The vector can be, for example, a plasmid vector or a viral vector of single-stranded or double-stranded RNA or DNA. Such vectors can be introduced into cells by well known techniques for the introduction of DNA and RNA into cells. Viral vectors may be replication competent or replication defective. In the latter case, viral spread will generally occur only in complementary host cells. As used herein, the term "host cell" or "host" is used to mean a cell of the invention that is harboring one or more polynucleotides of the invention Thus, at a minimum, the vectors should include the polynucleotides of the invention. Other components of the vector may include, but are not limited to, selectable markers, chromatin modification domains, additional promoters that promote the expression of other polypeptides that may also be present on the vector (eg, a lethal polypeptide), genomic integration, recombination sites, and molecular insertion pivots. The vectors can comprise any number of these additional elements, whether or not within the polynucleotides, such that the vector can be tailored to the specific goals of the desired therapeutic methods.

In one embodiment of the invention, the vectors that are introduced into the cells further comprise a "selectable marker gene" which, when expressed, indicates that the construction of the gene change of the invention has been integrated into the genome of the invention. host cell. In this way, the selection gene can be a positive marker for genomic integration. While not critical to the methods of the invention, the presence of a selectable marker gene allows the practitioner to select a population of living cells where the vector construct has been integrated into the genome of the cells. Thus, certain embodiments of the invention comprise the selection of cells where the vector has been successfully integrated. As used herein, the term "select" or variants thereof, when used in conjunction with cells, is intended to mean standard, well-known methods for choosing cells with a specific constitution or genetic phenotype. Typical methods include, but are not limited to, culturing cells in the presence of antibiotics, such as G418, neomycin, and ampicillin. Other examples of selectable marker genes include, but are not limited to, genes that confer resistance to dihydrofolate reductase, hygromycin, or mycophenolic acid. Other selection methods include, but are not, a selectable marker gene that allows the use of thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase or adenine phosphoribosyltransferase as selection agents. Cells comprising a vector construct that includes an antibiotic resistance gene or genes could then be able to tolerate the antibiotic in culture. Similarly, cells that do not comprise a vector construct that includes one or more antibiotic resistance genes may not be able to tolerate the antibiotic in culture.

As used herein, a "chromatin modification domain" (CMD) refers to nucleotide sequences that interact with a variety of proteins associated with the maintenance and / or alteration of the chromatin structure, such as, but not limited to DNA isolators. See Ciavatta et al, Proc. Nat '1 Acad. Sci U.S. A., 103: 9958 (2006). Examples of CMDs include, but are not limited to, the chicken ß-globulin isolator and the chicken hypersensitive site 4 (cHS4). The use of different CMD sequences between one or more gene programs (eg, a promoter, the coding sequence, and the regulatory region 31), for example, can facilitate the use of differential CMD DNA sequences, such as "mini homology arms" in combination with various microorganisms or in vitro recombination technologies to "exchange" gene programs between existing multigene and monogenic shuttle vectors. Other examples of chromatin modification domains are known in the art or can be easily identified.

The polynucleotide nucleic acid coding regions in the vector of the invention can be associated with additional coding regions that code for signal or secretory peptides, which direct the secretion of an immunomodulator, for example, TNF-alpha. According to the signal hypothesis, the proteins secreted by mammalian cells have a signal peptide or secretory guide sequence that is cleaved from the mature protein once the export of the growing protein chain through the endoplasmic reticulum has begun. thick. Polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the full length or "full length" polypeptide to produce a segregated or "mature" form of the polypeptide.

In one embodiment, a vector of the invention comprises a polynucleotide encoding a gene change, wherein the polynucleotide comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one sequence of transcription factor codes for a ligand-dependent transcription factor, and (2) a polynucleotide that codes for one or more proteins that have the function of an immunomodulator operably linked to a promoter that is activated by the transcription factor dependent on ligand, wherein the polynucleotide encoding one or more proteins that have the function of an immunomodulator further comprises a nucleic acid sequence encoding a signal peptide. In another embodiment, the signal peptide increases the secretion of the immunomodulator, e.g., TNF-alpha, encoded by the vector compared to a vector comprising the signal peptide gene native to the immunomodulator, e.g., signal peptide gene. wild type of TNF-alpha. In particular, the signal peptide used in the invention can be optimized by codon. In a specific embodiment, the signal peptide is encoded by the wild-type signal peptide gene of IL-2. In a further specific embodiment, the signal peptide is encoded by the codon-optimized IL-2 signal peptide gene.

The vector of the invention may comprise several regulatory regions, for example, 5 'untranslated region (5'UTR), 3' UTR, or both. The present invention also relates to the use of several regulatory regions to induce improved processes of secretion, protein translation, post-translation, mRNA transcription, or post-transcription. As used herein, the "5'-untranslated region" or "5'UTR" of a gene is to be understood as that part of a gene that is transcribed into a primary RNA transcript (pre-mRNA) and part which is located in the 5 'direction of the coding sequence. The primary transcript is the initial mRNA product, which contains introns and exons, produced by DNA transcription. Many primary transcripts must undergo RNA processing to form the physiologically active DNA species. Processing in a mature mRNA may comprise trimming the ends, removing introns, trimming and / or cleaving the individual rRNA molecules from their precursor RNAs. The 5'UTR of an mRNA in this manner is that part of the mRNA that is not translated into the protein and which is located in the 5 'direction of the coding sequence. In a genomic sequence, 5'UTR is typically defined as the region between the translation start site and the start codon. The 5 'untranslated regions (5' UTR) of the vertebrate mRNAs can be few tens of bases at several hundred bases in length (Crowe et al, 2006 BMC Genomics 7:16). The 5'UTR used herein may be presented naturally or modified to contain one or more nucleic acid sequences of a non-contiguous nature (chimeric sequences), and / or may encompass substitutions, insertions, and deletions and combinations thereof. In one embodiment, the 5'UTR sequence is derived from the wild-type TNF-alpha sequence or the 5U2 sequence. In another embodiment, the 5'UTR sequence is 5'UTR of 5U2. In some embodiments, 5'UTR induces improved protein expression, eg, transcription, pre-transcription, or post-transcription of mRNA.

The 3 'untranslated region (UTR) used in the invention refers to DNA sequences located in the 3' (3 ') direction of a coding sequence and can comprise polyadenylation and [poly (A)] recognition sequences and other sequences coding for regulatory signals capable of affecting gene expression or AR m processing. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3 'end of the mRNA precursor. Any suitable polyadenylation sequence can be used, including a synthetic optimized sequence, as well as the polyadenylation sequence of BGH (for Bovine Growth Hormone), polyoma virus, TK (Thymidine-Kinase), EBV (for its acronym in English of Epstein Virus Barr), and papillomaviruses, including human papillomaviruses and BPV (for Bovine Papilloma Virus). In a particular embodiment, a 3 'regulatory region is the SV40e polyadenylation sequence (Human Sarcoma Virus 40). In another particular embodiment, a regulatory region 31 is the polyadenylation sequence of human growth hormone.

In certain embodiments, the signal peptide and / or the regulatory sequence alone or in combination can improve protein secretion, transcription or translation at least twice, three times, four times, five times, six times, seven times, eight times , nine times, 10 times, 50 times, 100 times, 200 times, 300 times, 400 times, or 500 times compared to a control, which does not contain the signal peptide and / or the regulatory region. The level of secretion of a protein, for example, TNF-alpha, can be normalized to the expression of protein encoded by a vector having a wild-type gene. In another specific embodiment of the present invention, the signal peptide and / or the regulatory region alone or in combination increases the productivity of the immunomodulator, eg, TNF-alpha, from about 5% to about 10%, of about 11% at about 20%, from about 21% to about 30%, from about 31% to about 40%, from about 41% to about 50%, from about 51% to about 60%, from about 61% to about 70%, of about 71% to about 80%, about 81% to about 90%, about 91% to about 100%, about 101% to about 149%, about 150% to about 199%, about 200% to about 299%, from about 300% to about 499%, or from about 500% to about 1000%. In a specific embodiment, the present invention comprises a vector that conditionally expresses an immunomodulator, for example, TNF-alpha, wherein the vector comprises 5 'UTR of 5U2, a codon-optimized nucleic acid sequence coding for the peptide of IL-2 signal, a coding region optimized by codon coding for an immunomodulator, for example, TNF-alpha, and a polyadenylation signal of SV40e or human growth hormone.

In a further embodiment, the vector of the invention comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 47 (Vector 43318), SEQ ID NO: 48 (Vector 43319), SEQ ID NO: 49 (Vector 43320) SEQ ID NO: 50 (Vector 43321), SEQ ID NO: 51 (Vector 43322) SEQ ID NO: 52 (Vector 43323), SEQ ID NO: 53 (Vector 43324) SEQ ID NO: 54 (Vector 43325), SEQ ID NO: 55 (Vector 43326) SEQ ID NO: 56 (Vector 43327), SEQ ID NO: 57 (Vector 43328) and SEQ ID NO: 58 (Vector 43329). In a still specific embodiment, the vector comprises a polynucleotide sequence of SEQ ID NO: 52 (vector 43323) OR SEQ ID NO: 58 (vector 43329).

Particular vectors for use with the invention are expression vectors that encode proteins or polynucleotides. In general, such vectors comprise cis-acting control regions, effective for expression in a host operably linked to the polynucleotide to be expressed. The appropriate trans action factors are supplied by the host, supplied by a complementary vector or supplied by the vector itself after introduction into the host.

A wide variety of expression vectors can be used to express proteins or polynucleotides. Such vectors include chromosomal, episomal vectors and virus derivatives, for example, vectors derived from bacterial plasmids, from bacteriophages, from yeast episomes, from yeast chromosomal elements, from viruses such as adeno-associated viruses, lentiviruses, vaculoviruses, papovaviruses, such as SV40, vaccinia virus, adenovirus, avian pox virus, virus and pseudorabies retrovirus, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage gene elements, such as cosmids and phagemids. All can be used for expression according to this aspect of the invention. In general, any vector suitable for maintaining, propagating or expressing polynucleotides or proteins in a host can be used for expression in this regard.

Viral vectors suitable in the invention include, but are not limited to, adenovirus-based vectors, retroviral vectors, herpes simplex virus (HSV) -based vectors, parvovirus-based vectors, eg, vector based in adeno-associated viruses (AAV, for its acronym in English), and AAV-adenoviral chimeric vectors. These viral vectors can be prepared using normal recombinant DNA techniques and described in, for example, Sambrook et al., Molecular Cloning, Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994).

In one embodiment, a viral vector of the invention is an adenoviral vector. Adenovirus (Ad) is a 36 kb double-stranded DNA virus that efficiently transfers DNA in vivo to a variety of different types of target cells. The adenoviral vector can be produced in high titers and can efficiently transfer DNA to cells in replication and without replication. The genome of the adenoviral vector can be generated using any species, strain, subtype, mixture of species, strains, or subtypes, or chimeric adenovirus as the source of vector DNA. Adenoviral concentrate materials that can be used as a source of adenovirus can be amplified from serotypes 1 to 51 adenovirals, which are currently available from the American Type Culture Collection (ATCC, Manassas, Va.), Or from any other adenovirus serotype. available from any other source. For example, an adenovirus can be from subgroup A (for example, serotypes 12, 18, and 31), subgroup B (for example, serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (for example, serotypes 1, 2, 5, and 6), subgroup D (for example, serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviral serotype. Since the genome of human adenovirus serotype 5 (Ad5) has been completely cloned, the adenoviral vector of the invention is described herein with respect to serotype Ad5. The adenoviral vector can be any adenoviral vector capable of growing in a cell, which is somewhere significant (although not substantially necessary) derived from or based on the genome of an adenovirus. The adenoviral vector can be based on the genome of any suitable wild-type adenovirus. In certain embodiments, the adenoviral vector is derived from the genome of a wild type adenovirus of group C, especially of serotype 2 or 5. Adenoviral vectors are well known in the art and are described in, for example, US Pat. Nos. 5,559,099, 5,712,136, 5,731,190, 5,837,511, 5,846,782, 5,851,806, 5,962,311, 5,965,541, 5,981,225, 5,994,106, 6,020,191, and 6,113,913, International Patent Applications WO 95/34671, WO 97/21826, and WO 00/00628, and Thomas Shenk , "Adenoviridae and their Replication", and MS Horwitz, "Adenoviruses," Chapters 67 and 68, respectively, in Virology, BN Fields et al., Eds. , 3d ed., Raven Press, Ltd., New York (1996).

In other embodiments, the adenoviral vector is deficient in replication. The term "replication deficient" used herein means that the adenoviral vector comprises a genome that lacks at least one gene function essential for replication. A deficiency in the gene, gene function, or genomic or genomic region, as used herein, is defined as a deletion of sufficient genetic material from the viral genome to impart or cancel the function of the gene whose nucleic acid sequence was deleted in totally or in part. The essential gene functions for replication are those gene functions that are required for the replication (ie, propagation) of an adenoviral vector deficient in replication. The essential gene functions for replication are encoded, for example, by the early adenoviral regions (e.g., the El, E2, and E4 regions), the late regions (e.g., the L1-L5 regions), genes comprised in viral packaging (e.g., the IVa2 gene) ), and RNAs associated with viruses (for example, VA-RNA I and / or VA-RNA II). In still other embodiments, the adenoviral vector deficient in replication comprises an adenoviral genome deficient in at least one gene function essential to replication of one or more. regions of an adenoviral genome (eg, two or more regions of an adenoviral genome as a result of a multiplicity of adenoviral vectors deficient in replication). The one or more regions of the adenoviral genome is selected from the group consisting of the El, E2, and E4 regions. The adenoviral vector deficient in replication may comprise a deficiency in at least one essential gene function to replication of the El region (denoted an adenoviral vector deficient in El), particularly a deficiency in the gene function essential to the replication of each of the region The adenoviral adenovirus and the adenoviral E1B region. In addition to that deficiency in the El region, the recombinant adenovirus may also have a mutation in the major late promoter (MLP), as discussed in International Patent Application WO 00/00628. In a particular embodiment, the vector is deficient in at least one essential gene function in the replication of the El region and at least part of the non-essential E3 region (eg, an Xba I deletion of the E3 region) (denoted a vector adenoviral deficient in E1 / E3).

In certain embodiments, the adenoviral vector is "multiply deficient", meaning that the adenoviral vector is deficient in one or more gene functions required for viral replication in each of two or more regions of the adenoviral genome, eg, the adenoviral vector deficient in The deficient E1 / E3, mentioned above, may be additionally deficient in at least one gene function essential to the replication of the E4 region (denoted an adenoviral vector deficient in E1 / E4). A suppressed adenoviral vector from the entire E4 region can produce a lower host immune response.

Alternatively, the adenoviral vector lacks essential gene functions for replication in whole or in part of the El region and all or part of the E2 region (denoted a viral vector deficient in E1 / E2). Adenoviral vectors lacking the gene functions essential to replication in whole or in part of the El region, in whole or in part of the E2 region, and in whole or in part of the E3 region are also contemplated herein. If the adenoviral vector of the invention is efficient in the gene function essential to the replication of the E2A region, the vector does not comprise a complete deletion of the E2A region, which is less than about 230 base pairs in length. In general, the E2A region of the adenovirus codes for a DBP (DNA binding protein), a polypeptide required for DNA replication. DBP is composed of 473 to 529 amino acids depending on the viral serotype. It is believed that DBP is a symmetric protein that exists as an ellipsoid prolate consisting of a globular Ct with an extended Nt domain. Studies indicate that the Ct domain is responsible for the ability of DBP to bind to nucleic acids, bind to zinc and fuse in DNA synthesis at the level of DNA strand elongation. However, it is believed that the Nt domain functions in late gene expression both at the transcriptional and post-transcriptional levels, is responsible for the efficient nuclear localization of the protein, and may also be included in the improvement of its own expression. Deletions in the Nt domain between amino acids 2 to 38 have indicated that this region is important for DBP function (Brough et al, Virology, 196, 269-281 (1993)). While deletions in the E2A region coding for the Ct region of DBP have no effect on viral replication, deletions in the E2A region encoding amino acids 2 to 38 of the Nt domain of DBP impart viral replication. In one embodiment, the adenoviral vector multiply deficient in replication contains this portion of the E2A region of the adenoviral genome. In particular, for example, the desired portion of the E2A region to be retained is that portion of the E2A region of the adenoviral genome that is defined by the 5 'end of the E2A region, specifically at positions Ad5 (23816) a Ad5 (24032) of the E2A region of the adenoviral genome of serotype Ad5.

The adenoviral vector may be deficient in the gene functions essential to the replication of only the early regions of the adenoviral genome, only the late regions of the adenoviral genus, and both the early and late regions of the adenoviral genome. The adenoviral vector can also have essentially the complete or removed adenoviral genome, in which case at least either the viral inverted terminal repeats (ITR) and one or more promoters or the viral ITRs and a packaging signal they are left intact (ie, an adenoviral amplicon). The larger the region of the adenoviral genome that is removed, the larger the piece of exogenous nucleic acid sequence that can be inserted into the genome. For example, given that the adenoviral genome is 36 kb, when leaving the viral ITRs and one or more promoters intact, the exogenous insertion capacity of the adenovirus is approximately 35 kb. Alternatively, a multi-deficient adenoviral vector containing only one ITR and a packaging signal effectively allows the insertion of an exogenous nucleic acid sequence of approximately 37-38 kb. Of course, the inclusion of a spacer element in any or all of the efficient adenoviral regions will decrease the ability of the adenoviral vector for large insertions. Suitably replicating deficient adenoviral vectors, including multiple deficient adenoviral vectors, are described in U.S. Patent Nos. 5,851,806 and 5,994,106 and International Patent Applications WO 95/34671 and WO 97/21826. In another embodiment, the vector for use in the present inventive method is that described in International Patent Application PCT / US01 / 20536.

It should be appreciated that the deletion of different regions of the adenoviral vector can alter the mammalian immune response. In particular, the suppression of different regions can reduce the inflammatory response generated by the adenoviral vector. Additionally, the adenoviral vector cover protein can be modified to decrease the ability or inability of the adenoviral vector to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509 .

The adenoviral vector, when multiply deficient in replication, especially in gene functions essential to the replication of the El and E4 regions, may include a separating element to provide viral growth in a complementary cell line similar to that achieved by adenoviral vectors individually deficient in replication, particularly an adenoviral vector comprising a deficiency in the El region. The spacer element can contain any sequence or sequences that are of the desired length. The sequence of the separating element can be coding or non-coding and native and non-native with respect to the adenoviral genome, but it does not restore the essential function to replication to the deficient region. In the absence of a separator, the production of fiber protein and / or the viral growth of the adenoviral vector multiply deficient in replication is reduced compared to that of an adenoviral vector individually deficient in replication. However, inclusion of the separator in at least one of the deficient viral regions, preferably the E4 region, can counteract this decrease in viral growth and fiber protein production. The use of a separator in an adenoviral vector is described in U.S. Patent No. 5,851,806.

The construction of adenoviral vectors is well understood in the art. Adenoviral vectors can be constructed and / or purified using the methods set forth, for example, in U.S. Patent No. 5,965,358 and International Patent Applications WO 98/56937, WO 99/15686, and WO 99/54441. The production of adenoviral gene transfer vectors is well known in the art, and comprises the use of normal molecular biology techniques such as those described, for example, in Sambrook et al., Supra, Watson et al, supra, Ausubel et al. , supra, and in several of the other references mentioned herein.

Replication-deficient adenoviral vectors are typically produced in complementary cell lines that provide gene functions not present in the adenoviral vectors deficient in replication, but which are required for viral propagation, at appropriate levels in order to generate high titers of concentrated viral vector material in one embodiment, a cell line complements at least one and / or all of the replication essential gene functions not present in an adenovirus efficient in replication. The complementary cell line can complement a deficiency in at least one essential gene function to replication encoded by early regions, late regions, viral packaging regions, RNA regions associated with viruses, or combinations thereof, including all adenoviral functions ( for example, to allow the propagation of adenoviral amplicons, comprising minimal adenoviral sequences, such as only inverted terminal repeats (ITR) and the packaging signal or only ITR and an adenoviral promoter). In another embodiment, the complementary cell line complements a deficiency in at least one gene function essential to replication (e.g., two or more gene functions essential to replication) of the El region of the adenoviral genome, particularly a deficiency in a gene function essential to the replication of each of the E1A and E1B regions. further, the complementary cell line can complement a deficiency in at least one essential gene function to replication of the E2 regions (particularly with respect to the adenoviral DNA polymerase and terminal protein) and / or E4 of the adenoviral genome. Desirably, a cell that completes a deficiency in the E4 region comprises the E4-ORF6 gene sequence and produces the E4-ORF6 protein. That cell desirably comprises at least ORF6 and another ORF of the E4 region of the adenoviral genome. The cell line is preferably further characterized in that it contains the complementary genes in a non-overlapping manner with the adenoviral vector, which minimizes, and practically eliminates, the possibility of the vector genome recombining with the cellular DNA. Accordingly, the presence of replication competent adenoviruses (RCA) is minimized if not avoided in the concentrated material of the vector, which is therefore suitable for certain therapeutic purposes, especially gene therapy purposes. The lack of RCA in the concentrated vector material prevents the replication of the adenoviral vector in non-complementary cells. The construction of complementary cell lines comprises normal techniques of molecular biology and cell culture, such as those described by Sambrook et al, s pra, and Ausubel et al, supra. Complementary cell lines for producing the gene transfer vector (eg, adenoviral vector) include, but are not limited to, 293 cells (described, for example, in Graham et al, J. Gen. Virol., 36, 59- 72 (1977)), PER.C6 cells (described in, for example, International Patent Application WO 97/00326, and U.S. Patent Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (described in, for example. , International Patent Application WO 95/34671 and Brough et al, J Virol., 71, 9206-9213 (1997)). The insertion of a nucleic acid sequence into the adenoviral genome (e.g., the El region of the adenoviral genome) can be facilitated by known methods, for example, by the introduction of a single restriction site at a certain position of the adenoviral genome at .

The retrovirus is an RNA virus capable of infecting a wide variety of host cells. In infection, the retroviral genome integrates into the genome of its host cell and replicates along with the host cell DNA, thereby consistently producing viral RNA and any nucleic acid sequence incorporated into the retroviral genome. As such, long-term expression of a therapeutic factor can be achieved when retroviruses are used. The retroviruses contemplated for use in gene therapy are relatively non-pathogenic retroviruses, although there are some pathogens. When pathogenic retroviruses, eg, human immunodeficiency virus (HIV) or human T-cell lymphotropic virus (HTLV) are employed, care must be taken in altering the viral genome to eliminate host toxicity. . Additionally, a retroviral vector can be manipulated to render the virus replication deficient. As such, retroviral vectors are considered particularly useful for stable gene transfer in vivo. Lentiviral vectors, such as HIV-based vectors, are examples of retroviral vectors used for gene distribution. Different from other retroviruses, HIV-based vectors are known to incorporate their passenger genes into non-dividing cells and therefore, may be of use in the treatment of persistent forms of disease.

A viral vector based on HSV is suitable for use as a gene transfer vector for introducing a nucleic acid into numerous cell types. The mature HSV virion consists of an icosahedral capsid enveloped with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. Most replication-deficient HSV vectors contain a deletion to remove one or more intermediate-early genes to prevent replication. The advantages of the HSV vector are its ability to enter a latent state that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts up to 25 kb. Of course, the ability of HSV to promote long-term production of exogenous protein is potentially disadvantageous in terms of short-term treatment regimens. However, one skilled in the art has the understanding necessary to determine the appropriate vector for a particular situation. HSV-based vectors are described, for example, in U.S. Patent Nos. 5,837,532, 5,846,782, 5,849,572, and 5,804,413, and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583.

AAV vectors are viral vectors of particular interest for use in gene therapy protocols. The AAV is a DNA virus, which is not known to cause human disease. The AAV genome is comprised of two rep and cap genes, flanked by inverted terminal repeats (ITR), which contain recognition signals for DNA replication and packaging of the virus. The AAV requires co-infection with an auxiliary virus (ie, an adenovirus or a herpes simplex virus) or expression of ancillary genes, for efficient replication. The AAV can be propagated in a wide array of host cells including human, simian and rodent cells, depending on the helper virus used. An AAV vector used for administration of a nucleic acid sequence typically has about 96% of the parent genome deleted, such that only the ITRs remain. This eliminates the immunological or toxic side effects due to the expression of viral genes. If desired, the AAV rep protein can be co-administered with the AAV vector to allow integration of the AAV vector into the genome of the host cell. Host cells comprising an integrated genome of AAV show no change in morphology or cell growth (see, for example, U.S. Patent No. 4,797,368). As such, prolonged expression of therapeutic factors of AAV vectors may be useful in the treatment of persistent and chronic diseases.

The polynucleotide sequence in the expression vector is operably linked to the appropriate expression control sequence (s) that include, for example, a promoter to direct transcription of the mRNA. Representatives of the additional promoters include, but are not limited to, constitutive promoters and tissue-specific or inducible promoters. Examples of constitutive eukaryotic promoters include, but are not limited to, the promoter of the mouse metallothionein I gene (Hamer et al., J. Mol. Appl. Gen. 7: 273 (1982)); the TK promoter of Herpes virus virus (McKnight, Cell 57: 355 (1982)); the SV40 early promoter (Benoist et al., Nature 290: 304 (1981)); and the vaccinia virus promoter. Additional examples of promoters that could be used to boost expression of a protein or polynucleotide include, but are not limited to, tissue-specific promoters and other endogenous promoters for specific proteins, such as the albumin promoter (hepatocytes), a Proinsulin promoter (pancreatic beta cells) and the like. In general, the expression constructs will contain sites for transcription, initiation termination and, in the transcribed region, a ribosome binding site, for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation start AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately placed at the end of the polypeptide to be translated.

In addition, the constructions may contain control regions that regulate, as well as engender the expression. In general, such regions will operate by controlling transcription, such as receptor binding sites and enhancers, among others.

Examples of eukaryotic vectors include, but are not limited to pW-LNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Amersham Pharmacia Biotech; and pC VDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP available from Clontech. Many other vectors are well known and commercially available.

Particularly useful vectors, comprising molecular insertion pins for insertion and rapid elimination of gene program elements, are described in U.S. Patent Application Publication No. 2004/0185556, U.S. Patent Application No. 11 / 233,246 and the International Published Requests Nos. WO 2005/040336 and WO 2005/116231. An example of such vectors is the UltraVector ™ Production System (Intrexon Corp., Blacksburg, VA), as described in WO 2007/038276. As used herein, a "gene program" is a combination of genetic elements comprising a promoter (P), an expression sequence (E), and a 3 'regulatory sequence (3), such as "PE3" it's a gene program. The elements within the gene program can be easily exchanged between molecular pivots flanking each of the elements of the gene program. A molecular pivot, as used herein, is defined as a polynucleotide comprising at least two rare or non-common restriction sites, not variable, arranged in a linear fashion. In one embodiment, the molecular pivot comprises at least three rare or non-common restriction sites, not variables accommodated in a linear fashion. Typically, any molecular pivot may not include a rare or uncommon restriction site from any other molecular pivot within the same gene program. Cognate sequences of more than 6 nucleotides on which a given restriction enzyme acts, are termed "rare" restriction sites. However, there are restriction sites of 6 base pairs that appear more infrequently than would be statistically predicted, and these sites and the endonucleases that break them are called "uncommon" restriction sites. Examples of rare or uncommon restriction enzymes include, but are not limited to AsiS I, Pac I, Sbf I, Fse I, Ase I, MIu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, Sfo I, Sgr AI, AflIII, Pvu I, Ngo MIV, Ase I, FIp I, Pme I, Sda I, Sgf I, Srf I, Nru I, Acl I, Co. I, Csp45 I, Age I, Bstl 107 1, BstB I, Hpa I, Aat II, EcoR V, Nhe I, Spe I, Avi II, Avr II, Mfe I, Afe I, Fsp I, Kpn I, Sea I, BspE I, Nde I, Bfr I, Xho I, Pml I, ApaL I, Kas I, Xma I, BsrB I, Nsi I, Sac II, Sac I, BIp I, PspoM I, Pci I, Stu I, Sph I, BamH I, Bsu36 I, Xba I, BbvC I, Bgl II, Neo I , Hind III, EcoR I, BsrG I and Sse8781 I.

The vector may also comprise restriction sites for a second class of restriction enzymes called home endonucleases (HE) enzymes. HE enzymes have asymmetric, large restriction sites (12 to 40 base pairs), and their restriction sites are rare in nature. For example, the HE known as I-Scel has a restriction site of 18 base pairs (5 'AGGGATAACAGGGTAAT3' (SEQ ID NO: 28)), which is predicted to appear only once in each 7x1010 base pairs of random sequence . This rate of occurrence is equivalent to only one site in a genome that is 20 times the size of a mammalian genome. The rare nature of HE sites greatly increases the possibility that a genetic engineer can cut a gene program without disrupting the integrity of the gene program if the HE sites are included in the appropriate positions in a plasmid cloning vector.

The selection of appropriate vectors and promoters for expression in a host cell is a well-known procedure, and the techniques required for vector construction and introduction into the host, as well as their expression at the host, are routine skills in the art. .

The introduction of the polynucleotides into the cells may be a transient transfection, stable transfection, or it may be a specific insertion of the vector locus. Transient and stable transfection of the vectors within the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. , Basic Methods in Molecular Biology (1986); Keown et al, 1990, Methods Enzymol. 185: 527-37; Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y. These stable transfection methods result in the random insertion of the vector into the genome of the cell. In addition, the number of copies and the orientation of the vectors are also, generally speaking, random.

In one embodiment of the invention, the vector is inserted into a bio-neutral site in the genome. A bio-neutral site is a site in the genome where the insertion of polynucleotides interferes very little, if any, with the normal function of the cell. Bio-neutral sites can be analyzed using available bioinformatics. Many bio-neutral sites are known in the art, for example, the locus equivalent to ROSA. Other bio-neutral sites can be identified using routine techniques well known in the art. The characterization of the genomic insertion site (s) is carried out using methods known in the art. To control the position, the number of copies and / or the orientation of the polynucleotides when the vector is introduced into the cells, locus-specific insertion methods can be used. Locus-specific insertion methods are well known in the art and include but are not limited to, homologous recombination and recombinase-mediated genome insertion. Of course, if locus-specific insertion methods are to be used in the methods of the invention, the vectors may comprise elements that aid in this specific insertion of the locus, such as, but not limited to, homologous recombination. For example, the vectors may comprise one, two, three, four or more genomic integration sites (GISs). As used herein, a "genomic integration site" is defined as a portion of the vector sequence whose nucleotide sequence is identical or nearly identical to the portions of the genome within the cells, which allows the insertion of the vector into the cell. genome In particular, the vector may comprise two genomic insertion sites flanking at least the polynucleotides. Of course, GISs can flank additional elements, or even all the elements present on the vector.

In another embodiment, the specific insertion of the locus can be carried out by inserting genes specific for the recombinase site. In summary, bacterial recombinase enzymes, such as, but not limited to, PhiC31 integrase can act on sites of "pseudo" recombination within the human genome. These pseudo-recombination sites can be targeted for the specific insertion of the locus using the recombinases. The insertion of the gene, specific for recombinase, is described in Thiagarajan et al, Mol. Cell Biol. 21: 3926 (2001). Other examples of recombinases and their respective sites that can be used for the insertion of the recombinase site specific gene include, but are not limited to, serine recombinases such as R4 and TP901-1 and recombinase described in WO 2006/083253.

In a further embodiment, the vector may comprise a chemoresistance gene, eg, the mdrl gene, multidrug resistance, hydrofolate reductase, 06-alkylguanine-DNA-alkyltransferase. The chemoresistance gene may be under the control of a constitutive (eg, CMV) or inducible (eg, RheoSwitch ™) promoter. In this embodiment, if it is desired to treat a disorder in a subject while maintaining the modified cells within the subject, a clinician may apply a chemotherapeutic agent to destroy the diseased cells, whereas the modified cells could be protected from the agent due to the expression of an appropriate chemoresistance gene, and may continue to be used for the treatment, improvement or prevention of a disease or disorder. By placing the chemoresistance gene under an inducible promoter, unnecessary expression of the chemoresistance gene can be avoided, it will still be available in case continuous treatment is needed. If the modified cells themselves become diseased, they could still be destroyed by inducing the expression of a lethal polypeptide as described below.

The methods of the invention are carried out by introducing the polynucleotides encoding the gene change, and the exogenous gene within the cells of a subject. Any known method for introducing a polynucleotide into a cell, known in the art, such as those described above, can be used.

When the polynucleotides are to be introduced into the cells ex vivo, the cells can be obtained from a subject by any technique known in the art, including, but not limited to, biopsies, scrapings, and surgical tissue removal. The isolated cells can be cultured for a sufficient amount of time to allow the polynucleotides to be introduced into the cells, for example, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, hours or more . Methods for cultivating primary cells for short periods of time are well known in the art. For example, cells can be grown in plates (e.g., in microwell plates) adhered or in suspension.

For ex vivo therapeutic methods, the cells are isolated from a subject and cultured under suitable conditions to introduce the polynucleotides into the cells. Once the polynucleotides have been introduced into the cells, the cells are incubated for a sufficient period of time to allow the ligand-dependent transcription factor to be expressed, for example 0.5, 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 12, 18, or 24 hours or more. At some point after the introduction of the polynucleotides into the cells (either before or after significant levels of the ligand-dependent transcription factor are expressed), the cells are reintroduced into the subject. The reintroduction can be carried out by any method known in the art, for example, intravenous infusion, or direct injection into a tissue or cavity. In one embodiment, the presence of the polynucleotides in the cells is determined before introducing the cells back into the subject. In yet another embodiment, the cells containing the polynucleotides are selected (eg, based on the presence of a selectable marker in the polynucleotides) and only those cells containing the polynucleotides are reintroduced into the subject. After the cells are reintroduced to the subject, the ligand is administered to the subject to induce the expression of the therapeutic polypeptide or the therapeutic polynucleotide. In an alternative embodiment, the ligand can be added to the cells even before the cells are reintroduced to the subject, such that the therapeutic polypeptide or the therapeutic polynucleotide is expressed before reintroduction of the cells. The ligand can be administered by any suitable method, either systemically (eg, orally, intravenously) or locally (eg, intraperitoneally, intrathecally, intraventricularly, direct injection into the tissue or organ where the cells are reintroduced). Optimal synchronization of ligand administration can be determined for each cell type and disease or disorder using only routine techniques.

The in vivo therapeutic methods of the invention involve the direct in vivo introduction of the polynucleotides into the cells of the subject. The polynucleotides may be introduced into the subject systemically or locally (e.g., at the site of the disease or disorder). Once the polynucleotides have been introduced into the subject, the ligand can be administered to induce the expression of the therapeutic polypeptide or the therapeutic polynucleotide. The ligand can be administered by any suitable method, either systemically (eg, orally, intravenously) or locally (eg, intraperitoneally, intrathecally, intraventricularly, direct injection into the tissue or organ where the disease or disorder is occurring). Optimal synchronization of ligand administration can be determined for each cell type and disease or disorder using only routine techniques.

For in vivo use, the ligands described herein can be collected in pharmaceutically acceptable carriers, such as, for example, solutions, suspensions, tablets, capsules, ointments, elixirs, and injectable compositions. The pharmaceutical compositions may contain from 0.01% to 99% by weight of the ligand. The compositions may be in single or multiple dose forms. The amount of the ligand in any particular pharmaceutical composition will depend on the effective dose, i.e., the dose required to promote the desired gene expression or suppression.

Suitable routes of administration of pharmaceutical preparations (including oral and sublingual administration), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intratumoral, intradermal, intrathecal and epidural) or by nasogastric tube. It will be understood by those skilled in the art that the preferred route of administration will depend on the condition being treated and may vary with factors such as the condition of the patient.

As used herein, the term "rAD RheoIL12" refers to an adenoviral polynucleotide vector harboring the IL-12 gene under the control of a gene change of the RheoSwitch ™ (RTS) therapeutic system, which is capable of producing IL-12 protein in the presence of the activation ligand. As used herein, the term "rAd.cIL12" refers to an adenoviral polynucleotide control vector that contains the IL-12 gene under the control of a constitutive promoter.

As used herein, the term "IL-12p70" refers to the IL-12 protein, which naturally has two subunits commonly referred to as p40 and p35. The term IL-12p70 encompasses the fusion proteins comprising the two subunits of IL-12 (p40 and p35), wherein the fusion protein can include the linker amino acids between the subunits.

As used herein, the term "a protein having the function of an immunomodulator" refers to a protein having at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70 %, 80% or 90%) of some bioactivity of an immunomodulator selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL- 10R or a subunit thereof, DN, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, CCL3 (MIP- Ia), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL 12 (SDF -I), CCL21 (6Ckine), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGBl, Flt3L (IFN-beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1 , TGFbRII DN, ICOS-L and S100 Similarly, the term "a protein having the function of IL-12" refers to a protein that has at least 20% (for example, at least 30%, 40%, 50%, 60%, 70%, 80% or 90%) of some bioactivity of human IL-12. The bioactivities of such immunomodulators are well known. See the following Table. Table 6. Immunomodulators and their functions Immunomodulator Function Interleukin-2 (IL-2) IL-12 is a family of cytokines that is produced by T cells in response to antigenic or mitogenic stimulation, this protein is required for the proliferation of T cells and other activities crucial for the regulation of the immune response. IL-2 can stimulate B cells, monocytes, killer cells activated by lymphokine, natural killer cells, and glioma cells.

Interleukin-3 (IL-3) IL-3 stimulates the proliferation of progenitor cells hematopoietic pluripotent. It is secreted by activated T cells to support the growth and differentiation of T cells from the bone marrow into an immune response. The combined era of the intratumoral Ad-mIL-3 gene, in combination with radiotherapy, showed that it significantly suppresses tumor growth (Oh 2004).

Interleukin-4 (IL-4) IL-4 is a cytokine that participates in at least several processes of activation of B cells, as well as other cell types. This is a co-stimulator of DNA synthesis. It induces the expression of MHC class II molecules on resting B cells. Increases the secretion and suppression of IgE and IgGl on the cell surface. It also regulates the expression of low affinity Fe factor for IgE (CD23) on lymphocytes and monocytes.

Interleukin-5 (IL-5) IL-5 stimulates cell growth B and increases the secretion of immunoglobulin, and induces tumor suppression (Nakashima 1993, Wu 1992).

Interleukin-7 (IL-7) IL-7 is a cytokine that is a hematopoietic growth factor capable of stimulating the proliferation of lymphoid progenitors. This is important for proliferation during certain stages of B cell maturation.

The bioactivities of IL-12 are also known and include, without limitation, differentiation of intact T cells and Thl cells, stimulation of the development and function of T cells, production of interferon-gamma (iFN-gamma) and tumor necrosis factor alpha. (TNF-OI) from T cells and natural killer cells (NK), reduced IL-4 mediated suppression of IFN-gamma, increased cytotoxic activity of NK cells and CD8 + cytotoxic T lymphocytes, stimulation of expression of IL-12R-pi and IL-12R-p2, facilitation of the presentation of tumor antigens through the up-regulation of MHC I and II molecules, and anti-angiogenic activity. The term "a protein having the function of IL-12" encompasses mutants of the wild-type IL-12 sequence, wherein the wild-type sequence has been altered by one or more of amino acid addition, deletion or substitution, as well as the non-IL-12 proteins that mimic one or more of the bioactivities of IL-12.

As used herein, the terms "activation" or "activating" refer to any measurable increase in the cellular activity of a gene change resulting in the expression of a gene of interest (eg, selected from IL-1). , IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-IOR or a subunit of the same DN, IL-15, IL-18, IL-21 , IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, CCL3 (MIP-Ia), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA -alpha), CCR7, CCL 19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL 12 (SDF-I), CCL21 (6Ckine), OX40L, 4- IBBL, CD40, CD70, GITRL , LIGHT, b-Defensin, HMGB1, Flt3L, IFN-beta, TNF-alpha, dnFADD, TGF-alpha, PD-L1 RNAi, an antisense oligonucleotide of PD-Ll, TGFbRII DN, ICOS-L and S100.

As used herein, the terms "treating" or "treating" a disorder or disease, refer to the performance of a protocol, which may include the administration of one or more drugs or cells manipulated in vi tro to a mammal (human or non-human), in an effort to alleviate the signs or symptoms of the disorder. Thus, "treatment" or "treatment" should not necessarily be considered as requiring complete relief of signs or symptoms, does not require a cure, and specifically includes protocols that have only marginal effect on the subject.

As used herein, "immune cells" include dendritic cells, macrophages, neutrophils, mast cells, eosinophils, basophils, natural killer cells and lymphocytes (e.g. B and T cells).

As used herein, the terms "dendritic cells" and "DC" are used interchangeably.

As used herein, the term "therapeutic support cells" (TSC) are cells that can be modified (eg, transfected, subjected to electroporesis, etc.) with the vector of the invention to distribute one or more proteins which have the function of an immunomodulator and, optionally, a protein that has the function of IL-12, towards the tumor microenvironments. Such TSCs include, but are not limited to, stem cells, fibroblasts, endothelial cells, and keratinocytes.

As used herein, the terms "immune cells engineered in vitro" or "in vitro engineered population of immune cells" or "a population of engineered immune cells" or "immune cells expressing an immunomodulator" or "immune cells" which express IL-12", refer to immune cells, for example, dendritic cells, which conditionally express an immunomodulator and / or IL-12 as the case may be, under the control of a gene change, which can be activated by a activation ligand.

As used herein, the terms "TSC manipulated in vitro" or "in vitro manipulated population of TSC" or "a population of engineered TSC" or "TSC expressing an immunomodulator" or "TSC expressing IL-12"refer to therapeutic support cells, for example, stem cells, fibroblasts, endothelial cells and keratinocytes, which conditionally express an immunomodulator and / or IL-12, as the case may be, under the control of a gene change, which it can be activated by the activation ligand.

As used herein, the term "modified cell" refers to cells that have been altered by a process that includes, but is not limited to, transiation, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, precipitation with calcium phosphate and lipofection (fusion of lysosomes).

As used herein, the terms "MOI" or "Multiplicity of Infection" refer to the average number of adenoviral particles that infect a single cell in a specific experiment (eg, recombinant adenovirus or control adenovirus).

As used herein, the term "tumor" refers to all growth and proliferation of benign or malignant cells either in vivo or in vitro, whether they are cells and / or precancerous or cancerous tissues.

In another embodiment, the vector and methods of the present invention can be used to treat diseases.

In another embodiment, the vector and methods of the present invention can be used to treat a cancer. Non-limiting examples of cancers that can be treated according to the invention include breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, carcinoma primary brain, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, carcinoma of the small cell lung, Wilms tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma , renal cell carcinoma, endometrial carcinoma, suprarenal cortex carcinoma, malignant pancreatic insulinoma, carcinoma carcinoi of malignant, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, disease of Hodgkin, non-Hodgkin's lymphoma, soft tissue sarcoma, mesothelioma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, and the like. In another embodiment, the vector and methods of the present invention can be used to treat a metabolic relationship disorder, including, but not limited to, a disorder selected from the group consisting of dyslipidemia, atherosclerosis, insulin resistance, diabetes ( for example, type I diabetes, type II diabetes, MODY, and gestational diabetes), obesity, impaired glucose intolerance, atheromatous disease, hypertension, heart disease (including, but not limited to, coronary heart disease, stroke, heart failure) , coronary insufficiency, and high blood pressure), hyperlipidemia, glucose intolerance, insulin resistance, hyperglycemia, hyperinsulinemia, metabolic syndrome X (or syndrome X or insulin resistance syndrome, or Reaven syndrome, or metabolic cardiovascular risk syndrome) ), hypertension, chronic fatigue, accelerated aging, degenerative disease, endocrine deficiencies of aging, Gml gangliosidosis, Morquio-B disease, Krabbe disease, Fabry disease, Gaucher disease, Tay-Sachs disease, Sandhoff disease, fucosidosis, carbohydrate metabolism disorders (eg, glycogen storage disease), disorders of amino acid metabolism (e.g., phenylketonuria, maple syrup urine disease, glutaric acidemia type 1), organic acid metabolism disorders (e.g., alkaptonuria), fatty acid oxidation disorders, and mitochondrial metabolism (e.g., deficiency) of medium chain acyl dehydrogenase), porphyrin metabolism disorders (e.g., acute intermittent porphyria), purine or pyrimidine metabolism disorders (e.g., Lesch-Nyhan disease), steroid metabolism disorders (e.g. congenital adrenal hyperplasia), mitochondrial function disorders (eg, Kearns-Sayre syndrome), and disorders of peroxisomal function (for example, Zellweger syndrome).

In another embodiment, the vector and methods of the present invention can be used to treat an autoimmune disorder, including, but not limited to, disorder selected from the group consisting of chronic active autoimmune hepatitis of Achlorhydra, acute disseminated encephalomyelitis, hemorrhagic leukoencephalitis. Acute, Addison's disease, gammaglobulinemia, Agamma-globulinaemia, Alopecia areata, Amyotrophic lateral sclerosis, Ankylosing spondylitis, Anti-GBM / TBM nephritis, Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopic allergy, Atopic dermatitis, Aplastic anemia, Autoimmune cardiomyopathy, Anemia Autoimmune hemolytic disease, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune progesterone dermatitis, I, II and III autoimmune syndrome, Purple thrombocyte autoimmune pneumonia, autoimmune uveitis, Balo's disease / Balo's concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaff's encephalitis, Blau's syndrome, Bullous pemphigoid, Castleman's disease, Chronic Fatigue Immune Dysfunction Syndrome, chronic inflammatory demyelinating polyneuropathy , chronic recurrent multifocal ostomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, celiac disease, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, angiitis cutaneous leukocytoclastic, Dego's disease, Dermatitis herpetiformis, Dermatomyositis, Type 1 diabetes mellitus, Diffuse cutaneous systemic sclerosis, Dressler's syndrome, Discoid lupus erythematosus, eczema, Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysis bulosa acquisita, Erythema nodosum, Cryoglobulinemia me Essential zclada, Evan syndrome, Progressive ossifia fibrosis, Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis, Goodpasture syndrome, Graves disease, Guillain-Barre syndrome (GBS), encephalitis of Hashimoto, Hashimoto's thyroiditis, Hemolytic anemia, Henoc purple - Schonlein, Herpes gestationis, Hughes syndrome (or antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy (or Berger), myositis by inclusion bodies, demyelinating polyneuropathy of ory, Juvenile idiopathic arthritis, Juvenile rheumatoid arthritis, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, linear IgA disease (LAD). ), Lou Gehrig's disease, hepatitis, lu poide lupus erythematosus, Majeed syndrome, Ménière's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, Mucha-Habermann's disease, Muckle-Wells syndrome, multiple myeloma, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optics (also Devic's disease), ocular scar pemphigoid, Ord's thyroiditis, palindromic rheumatism, PANDAS (pediatric autoimmune neuro-psychiatric disorders associated with Streptococcus), paraneoplastic cerebellar degeneration, paraneoplastic cerebellar degeneration, Parry Romberg's syndrome, Parsonnage-Turner syndrome , Planitis de Pars, Pemphigus, Pemphigus vulgaris, Pernicious anemia, Perivenous encephalomyelitis, POEMS syndrome, Polyarthritis nodosa, Polimialgia rheumatica, Polymyositis, Primary biliary cirrhosis, psoriasis, Psoriatic arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen encephalitis, Raynaud Phenomenon, Pol relapsing icondritis, Reiter syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, Schmidt's syndrome, Schnitzler syndrome, Scleritis, Sjögren's syndrome, Spondyloarthropathy, Sticky blood syndrome, Still's disease, Subacute bacterial endocarditis (SBE), Susac syndrome, Sweet's syndrome, Sydenham's korea, Sympathetic ophthalmia, Takayasu's arteritis , Temporal arteritis, Tolosa-Hunt syndrome, Transverse myelitis, Ulcerative colitis, undifferentiated connective tissue disease, Non-differentiated spondyloarthropathy, vasculitis, Wegener's granulomatosis, Wilson's syndrome, and Wiskott-Aldrich syndrome.

In another embodiment, the vector and methods of the present invention can be used to treat an ocular disorder that includes, but is not limited to, a disorder selected from the group consisting of glaucoma that includes Open Angle Glaucoma (e.g., Primary Glaucoma). Open Angle, Pigmentary Glaucoma, Exfoliating Glaucoma, Low Tension Glaucoma), Angle Closure Glaucoma (also known clinically as Closed Angle Glaucoma, Narrow Angle Glaucoma, Pupillary Block Glaucoma and Ciliary Block Glaucoma) (for example, Acute Angle Closure Glaucoma and Chronic Angle Closure Glaucoma), Aniric Glaucoma, Congenital Glaucoma, Juvenile Glaucoma, Lens Induced Glaucoma, Neovascular Glaucoma (for example, using decoy vectors of Vascular Endothelial Growth Factor (VEGF, for its acronyms), Pigment Derived Growth Factor (PDGF), Endostatin, Angiostatin, or Ang iopoietin-1), Post-Traumatic Glaucoma, Steroid-Induced Glaucoma, Sturge-Weber Syndrome Glaucoma, and Uveitis-Induced Glaucoma, Diabetic Retinopathy (for example, using decoy vectors of VEGF, PDGF, Endostatin, Angiostatin, or Angiopoietin-1), macular degeneration (eg, decoy vectors of VEGF, PDGF, Endostatin, Angiostatin, Angiopoietin-1, Member 4 of the Subfamily A of the ATP binding cassette), macular degeneration (eg using vectors decoy compounds of VEGF, PDGF, Endostatin, Angiostatin, Angiopoietin-1, Member 4 of Cassette A from Cassette to ATP), choroidal neovascularization (eg, using decoy vectors of VEGF, PDGF, Endostatin, Angiostatin, or Angiopoietin-1), vascular leakage, and / or retinal edema, bacterial conjunctivitis, fungal conjunctivitis, viral conjunctivitis, uveitis, keratic precipitates, macular edema (for example, using vectors c decoy adjuncts of VEGF, PDGF, Endostatin, Angiostatin, or Angiopoietin-1), response to inflammation after intraocular lens implantation, uveitis syndromes (eg, chronic iridocyclitis or endophthalmitis), retinal vasculitis (eg, as seen in rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, progressive systemic sclerosis, polyarteritis nodosa, Wegener's granulomatosis, temporal arteritis, Adamantiades Bechcet's disease, Sjórgen syndrome, cyclic polychondritis and spondylitis associated with HLA-B27), sarcoidosis , Eales disease, acute retinal necrosis, Vogt Koyanaki Harada syndrome, ocular toxoplasmosis, radiation retinopathy, proliferative vitreoretinopathy, endophthalmitis, ocular glaucoma (for example, inflammatory glaucoma), optic neuritis, ischemic optic neuropathy (for example, vectors composed of unit 4 of NADH dehydrogenase Alotopic), orbitopathy aso thyroid gland, pseudotumor orbital, pigment dispersion syndrome (of pigmentary glaucoma), scleritis, choroidopathies due to episcleritis (eg, "white spot syndrome" including, but not limited to, acute multifocal posterior placoid), retinopathies ( for example, cystoid macular edema, central serous choroidopathy and presumed ocular histoplasmosis syndrome (e.g., vectors composed of Gluten-Derived Neurotropic Factor, Periferin-2), retinal vascular disease (e.g., diabetic retinopathy, Coat disease and retinal arterial macroaneurysm), retinal artery occlusions, retinal vein occlusions, retinopathy of prematurity, retinitis pigmentosa (for example, vectors composed of retinal pigment-specific 65 kDa protein), familial exudative vitreoretinopathy (FEVR), idiopathic polypoid choroidal vasculopathy, Epidermal macular membranes and cataracts.

In another embodiment, the vector and methods of the present invention can be used to treat a blood disorder that includes, but is not limited to, a blood disorder selected from the group consisting of anemia, bleeding disorders and coagulation (e.g., disseminated intravascular coagulation (DIC), hemophilia, Henoch-Schonlien purpura, hereditary hemorrhagic telangiectasia, thrombocytopenia (ITP, TTP), thrombophilia, von Willebrand disease), leukemias (eg acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia), lympholas (eg, Hodgkin's lymphoma, non-Hodgkin's lymphoma), myeloproliferative disorders (e.g., myelofibrosis, polycythemia vera, thrombocythemia), cellular plasma disorders (e.g., macroglobulinemia, monoclonal gammopathies of undetermined significance, multiple lieloma), splenic disorders, white blood cell disorders (e.g., basophilic disorder, eosinophilic disorder) , lymphocytopenia, monocyte disorders, neutropenia, neutrophil leukocytosis ofílica), thrombosis, deep vein thrombosis (DVT), hemochromatosis, menorrhagia, sickle cell disease and thalassemia.

In another embodiment, the vector and methods of the present invention can be used to treat a neurological disorder including, but not limited to, neurological disorders selected from the group consisting of Gaucher's disease, Parkinson's disease, Alzheimer's disease, sclerosis Amyotrophic lateral (ALS), multiple sclerosis (MS), Huntington's disease, Fredrich's ataxia, moderate cognitive impairment, cerebral amyloid angiopathy, Parkinson's disease, Lewy body disease, multiple system atrophy (MSA) of frontotemporal dementia (ALS) FTD), progressive supranuclear palsy, and movement disorders (including ataxia, cerebral palsy, choreoathetosis, dystonia, Tourette's syndrome, cernicterus) and seizure disorder, and leukodystrophies (including adrenoleukodystrophy, metachromatic leukodystrophy, Canavan's disease, Alexander's disease, Pelizaeus-Merzbacher disease), neural ceroid lipofuscoses, ataxia tela ngectasia, Rett syndrome, alpha-synucleinopathy (eg, Lewy body disease, multiple system atrophy, Hallervorden-Spatz disease, or frontotemporal dementia), Type C Niemann-Pick disease (NPCD), spinocerebellar ataxia type 1, type 2 and type 3, and pallidoluisiana Dentatorubral atrophy (DRLPA).

In another embodiment, the vector and methods of the present invention can be used to treat a pulmonary disorder including, but not limited to, a pulmonary disorder selected from the group consisting of asthma, atelectasis, bronchitis, COPD (chronic obstructive pulmonary disease) ), emphysema, lung cancer, mesothelioma, pneumonia, asbestosis, aspergilloma, aspergillosis, acute invasive aspergillosis, bronchiectasis, bronchiolitis obliterans organizing pneumonia (BOOP), eosinophilic pneumonia, necrotising pneumonia, ral effusion, pneumoconiosis, pneumothorax, pulmonary actinomycosis , alveolar pulmonary proteinosis, pulmonary anthrax, pulmonary arteriovenous malformation, pulmonary fibrosis, pulmonary embolism, pulmonary X-histiocytosis (eosinophilic granuloma), pulmonary hypertension, pulmonary edema, pulmonary hemorrhage, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive disease, rheumatoid lung disease , sarcoidosis, radiation fibrosis pneumonitis, hypersensitivity pneumonitis, acute respiratory distress syndrome (ARDS), respiratory effort syndrome in infants, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia, lymphangioleiomyomatosis, lung histiocytosis of Langerhans cells, pulmonary alveolar proteinosis, sinusitis, tisilitis, otitis media , pharyngitis, laryngitis, pulmonary hamartoma, pulmonary sequestration, congenital cystic adenomatoid malformation (CCAM), and cystic fibrosis.

In another embodiment, the vector and methods of the present invention can be used to treat a rheumatologic disorder including, but not limited to, a rheumatic disorder selected from the group consisting of systemic lupus erythematosus, dermatomyositis, scleroderma, systemic necrotizing arteritis, cutaneous necrotizing venulitis, rheumatoid arthritis, Sjögren's syndrome, Raynaud's phenomenon, Reiter's syndrome, arthritis, psoriatic arthritis, seronegative spondyloarthropathies, Sjogren's syndrome, systemic sclerosis, dermatomyositis / polymyositis, mixed connective tissue disease and ankylosing spondylitis.

In another embodiment, the vector and methods of the present invention can be used to treat an infectious disease in a human which includes, but is not limited to, an infectious disease selected from the group consisting of fungal diseases such as dermatophytosis (e.g. trichophytosis, infections with tinea or dermatophytosis), athlete's foot, paronychia, pityriasis versicolor, erythrasma, intertrigo, fungal rash due to diapers, candida vulvitis, candida balanitis, otitis externa, candidiasis (cutaneous and mucocutaneous), chronic mucocanthiasis (for example, thrush and vaginal candidiasis), cryptococcosis, geotrichosis, trichosporosis, aspergillosis, penicilliosis, fusariosis, zygomycosis, sporotrichosis, chromomycosis, coccidioidomycosis, histoplas-mosis, blastomycosis, paracoccidioidomycosis, pseudales-queriosis, mycetoma, fungal keratitis, otomycosis, pneumocystosis, and fungemia , infections with Acinetobacter, actinomycosis, African sleeping sickness, AIDS (acquired immunodeficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, atrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis (BV), Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black stone, Blastocystis hominis infection, Borrelia infection, botulism (botulism in infants), Brazilian hemorrhagic fever, brucellosis, infection by Burkhold eria, Buruli ulcer, infection by Calicivirus (Norovirus and Sapovirus), candidiasis, cat scratch disease, cellulitis, Chagas disease (trypanosomiasis americana), canker, chickenpox, chlamydia, cholera, chromoblastomycosis, clonorchiasis, Clostridium difficile, Coccidioidomycosis, Colorado tick fever (CTF), common cold (acute viral rhinopharyngitis); acute coryza), Creutzfeldt-Jakob disease (CJD), cryptococcosis, cryptosporidiosis, larva migratoria (CLM), dengue fever, Dientamoebiasis, Diphtheria, Difilobrosis, Difilobrosis, Dracunculiasis, fever Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, enterobiasis (intestinal worm infection), JJnterococcus infection, Enterovirus infection, epidemic typhus, Erythema infeccioso, exanthema subitum, Fasciolopsiasis, Fasciolosis, fatal familial insomnia (IFF), Filariasis, Fusobacterium infection, GAS gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Stráussler-Scheinker syndrome (GSS), Giardiasis Glanders, Gnathostomiasis, Gonorrhea, Inguinal granuloma (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influe, disease of hand, feet and mouth (HFMD, for its acronym in English), pulmonary syndrome by hantavirus (HSP), Helicobacter pylori infection, ic-uremic syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), Hepatitis A, B, C, D, E, Herpes Simple, Histoplasmosis, hookworm infection, bocavirus infection, human ehrlichiosis ewing, human granulocytic anaplasmosis (HGA), human granulocytic anaplasmosis (HGA), human monocytic ehrlichiosis, human papillomavirus (HPV) infection, human parainfluevirus, Hymenolepiasis, Epstein-Barr virus infectious mononucleosis (Mono), Influe Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (legionnaire's disease), Legionellosis (fever) of Pontiac), leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (Lyme borreliosis), lymphatic filariasis (Elephantiasis), lymphocytic choriomeningitis, malaria, fever, Marburg haemorrhagic fever (MHF), Measles, melioidosis (Whitmore's disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscum contagiosum (MC), Mumps, Murine typhus (endemic typhus), mycoplasmic pneumonia, mycetoma, Myiasis, neonatal conjunctivitis (Ophthalmia neonatorum), (New) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, onchocerciasis (river blindness), paracocci-dioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (head lice), Pediculosis corporis (body lice), Pediculosis pubis (pubic louse, crabs), pelvic inflammatory disease (PID), Pertussis (hooping cough), plague, pneumococcal infection, pneumocystis pneumonia (PCP, for short) in English), pneumonia, poliomyelitis, poliomyelitis, infection by Prevotella, meningoencephalitis by amoebae mary (PA), leukoencephalopathy progressive multifocal disease, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus infection, Rinosporidiosis, Inovirus infection, rickettsial infection, rickettsial pox, Rift Valley fever (RVF), Rocky mountain spotted fever (R SF), rotavirus infection, rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, schistosomiasis, sepsis, shigellosis (bacillary dysentery), herpes (herpes zoster), smallpox (Variola) sporotrichosis, staphylococcal food poisoning, infection staphylococcal, strongyloidiasis, syphilis, taeniasis, Tanus (Lockjaw), Tinea barbae (Barber's itch), Tinea capitis (Ringworm of the scalp), Tinea corporis (ringworm of the body), Tinea cruris (itch of yóquey), Tinea manuum (tinea of the hand), Tinea nigra, Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Visceral Migrant Larva (VLM)), Toxoplasmosis, Trichinosis , Trichomoniasis, Trichuriasis (trichocephalic infection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white stone (white line), Yersinia pseudotuberculosis infection, Yersiniosis, fever yellow, and Zygomycosis.

In another embodiment, the vector and methods of the present invention can be used to treat one or more diseases in a mammal. In one aspect, the mammal is a human. In another aspect, the mammal is a non-human animal. A variety of diseases that can be treated using the teachings of the present invention can be easily contemplated. These diseases include, but are not limited to, chronic kidney disease, osteoarthritis, oncology, viral upper respiratory infection, feline plasma cell stomatomies, feline eosinophilic granulomas, feline leukemia virus infection, canine distemper infection, systemic fungal infections, cardiomyopathy, mucopolysaccharidosis VII, and infectious diseases.

In one aspect, the disease being treated is an infectious disease in an animal, and this infectious disease includes, but is not limited to, bovine respiratory disease, porcine respiratory disease, avian influenza, avian infectious bronchitis, bovine spongiform encephalopathy, canine leishmaniosis. , chronic atrophy disease, acquired human immunodeficiency virus (HIV), hepatitis, hepatitis A, hepatitis B, hepatitis C, classical swine fever, Echinococcus, Enzootic pneumonia, FIP, foot and mouth disease, Jaagsiekte, Maedi-Visna, mastitis in animals, Microsporum canis, Orf (animal disease), pes petits ruminants, smallpox diseases, feather and Psittacid diseases, rabies, Mediterranean fever (Brucellosis) or Bang disease or malt fever, rippling fever, contagious abortion , epizootic abortion, food poisoning by Salmonella, enteric paratyphosis, bacillary dysentery, Pseudotuberculosis, plague, fever pestilen Tuberculosis, vibrios, spiral disease, Weil's disease (leptospirosis) or canicola fever, hemorrhagic jaundice (Icterohaemorrhagia leptospira), dairy worker fever (L. hardjo), cyclical fever, tick-borne cyclic fever, spirocetal fever, vagabond fever, hunger fever, Ly e arthritis, Bannworth syndrome (Lyme disease), meningopolineuritis transmitted by tick, chronic erythema migrans, vibriosis, Colibacteriosis, colitoxemia, white diarrhea, intestinal gut edema, enteric paratyphosis, staphylococcal food toxicosis, staphylococcal gastroenteritis, canine crown virus (CCV) or canine parvovirus enteritis, feline infectious peritonitis virus, transmissible gastroenteritis virus (TGE) , for its acronym in English), Hagerman Redmouth disease (ERMD, for its acronym in English), infectious hematopoietic necrosis (IHN, for its acronym in English), pleuropneumonia of Actinobacilo (haemofilo), Hansen's disease, Streptotricosis, fungal dermatitis of Sheep, Pseudoglanders, hitmore disease, Francis disease, deer fly fever, fie rabbit fever, O'Hara disease, Streptobacillary fever, Haverhill fever, epidemic arthritic erythema, sodoku, biotransport fever, haemorrhagic septicemia, ornithosis, fever, chlamydiosis, North American blastomycosis, Chicago disease, Gilchrist's disease, fever by Cat Scratch, Benign Linforeticulosis, Benign Non-Bacterial Lymphadenitis, Bacillary Angiomatosis, Peliosis Hepatis Bacillary, Search Fever, Balkan Influenza, Balkan Flu, Slaughter Fever, Tick-borne Fever, Pneumorickettsiasis, Typhus by American Tick, Typhoid-transmitted Typhoid tick, vesicular rickettsiasis, Kew Gardens spotted fever, flea-transmitted typhoid fever, endemic typhoid fever, urban typhoid, ringworm, dermatophytosis, ringworm, trichophytosis, microsporosis, yóquey pruritus, athlete's foot, Sporothrix schenckii, dimorphic fungus, cryptococcosis and histoplasmosis, benign epidermal monkeypox, BEMP, Herpesvi simian rus, simian disease B, Venezuelan equine encephalitis, lethargic encephalitis Type C, Yellow fever, Black vomit, Hantavirus pulmonary syndrome, Korean hemorrhagic fever, Epidemic nephropathy, Epidemic hemorrhagic fever, Hemorrhagic nephrosonephritis, Lymphocytic choriomeningitis, Californian encephalitis / Crosse encephalitis, African hemorrhagic fever, Vervet or green monkey disease, Hydrophobia, Rabies, infectious hepatitis, epidemic hepatitis, epidemic jaundice, Rubella, Morbilli, swine and equine influenza, avian pest, Newcastle disease, Piroplasmosis, Toxoplasmosis, African sleeping sickness, Gambia trypanosomiasis, Rhodesian trypanosomiasis, Chagas disease, Chagas-Mazza, South American trypanosomiasis, Entamoeba histolytica, Balantidial dysentery, cryptosporidiosis, giardiasis, cutaneous leishmaniasis: chiclero ulcer, espundia, pianbols, uta, and buba (in the Americas); oriental ulcer, button of Aleppo (in the Old World) and button of Baghdad, button of Delhi, ulcer of Bauru, visceral leishmaniasis: kala-azar, Microsporidiosis, anisakiasis, trichinosis, angios-trongilosis, eosinophil meningitis or meningoencephalitis (A. cantonensis ), abdominal angiostrongylosis (A. costaricensis), hookworms, necatoriasis, hookworm disease, Capillariasis, Brugiasis, Toxocariasis, Oesophagostomiasis, Strongyloidiasis, Trichostrongylosis, Ascariasis, Difilobotriasis, Esparganosis, Hydatidosis, Hydratic disease, Echinococcal granulosis, Cystic hydration disease, solitary infection, Schistosoma and the like.

The treatment of malignant diseases caused by infectious pathogens is also contemplated. Examples of these diseases include, but are not limited to, osteosarcoma, leukemia, lymphoma, Burkitt's lymphoma caused by EBV, Rous sarcoma caused by Rous retrovirus, Kaposi's sarcoma caused by herpes virus type 8, adult T cell leukemin caused by HTLV-I retrovirus, or hairy cell leukemia caused by HTLV-II, and many other tumors and leukemias caused by infectious agents and viruses.

In one embodiment, the one or more proteins used to treat one or more of the foregoing diseases includes, but is not limited to, erythropoietin, ghrelin, osteoprotegerin, RANKL, RANKL decoy, TNF-OI antagonists, an IL- antagonist. 1, G-CSF, GM-CSF, IFN-a, IFN- ?, angiostatin, endostatin, TNF-, PP1DCY-LSRLOC, β-glucuronidase, and IL-12. In another embodiment, the one or more proteins of the invention include, but are not limited to, IL-1, IL-2, IL-12, IL-3, IL-4, IL-5, IL-7, IL- 8, IL-9, IL-10R DN or a subunit thereof, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN- gamma, IFN-alpha 1, IFN alpha 2, IL-15-R-alpha, CCL3 (MIP-la), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6China), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1 , Flt3L, IFN-beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1 RNAi, an antisense oligonucleotide of PD-L1, TGFbRII DN, ICOS-L, and S100.

In one embodiment, the vector administered to the mammal afflicted with one or more of the diseases described is an adenoviral vector. In one embodiment, the vector comprises a polynucleotide that codes for a gene change. In one aspect, gene change is a gene change based on EcR. In another embodiment, the polynucleotide encoding a gene change comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by the The first transcription factor sequence and the second sequence of the transcription factor interact to form a protein complex that functions as a ligand-dependent transcription factor. In one aspect, the ligand is a diacylhydrazine. In another aspect, the ligand is selected from RG-115819, RG-115932, RG-115830. In yet another aspect, the ligand is an amidoketone or an oxadiazoline.

In another embodiment, the present invention can be used to treat one or more lysosomal storage diseases in a mammal. In one aspect, the mammal is a human. In another aspect, the mammal is a non-human animal. Examples of lysosomal storage diseases that can be treated according to the invention include, but are not limited to, Pompe disease / Type II glycogen storage disease, Gaucher disease (type I, type II, type III), Fabry disease, mucopolysaccharidosis (Hunter syndrome), mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), mucopolysaccharidosis I, metachromatic leukodystrophy, neuronal ceroid lipofuscinosis or CLN6 disease (late atypical infantile CLN5 disease, late-onset, early juvenile, syndrome of Finnish variant, late childhood CLN2 / TPP1 disease, Jansky-Bielschowsky disease, adult onset NCL / CLN4 disease / Kufs, childhood late CLN8 variant / northern epilepsy, CLN1 / PPT infant / Santavuori disease Haltia, Beta-mannosidosis), NCL / CLN3 juvenile / Batten-Spielmeyer-Vogt disease, Sanfilippo type A syndrome, Sanfilippo type B syndrome, Sanfilippo type C, Sanfilippo type D syndrome, MPSI Hurler syndrome, Niemann-Pick disease (Type A, Type B, Type C, Type D), Activator deficiency / Gangliosidosis of GM2, Alpha-mannosidosis, Aspartylglucosaminuria, cholesteryl ester storage disease, Chronic hexosaminidase deficiency, Cystinosis, Danon's disease, Farber's disease, Fucosidosis, Galactosialidosis (Goldberg's syndrome), GM1 gangliosidosis (Infantile, Late infant / Juvenile, Adult / chronic), disease of Cells I / Mucolipidosis II, Sickle Free Infant Sickle Storage Disease / ISSD, Hexosaminidase A Youth Deficiency, Krabbe's Disease (Early Childhood Start, Late Start), Mucopolysaccharidosis Disorders (Pseudo-Hurler / Mucolipidosis IIIA Polydistrophy, Scheie syndrome MPS I Hurler-Scheie Morquio type A / MPS IVA, Morquio type B / MPS IVB, MPS IX deficiency hyaluronidase, Sly syndrome (MPS VII), Mucolipidosis I / Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV), Multiple sulfatase deficiency, pycnodysostosis, Sandhoff disease / Adult onset / GM2 gangliosidosis, Sandhoff / gangli disease GM2 Ossidosis, Infantile, Sandhoff's disease / GM2 gangliosidosis, Juvenile, Schindler's disease, Salla's disease, infant sialic acid storage disease, Tay-Sachs / GM2 gangliosidosis, Wolman's disease, Asparilglucosaminuria and prosaposin.

It will be appreciated that Sanfilippo Type A syndrome is synonymous with Sanfilippo syndrome Type A / MPS IIIA, Sanfilippo syndrome Type B is synonymous with Sanfilippo syndrome Type B / MPS IIIB, Sanfilippo syndrome Type C is synonymous with Sanfilippo syndrome type C / MPS IIIC, Sanfilippo syndrome type D is synonymous with Sanfilippo syndrome type D / MPS IIID.

In one embodiment, the one or more proteins expressed by the vector of the invention used to treat one or more of the above lysosomal storage diseases include, but are not limited to, α-galactosidase A, arylsulfatase A, α-glucosidase, β -glucosidase, glucocerebrosidase, CLN6 protein, juvenile associated with CLN3, N-sulfoglucosamine-sulfohirolase (SGSH), aN-acetylglucosaminidase, acetyl-CoA-glucosaminide acetyltransferase, N-acetylglucosamine-6-sulphatase, aL-iduronidase, arylsulfatase B, acid sphingomyelinase, and yururonate sulfatase.

In one embodiment, the vector administered to the mammal afflicted with one or more of the described lysosomal storage diseases is an adenoviral vector. In one embodiment, the vector comprises a polynucleotide that codes for gene change. In one aspect, gene change is a gene change based on EcR. In another embodiment, the polynucleotide encoding gene change comprises a first sequence of transcription factor under the control of a first promoter and a second sequence of transcription factor under the control of a second promoter, wherein the proteins encoded by the first Transcription factor sequence and the second sequence of transcription factor interact to form a protein complex that functions as a ligand-dependent transcription factor. In one aspect, the ligand is a diacylhydrazine. In another aspect, the ligand is selected from RG-115819, RG-115932, and RG-115830. In yet another aspect, the ligand is an amidoketone or oxadiazoline.

In another embodiment, the present invention can be used to treat one or more liver diseases in a mammal. In one aspect, the mammal is a human. In another aspect, the mammal is a non-human animal. In one aspect, the liver disease is hepatitis B. In another aspect, the liver disease is hepatitis C. In one embodiment, the protein expressed by a vector of the invention is IFN-. In another embodiment, the protein expressed by the vector of the invention is one or more of the hepatic diseases comprising ceruloplasmin.

A non-limiting example of a human liver chimeric mouse model for hepatitis B and C virus infection and treatment is described in Bissig, KD et al., J, Clin. Investigation 120: 924 (2010). Another non-limiting example of a human hepatocyte model is the humanized mouse system marketed by YecurisMRm (Portlan, OR).

A non-limiting example of an encephalitis model useful for evaluating antiviral / anti-infective treatment is described in O'Brien, L. et al, J. General Virology 50: 874-882 (2009).

Non-limiting examples of influenza models useful for evaluating antiviral / anti-infective treatment are described in Beilharz, M.W. et al, Research Biochemical Biophysical Communications 355: 740-744 (2007), and Koerner, I. et al, J Virology, 31: 2025-2030 (2007).

In one embodiment, the vector administered to the mammal afflicted with one or more of the liver diseases described is an adenoviral vector. In another embodiment, the vector is not an adenoviral vector. In another embodiment, the vector is a plasmid. In one embodiment, the vector comprises a polynucleotide that codes for a gene change. In one aspect, gene change is a gene change based on EcR. In another embodiment, the polynucleotide encoding a gene change comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by the The first transcription factor sequence and the second transcription factor sequence interact to form a protein complex that functions as a ligand-dependent transcription factor. In one aspect, the ligand is a diacylhydrazine. In another aspect, the ligand is selected from RG-115819, RG-115932, and RG-115830. In yet another aspect, the ligand is an amidoketone or oxadiazoline.

The invention provides engineering engineering of cells, eg, immune cells and TSC, to conditionally express a protein having the function of immunomodulator and optionally, IL-12 and therapeutic uses and / or applications for the treatment of cancer. or tumors or both. The in vitro manipulated immune cells and TSC conditionally expressing a protein having the function of an immunomodulator and optionally IL-12, are a safe improvement over the constitutive production of the protein (s). Additionally, the ability to control the timing and level of the immunomodulator and optionally the expression of IL-12, provides improved control of treatment efficacy. Therefore, immune cells manipulated in vitro and TSC can be formulated in pharmaceutical compositions as pharmaceuticals for the treatment of a cancer or a tumor in a human or non-human organism. Alternatively, in vitro manipulated populations of immune cells, TSCs or subgroups thereof, can be used as vehicles to conditionally distribute an immunomodulator and optionally the production of IL-12 protein to a specific area (normal tissue, cancer, or tumor) in the body of a human or non-human organism. The immune cells may be autologous or they may be non-autologous dendritic cells. Dendritic cells can be isolated from bone marrow or from peripheral blood circulation. In human patients, populations of dendritic cells can be isolated via a leukophoresis procedure, where a fraction of white blood cells is isolated and removed and other blood components are reinfused to the patient.

In yet another embodiment, dendritic cells can be prepared by transfecting human hematopoietic stem cells, with a vector of the invention expressing a protein having the function of a munomodulator and optionally a protein having the function of IL-12. , and differentiating the transfected stem cell to give a dendritic cell. See United States Patent 6,734,014.

In one embodiment, an adenoviral nucleic acid vector containing a gene change is provided, wherein the coding sequences for VP16-RXR and Gal4-EcR are separated by the sequence from the site of entry to the internal ribosome EMCV (IRES), are inserted into the adenoviral shuttle vector under the control of the human ubiquitin C promoter. The coding sequences for the p40 and p35 subunits of IL-12 separated by an IRES sequence, and placed under the control of a synthetic inducible promoter, are inserted upstream of the ubiquitin C promoter.

In another embodiment, the invention provides a shuttle vector that possesses transcription units (VP16-RXR and Gal4-EcR) for the fusion proteins, and inducible IL-12 subunits recombined with the adenoviral backbone (AdEasil) in E cells. coli BJ5183. After checking the recombinant clone, the plasmid possessing the rAd.RheoIL12 genome is developed in and purified from the XLIO-Gold cells, digested from the plasmid backbone and packaged by transfection into HEK 293 cells or CHO cells.

Purification of the vector to improve the concentration can be achieved by any suitable method, such as density gradient purification (e.g., cesium chloride (CsCL)) by chromatography techniques (e.g., column or batch chromatography). For example, the vector of the invention can be subjected to two or three steps of purification by density gradient. The vector, for example, an adenoviral deficient replication vector, is desirably purified from cells infected with the replication deficient adenoviral vector using a method comprising lysate adenovirus infected cells, applying the lysate to a chromatography resin, eluting the adenovirus from the chromatography resin, and collect a fraction containing adenovirus.

In a particular embodiment, the resulting primary viral reserve is amplified by reinfection of HEK 293 cells or CHO cells and is purified by centrifugation by CsCl density gradient.

In one embodiment, the immunomodulator and / or the IL-12 gene is a wild-type gene sequence. In yet another embodiment, the immunomodulator and / or the IL-12 gene is a modified gene sequence, for example, a chimeric sequence or a sequence that has been modified to use preferred codons.

In one embodiment, the immunomodulator and / or the IL-12 gene is the human wild-type sequence. In yet another embodiment, the sequence is at least 85% identical to the wild-type human sequence, for example, at least 90%, 95%, or 99% identical to the human wild-type sequence. In a further embodiment, the gene sequence encodes the human polypeptide. In yet another embodiment, the gene encodes a polypeptide that is at least 85% identical to the wild-type human polypeptide, for example, at least 90%, 95%, or 99% identical to the human wild-type polypeptide.

In one embodiment, the IL-12 gene is the wild-type mouse IL-12 sequence. In yet another embodiment, the sequence is at least 85% identical to wild-type mouse IL-12, for example, at least 90%, 95%, or 99% identical to wild-type mouse IL-12. In a further embodiment, the IL-12 gene sequence encodes the mouse IL-12 polypeptide. In yet another embodiment, the gene encodes a polypeptide that is at least 85% identical to wild-type mouse IL-12, for example, at least 90%, 95%, or 99% identical to IL-12 from wild type mouse.

DC can be isolated from bone marrow of humans, mice or other mammals. Dendritic cells can be isolated from the blood of humans, mice or other mammals. In human patients, populations of dendritic cells can be isolated via a leukophoresis procedure as is known in the art, where a fraction of white blood cells is isolated and removed, and other blood components are reinfused to the patient. In one embodiment, DCs are derived from murine bone marrow as previously described (Tatsumi et ah, 2003). Briefly, wild-type mouse bone marrow (BM) or EGFP Tg is cultured in conditioned medium (CM) supplemented with 1000 units / ml of murine, recombinant, and recombinant milo-granulocyte colony stimulating factor (MUC-4) ( Peprotech, Rocky Hill, NJ) at 37 ° C in a humidified 5% C02 incubator for 7 days. CDIIIc + DCs are then isolated, for example, using specific MACSTM spheres, following the manufacturer's instructions (Miltenyi Biotec, Auburn, CA). DC CDc + produced in this way more than > 95% pure based on the morphology and coexpression of CDllb, CD40, CD80, and MHC antigens of class I and class II.

One embodiment of the invention provides engineered and engineered TSC cells, which conditionally express a protein having the function of an immunomodulator and optionally IL-12 suitable for therapeutic applications for the treatment of cancer, or tumors or both, as gene therapy in human or non-human organisms. In one embodiment, the invention provides the engineered immune cells, and the manipulated TSCs containing the gene change.

In another embodiment, the invention provides engineered cells and engineered TSCs containing at least a portion of an ecdysone receptor. In yet another embodiment, the invention provides the engineered engineered immunomodulatory cells, and the TSCs containing a gene change based on the ecdysone receptor. In yet another embodiment, the invention provides the engineered cells and the engineered TSCs containing RheoSwitch. In yet another embodiment, the invention provides a kit comprising engineered and engineered TSC immune cells containing a gene change and a ligand that modulates gene change. In yet another embodiment, the kit further comprises a diacylhydrazine ligand. In yet another embodiment, the kit further comprises RG-115830 or RG-115932.

In one embodiment, the invention provides a population engineered by immune cells and TSC. In one embodiment, DCs cultured 7 days are treated with recombinant adenovirus that encodes an immunomodulator and / or IL-12 driven outside of a constitutive or inducible promoter, or are infected with the control adenoviral vector, or negative control (rAdiJj5), over a multiplicity of infection interval (MOIs). After 48 hours, the infected DCs are harvested and analyzed for the phenotype and for the production of an immunomodulator and / or IL-12 using the specific ELISA kit (BD-PharMingen, San Diego, CA), with a lower level of detection of 62.5 pg / ml.

In another embodiment, the invention provides an in vitro engineered population of imminent cells, and TSCs comprising a vector, eg, a DNA vector, having a gene change capable of conditionally expressing a protein having the function of a immunomodulator and / or IL-12, and further comprising activation of the ligand. In a further embodiment, the invention provides a method for treating cancer, for example, melanoma or glioma, by administering the DCs engineered to a patient, and then administering an activating ligand, such as RG-115819, RG-115830 or RG-115932, to the patient. The patient can be a human or an animal with cancer. The treatment method and the products, the engineered cells, the kits and the ligands have application in human therapy and veterinary animal therapy. Therefore, the products and methods with contemplated to be used for human and animal veterinary purposes.

Thus, in one embodiment, the polynucleotide expressing the immunomodulator, for example, TNF-alpha, and activating ligand are co-administered to a patient having a cancer. The activating ligand is generally administered for several days, for example, before and after administration of the polynucleotide. If systemic toxicity develops due to the immunomodulator, for example, TNF-alpha, then the administration of activating ligand can be reduced or eliminated in an effort to attenuate side effects.

In another embodiment, the polynucleotide expressing the immunomodulator, e.g., TNF-alpha, and the activating ligand are co-administered to a patient suffering from one or more lysosomal storage diseases, or one or more liver diseases. The activating ligand is generally administered for several days, for example, before and after administration of the polynucleotide. If systemic toxicity develops, then the administration of the activating ligand can be reduced or eliminated in an effort to attenuate the side effects.

In certain embodiments, the invention provides a method for reducing the size of a tumor, which comprises administering an adenoviral vector, comprising a polynucleotide that conditionally expresses an immunomodulated, e.g., TNF-alpha, and administering an activating ligand. A method for preventing the formation of a tumor is also provided, which comprises administering an adenoviral vector, comprising a polynucleotide that conditionally expresses an immunomodulator, e.g., TNF-alpha, and administering an activating ligand. In some embodiments, the invention provides a method for reducing or ameliorating one or more symptoms of a neoplastic disorder comprising administering an adenoviral vector, comprising a polynucleotide that conditionally expresses an immunomodulator, e.g., TNF-alpha, and administering a ligand activator. In particular, the composition comprising the vector, for example, adenoviral vector, which conditionally expresses an immunomodulator can reduce, prevent, or improve systemic toxicity in the treated subject compared to a vector that constitutively expresses the immunomodulator.

In certain embodiments, the invention provides a method for treating one or more diseases or one or more lysosomal storage diseases, or one or more liver diseases, in mammals, which comprises administering an adenoviral vector, comprising a polynucleotide that expresses conditionally one or more proteins and administer an activating ligand. In some embodiments, the invention provides a method for reducing or improving one or more symptoms of one or more diseases or one or more lysosomal storage diseases, or one or more liver diseases in mammals, which comprises administering an adenoviral vector, comprising a polynucleotide that conditionally expresses an immunomodulator, e.g., TNF-alpha, and administer an activating ligand.

Protein-based brands reduce or eliminate the need for highly specific post-translational modifications for effective targeting. Useful protein-based tags include, but are not limited to, targeting by IGF2 (IGF2 engineering (GILT) / IGF2), target selection of transferrin receptor (trans-errine, peptides that target TfR), and protein Tat (in which cell surface heparin sulfate proteoglycans (HSPG) mediate the internalization of Tat).

Other proteins that are targeted to the lysosome that can be used as a label include, but are not limited to, vitamin D binding protein, folate binding protein, lactotransferrin, sex hormone binding globulin, transthyretin, saposin, retinol binding protein, lipoprotein B Apo, lipoprotein E Apo, prolactin, receptor-associated protein (in one embodiment, without the HNEL sequence), native transferrin, and mutant transferrin (for example, the mutant K225E / R651A or the mutant K225E / K553A).

In one embodiment, the expression construct also encodes for one or more of the indicator sequences, a location mark sequence, and a detection mark sequence. Additionally, it will be appreciated that the composition of the invention or methods for using the composition can be combined with any chemotherapeutic agents or agents (e.g., to provide a combined therapeutic regimen) that removes, reduces, inhibits or controls the growth of neoplastic cells. or tumors in vivo. As used herein, the terms "chemotherapeutic agent" or "chemotherapeutic" should be understood to mean any therapeutic compound that is administered to treat or prevent the growth of tumors in vivo. In particular, chemotherapeutic agents compatible with the invention comprise "traditional" chemotherapeutic agents such as small molecules and, more recently, developed biological products such as antibodies, cytokines, antisense molecules, etc., which are used to reduce or delay the growth of malignant cells. .

In one aspect, the invention provides a pharmaceutical composition suitable for administration to a human or a non-human comprising a population of immune cells engineered in vitro or TSC or a vector, eg, an adenoviral vector, which expresses a protein which has the function of an immunomodulator, for example, TNF-alpha, and / or IL-12, wherein the formulation is suitable for administration by intratumoral administration. In another embodiment, a composition, e.g., pharmaceutical composition, comprises a vector that conditionally expresses an immunomodulator, e.g., TNF-alpha. In some embodiments, the composition comprises approximately 1 xlO5 or more particle units (pu) of the gene transfer vector. A "particle unit" is an individual vector particle. In certain embodiments, the composition comprises about 1 x 10 6 units of gene transfer vector particles (e.g., about 1 x 107 or more particle units, about 1 x 108 or more particle units, or about 1 x 109 or more). more particle units). In other embodiments, the composition comprises from about 1 x 1010 or more pu, 1 x 1011 or more pu, 1 x 1012 or more pu, 1 x 1013 or more pu, 1 x 1014 or more pu, or 1 x 1015 or more pu of the gene transfer vector, especially of a viral vector, such as an adenoviral vector deficient in replication. The number of particle units of the gene transfer vector in the composition can be determined using any suitable known method, such as by comparing the absorbance of the composition with the absorbance of a normal solution of the gene transfer vector (i.e. a solution of known concentration of gene transfer vector) as further described herein.

The invention further provides a pharmaceutical composition comprising an activating ligand, such as RG-115819, RG-115830 or RG-115932, wherein the composition is suitable for administration by intraperitoneal, oral or subcutaneous administration.

A composition of the invention, for example, a composition comprising an engineered DC, a vector (eg, an adenoviral vector), or an activating ligand, may additionally comprise a pharmaceutically acceptable carrier. The carrier can be any suitable carrier for engineered dendritic cells, gene transfer vector, or activating ligand. Suitable carriers for the composition are described in U.S. Patent No. 6,225,289. The carrier will typically be liquid, but it can also be solid, or a combination of liquid and solid components. The carrier is desirably a carrier (eg, excipient or diluent) to a pharmaceutically acceptable carrier (eg, physiologically or pharmacologically acceptable). Pharmaceutically acceptable carriers are well known and readily available. The choice of the carrier will be determined, at least in part, by the particular components in the composition and the particular method used to administer the composition. The composition may further comprise any other suitable component, especially to improve the stability of the composition and / or its end use. Accordingly, there is a wide variety of suitable formulations of the composition of the invention.

Formulations suitable for oral administration include (a) liquid solutions, such as an effective amount of the active ingredient dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing it contains a predetermined quantity of the active ingredient, such as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. The tablet forms may include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia gum, gelatin, colloidal silicon dioxide, croscarmellose sodium, talcum, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. The tablet forms may comprise the active ingredient in a flavor, usually sucrose and acacia gum or tragacanth, as well as tablets comprising the active ingredient in an inert base (such as gelatin and glycerin, or sucrose and acacia gum), and emulsions, gels, and the like which contain, in addition to the active ingredient, excipients as is known in the art.

For example, the composition comprising the vector, the population of the immune cells or TSC, or the cells engineered in vitro may comprise a buffering agent, for example, TRIS. In one embodiment, the composition may comprise TRIS and / or glycerin. In another embodiment, the composition also comprises acidifiers, anionic or nonionic surfactants, compatibility agents, and / or diluents.

Formulations suitable for administration by inhalation include aerosol formulations. Aerosol formulations can be placed in acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen and the like. They can also be formulated as non-pressurized preparations, for distribution of a nebulizer or an atomizer.

Formulations suitable for parenteral administration include sterile, isotonic aqueous and non-aqueous injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which fly isotonic to the formulation with the proposed recipient blood, and sterile aqueous and non-aqueous suspensions which they may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations may be presented in sealed single-dose or multi-dose containers, such as ampoules and flasks, and may be stored in a freeze-dried (lyophilized) condition that requires only the addition of a sterile liquid excipient, eg, water. , for injections, immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind described above.

Formulations suitable for anal administration can be prepared as suppositories by mixing the active ingredient with a variety of bases such as emulsifying bases or water soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or aspersion formulas not containing, in addition to the active ingredient, carriers as are known in the art to be appropriate.

In addition, the composition may comprise additional therapeutically or biologically active agents. For example, therapeutic factors useful in the treatment of a particular indication may be present. Factors controlling inflammation, such as ibuprofen or steroids, may part of the composition to reduce the swelling and inflammation associated with in vivo delivery of the gene transfer vector and physiological distress. The immune system suppressors can be administered with the method of the composition to reduce any immune response to the gene transfer vector, itself or associated with a disorder. Alternatively, immune enhancers may be included in the composition to promote the expression of the body's natural defenses against the disease. In addition, cytokines with the composition can be administered to attract immune effector cells to the tumor site.

In the particular embodiment described herein, the invention provides a method for treating a tumor, comprising the steps in the order of: to. administering intratumorally in a mammal a population of immune cells managed in vitro by engineering or TSC; Y b. administering to the mammal a therapeutically effective amount of an activating ligand.

In one embodiment, the activation ligand is administered substantially at the same time as immune cells or TSCs engineered in vitro, for example, within one hour before or after administration of the cells. In yet another embodiment, the activating ligand is administered at or within 24 hours after the administration of immune cells or TSCs manipulated in vitro. In another modality more, the activation ligand is administered at or within less than 48 hours after the immune cells or TSC manipulated in vitro. In yet another embodiment, the ligand is RG-115932. In yet another embodiment, the ligand is administered at a dose of about 1 to 50 mg / kg / day. In another embodiment, the ligand is administered in a dose of approximately 30 mg / kg / day. In yet another embodiment, the ligand is administered at a dose of about 7 to 28 mg / kg / day. In another modality, the ligate is administered daily for a period of 14 days. In yet another embodiment, approximately 1 x 106 to 1 x 108 cells are administered. In yet another embodiment, approximately 1 x 10 to 1 x 10 7 cells are administered.

In one embodiment, dendritic cells are engineered to conditionally express IL-2 and IL-12. IL-2 exerts potent immunoregulatory effects on effector and regulatory T, K and NK-T cells. It is expected that the expression of IL-2 and IL-12 in cells will result in the reciprocal upregulation of other receptors, and induce differences by complementary biological effects, by virtue of the separate signaling pathways. It is also expected that the combination of IL-2 and IL-12 will prolong the duration of immune stimulation and reduce the effective dose of cells that may be more tolerated by the animal. See Dietrich 2002, Wigginton 2002, 2001, 1996 and Koyama, 1997, McDermott and Atkins 2008; Berntsen et al 2008; Tarhini et al 2008; Heemskerk et al 2008; Horton et al. 2008. The polynucleotide sequences of IL-2 are available under accession numbers U25676 (human); M 008366 (mouse); NM_204153 (chicken); and NM_053836 (rat). The polynucleotide sequences of IL-12 are available under accession numbers NM 000882 (human IL12A); NM_002187 (human IL12B); NM_008351 (mouse IL12a); NM_008352 (mouse IL12b); NM_213588 (chicken IL12A); NM_213571 (chicken IL12B); NM 053390 (rat IL12a); and NM_022611 (rat IL12b). SEQ ID NOS: 13, 15, 21 and 23 encode human and mouse IL-12 and subunits thereof.

In another embodiment, dendritic cells are manipulated to conditionally express IL-18 and IL-12. IL-18 induces the production of IFN-gamma and promotes the development of helper T cells and the activation of K cells. In addition, IL-18 can intensify GM-CSF production and decrease the production of IL-10. It is expected that the expression of IL-18 and IL-12 will overcome the limitations observed when the cytokine is administered alone. It is expected that the expression of IL-12 and IL-18 in dendritic cells will stimulate more vigorous tumor antigen-specific Thl responses, than when dendritic cells are transduced with any cytokine alone.

The intratumoral injection of DC manipulated by engineering to secrete IL-12 and IL-18 mediates the highest levels of INF- production. and complete tumor rejection (Tatsumi 2003). See, Vujanovic, 2006. See also Coughlin, 1998, Subleski, 206, Tatsumi, 2003, and Sabel, 2007; Shiratori et al 2007; Lian et al 2007; Iinuma et al 2006. See above for IL-12 polynucleotide sequences. The polynucleotide sequences of IL-18 are available under accession numbers U90434 (human); NM_008360 (mouse); EU747333 (chicken); and AY258448 (rat).

In another embodiment, dendritic cells are engineered to conditionally express IL-15 and IL-12. IL-15 shares some biological activities with IL-2 which also makes it potentially useful for cancer therapies. IL-15 stimulates the proliferation of NK cells and activated T cells, and supports the expansion of effector T cells. It has been reported that the presentation of IL-15 synergized with IL-12 for the intensified production of IFN-gamma in NK cells. Koka, 2004; Basak 2008; Lasek et al 2004. The intratumoral distribution of IL-15 and IL-12 induced significant tumor regression in a melanoma model (Lasek 1999). See above for IL-12 polynucleotide sequences. SEQ ID NOS: 11 and 19 encode human and mouse IL-15. Figures 2 and 4 are plasmid maps for the expression system that can be used for human and mouse IL-12 and IL-15.

In yet another embodiment, the dendritic cells are engineered to conditionally express IL-21 and IL-12. IL-21 and its receptor share sequence homology with IL-2 and IL-15. IL-21 promotes the expansion and maturation of NK cells. The biological effects of IL-21 potentially synergize with IL-12 since the treatment of NK cells with IL-21 results in significant upregulation of the IL-12 receptor. In addition, IL-21 can enhance the transduction of the IL-12 signal and cooperate for the increased production of IFN-gamma. See above for the IL-12 polynucleotide sequences. IL-21 polynucleotide sequences are available under accession numbers AF254069 (human); NM_021782 (mouse); NM_001024835 (chicken); and NM_001108943 (rat). SEQ ID NOS: 6, 7, 8, 9, and 17 encode human and mouse IL-21. SEQ ID NOS: 1 and 2 are polynucleotide constructs coding for IL-12 and mouse and human IL-21. Figures 7 and 8 are plasmid maps for the expression systems that can be used to express IL-12 and IL-21, respectively.

In yet another embodiment, dendritic cells are engineered to conditionally express TNF-alpha and IL-12. TNF-alpha is a potent activator of immune cells and mediates antitumor properties. In addition, TNF-alpha can synergize with IL-12 for enhanced expression of IFN-gamma and the IL-12 receptor on T cells. In animal studies, the application of IL-12 and TNF-alpha resulted in infiltration tumor by DN8 + T cells, significant production of IFN-gamma, and subsequent tumor regression. See Sabel, 2003, 2004, 2007, Taniguchi, 1998, Lasek, 2000; and Xia et al. 2008. See above for IL-12 polynucleotide sequences. Polynucleotide sequences encoding TNF-alpha are available under accession numbers X02910 (human); N _013693 (mouse); and BC107671 (rat).

In another embodiment, dendritic cells are engineered to conditionally express IL-7 and IL-12. IL-7 is a member of the IL-2 family and is important for T-cell and B-cell lymphopoiesis. IL-7 regulates the homeostasis of the survival and proliferation of intact CD8 + T cells and memory. It has been proven that IL-7 intensifies the generation of CTL against tumors. In addition, IL-12 acts on CD8 + T cells to enhance proliferation mediated by IL-7. In addition, it has been reported that IL-7 and IL-12 synergistically intensify the cytotoxicity of CD8 + T cells. Mehrotra, 1995; Sharma et al 2003; Tirapu et al 2002. In this way, it is expected that the coexpression of IL-7 and IL-12 will provide more optimal antitumor responses. See above for the polynucleotide sequences that code for IL-12. The polynucleotide sequences encoding IL-7 are available under accession numbers J04156 (human); NM 008371 (mouse); NM_001037833 (chicken); and N _013110 (rat).

In yet another embodiment, the dendritic cells are engineered to conditionally express GM-CSF and IL-12. GM-CSF regulates the differentiation and proliferation of hematopoietic progenitor cells, and plays a particularly important role in the maturation of professional antigen-presenting cells (APCs) such as dendritic cells. GM-CSF also enhances the ability of dendritic cells to process and present antigen. GM-CSF functions differently than IL-12, and both promote significant antitumor responses in animal studies. The combination of IL-12 (activation of T cells) and GM-CSF (activation of dendritic cells) is expected to result in a more potent antitumor unit. In animal studies, GM-CSF in combination with treatment with IL-12 significantly suppressed tumor growth in multiple cancer models. Wang, 2001; Chang, 2007; Jean, 2004; Nair, 2006; Hill 2002; Small et al. 2007. In human trials, GM-CSF + IL-12 were successfully used to treat patients with myeloma, where the combined actions of both cytokines led to a reduction in circulating B cells. Rasmussen, 2003; Hansson, 2007; Abdalla, 2007. It is expected that the coexpression of GM-CSF and IL-12 in a single cell will avoid undesired systemic effects such as reductions in circulating B cells. See for polynucleotide sequences that code for IL-12. The polynucleotide sequences of GM-CSF are available under accession numbers MI 1734 (human); NM_009969 (mouse); EU520303 (chicken); NM_001037660 (rat Csf2ra); and NM_133555 (rat Csf2rb).

In yet another embodiment, dendritic cells are engineered to conditionally express a chemokine (e.g., CCL3 (MIP-la), CCL5 (RANTES), CCL7 (MCP3), XCLl (lymphotactin), CCL 19 (MIP-3b), CXCL9 (MIG), CXCLlO (IP-10), CXCL 12 (SDF-I), or CCL21 (6Ckine)) and IL-12. Chemokines are chemoattractant cytokines that regulate the trafficking and activation of leukocytes and other cell types under a variety of inflammatory and non-inflammatory conditions.

Inflammatory cytokines control the recruitment of leukocytes in inflation and tissue damage. Homeostatic chemokines perform household maintenance functions such as navigational leukocytes (eg, dendritic cells) to and within secondary lymphoid organs, as well as the bone marrow and the thymus during hematopoiesis. In animal studies, the intratumoral co-injection of two separate adenoviruses expressing IL-12 and CXCL10 led to 100% regression of the tumor nodules derived from the CT26 murine colorectal adenocarcinoma cell line. Narvaiza et al., 2000. Emtage et al, 1999, describe two recombinant double adenoviral vectors that express either IL2 and XCLl (lymphotactin) or IL-12 and XCLl. Intratumoral injection of vectors to breast adenocarcinoma tumors in mice, promoted potent antitumor responses and gave rise to protective immunity. In other animal studies, cotransduction of adenoviral vectors expressing IL-12 and CCL27 resulted in tumor regression and long-term specific immunity. Gao et al, 2007. Thus, it is expected that the coexpression of a chemokine and IL-12 according to the invention will result in synergistic antitumor activity.

In yet another embodiment, dendritic cells are engineered to conditionally express an anti-angiogenic cytokine (e.g., IP-10 and Mig) and IL-12.

IP-10 and Mig are chemoattractants for T cells and NK cells, and their ability to inhibit angiogenesis is dependent on NK cells. Studies in animals have shown that therapy in combination with two adenoviruses, one that expresses IP10 and another that expresses IL-12, resulted in marked antitumor synergy. Narvaiza et al, 2000. In other studies, adenoviral vectors expressing IP10 or MIG and / or IL-12 were administered intratumorally in a murine model of mammary adenocarcinoma and fibrosarcoma. It was found that the administration of IP-10 or MIG in combination with IL-12 resulted in considerable tumor regression and increased the survival time of the animals that had tumors, in comparison to IP10, MIG, IL-12 alone or treated animals with control, with the combination of IP-10 and IL-12 that is the most effective. Palmer, 2001. See also Mazzolini, 2003; and Huang 2004. Thus, it is expected that the coexpression of an anti-angiogenic cytokine and IL-12 will result in synergistic antitumor activity.

To demonstrate an effective IL-12-mediated gene therapy, a conditional cDNA expression system is used that someone turns on an immunomodulator and / or the production of IL-12 by the immune cells or TSC at various time points after the intratumoral injection. Based on the results in the aggressive B16 melanoma model in C57BL / 6 mice, the following conclusions are made: 1) high levels of IL-12 of DC.RheoIL12 are secreted in the presence of the activating ligand RG-115830, but not in absence of the ligand; 2) Intratumoral therapy based on DC.RheoIL12 is as effective as intratumoral therapy based on DC.CIL12, provided that RG-115830 is administered to the treated animals within 24 hours of the DC injection (and at later time points) of the provision of the ligand, the therapy with RG-115830 fails); 3) the expression of IL-12 in DC seems to prolong the survival of these cells in the tumor microenvironment, and is associated with higher numbers of intratumorally injected DC that migrate towards the lymphatic nodules of tumor drainage; and 4) the strongest immune correlation towards the result of the therapy is the level of tumor-specific CD8 + T cells cross-sorted by the therapy, and not the number of DC injected, supported by the tumor microenvironment. In general, these data suggest that DCIL-12-based therapies are probably successful based on their positive influence on the afferent (cross-priming) of CD8 + T cell effectors type 1, and not on subsequent efferent events, such as DC-mediated recruitment of injected antitumor T cells into the tumor microenvironment, etc.

Before the intratumoral injection, the cells (immune cells or TSC) can be treated with a factor to stimulate the activity of the cells. For example, cells can be treated with a costimulatory molecule such as the positive costimulatory molecule that includes 0X4OL, 4-IBBL, CD40, CD40L, GITRL, CD70, LIGHT or ICOS-L or a negative co-stimulatory molecule such as antibodies. anti-CTLA4, anti-PD-Ll or anti-PD-L2. For example, cells (e.g., immune cells or TSC) can be incubated with a cell expressing one or more costimulatory molecules, e.g., J588 lymphoma cells expressing the ligand molecule CD40. In yet another embodiment, the cells (immune cells or TSC) can be treated with an anti-immunosuppressive molecule (tolerance inhibitor) such as anti-TGF-beta antibodies (to inhibit TGF signaling within the microenvironment), anti-HIV antibodies. IL10, TGFbRII DN (to inhibit TGF signaling within cells modified with the gene), IL-IOR DN, dnFADD (to inhibit cell death pathways within cells), anti-SOCSl, siRNA or decoy antibodies ( to inhibit suppressor cytokine signaling within cells), or anti-TGFα antibodies.

Recombinant adenoviruses possessing the polynucleotide sequences shown in Figures 1-8 are produced. For example, hIL-21 is produced by cotransfection of the linearized hLL-21 expression vector by restriction digestion at a site upstream of the left ITR, and the appropriate adenoviral backbone (e.g., deleted at E3) in a cell line permissive such as HEK293 cells. The adenoviral vector possessing the murine immunomodulatory genes is used for the transduction of murine TSC dendritic cells for use in murine therapeutic models. For the therapeutic application in humans, a polynucleotide encoding the human homolog of the immunomodulatory gene is inserted into the appropriate vector. The adenoviral vector for human therapeutic application is produced under GMP conditions. The example of a treatment profile (clinical trial) for patients with stage III / IV melanoma is as follows: Treatment in this case involves intratumoral injection of transduced, adenoviral dendritic cells, and oral administration for 14 days. activator drug (ligand). Subjects are selected 30 days to a week before the clinical trial. Each subject is asked to sign an informed consent before any procedure is initiated. The investigator will inform all subjects of the nature of the objectives, duration, potential hazards, and procedures that will be performed during the test, and the possibility that their medical records may be reviewed by the FDA. The subjects (a total of 16 to 20) are randomly grouped into four groups. All groups will receive an intratumoral injection of up to 5x107 dendritic cells transduced approximately 3 hours after the first dose of oral administration of the ligand. The 4 different groups in the oral daily dose of the ligand received: exemplary group 1 = O.Olmg / kg; group 2 = 0.3 mg / kg; group 3 = 1 mg / kg; group 4 = 3 mg / kg. During the course of treatment, blood is drawn at the specified time intervals for the evaluation of the single dose and the resting pharmacokinetics of the activating drug and its major metabolites. Also, blood is drawn at the specified time points for the evaluation of humoral and cellular immune responses against the viral vector, the RTS components and the tumor. Urine is collected and blood is drawn at specific time points for serum chemistry, urinalysis and hematology (safety profile). Tumor biopsies of the drained lymph nodes are taken at the specified time points to evaluate the expression of the transgene and the immune response to the tumor as a result of the therapy. Criteria for early termination are established for patients in case of adverse events. And adverse events are recorded.

Patients are followed until 1, 2, 3 and 4 months for adverse events and the therapeutic outcome.

In another embodiment, a subject in need of treatment of a tumor is (a) administered with dendritic cells engineered to express an immunomodulator, eg, an immunomodulator described herein, either constitutively or conditionally, and (b) a vector that expresses an immunomodulator, for example an immunomodulator described herein, either constitutively or conditionally, is injected intratumorally into the subject. In a preferred embodiment, the dendritic cells are engineered to express an Ad-immunomodulatory vector, and particularly the Ad-RTS-immunomodulatory vector. In another preferred embodiment, the vector that is injected intratumorally into the subject is an Ad-immunomodulatory vector, and particularly the Ad-RTS-immunomodulatory vector.

In yet another embodiment, a subject in need of treatment of a tumor is (a) administered with dendritic cells engineered to express IL-12, either constitutively or conditionally, and (b) a vector expressing IL-12, whether constitutive or conditionally, it is injected intratumorally to the subject. In a preferred embodiment, the dendritic cells are manipulated to express an Ad-IL-12 vector, and particularly the Ad-RTS-IL-12 vector. In another preferred embodiment, the vector that is intratumorally injected to the subject is an Ad-IL-12 vector, and particularly the Ad-RTS-IL-12 vector.

In yet another embodiment, a subject in need of treatment of a tumor is (a) administered with the dendritic cells engineered to express IL-12, either constitutively or conditionally, and (b) the subject is administered with one or more chemotherapeutic agents anticancer In a preferred embodiment, dendritic cells engineered are engineered to express an Ad-IL-12 vector, and particularly Ad-RTS-IL-12. One or more anti-cancer chemotherapeutic agents may be administered before the engineered dendritic cells are administered, after the manipulated dendritic cells are administered, or concurrently with the administration of the engineered dendritic cells. In a preferred embodiment, the anti-cancer chemotherapeutic agent is paclitaxel, a paclitaxel derivative or analogue, temozolomide, a temozolomide derivative or analog, sunitinib, a sunitinib derivative or analog, gemcitabine, or a derivative or analog of gemcitabine.

In yet another embodiment, a subject in need of treatment of a tumor is (a) administered with the dendritic cells engineered to express IL-12, either constitutively or conditionally, (b) a vector expressing IL-12, whether constitutive or conditionally, the subject is injected intratumorally, and (c) the subject is administered with one or more anti-cancer chemotherapeutic agents. In a preferred embodiment, the dendritic cells are manipulated to express an Ad-IL-12 vector, and particularly the vector, Ad-RTS-IL-12. In another preferred embodiment, the vector that is intratumorally injected to the subject is an Ad-IL-12r vector, and particularly the Ad-RTS-IL-12 vector. One or more anti-cancer chemotherapeutic agents may be administered before the manipulated dendritic cells and the vector expressing IL-12 are administered, after the manipulated dendritic cells and the vector expressing IL-12 are administered, or concurrently with the administration of dendritic cells manipulated by engineering, and the vector that expresses IL-12. In a preferred embodiment, the anti-cancer chemotherapeutic agent is paclitaxel, a paclitaxel derivative or analogue, temozolomide, temozolomide, a temozolomide derivative or analogue, sunitinib, a sunitinib derivative or analog, gemcitabine, or a derivative or analog of gemcitabine. .

In yet another embodiment, a subject in need of treatment of a tumor is (a) administered with manipulated dendritic cells to express an immunomodulator, for example, an immunomodulator described herein, either constitutively or conditionally and (b) a vector that expresses an immunomodulator, eg, an immunomodulator described herein, whether constitutively or conditionally, is intratumorally injected to the subject. In a preferred embodiment, the dendritic cells are engineered to express an Ad-immunomodulatory vector, and particularly the Ad-RTS-immunomodulatory vector. In another preferred embodiment, the vector that is intratumorally injected into the subject is an Ad-IL-immunomodulatory vector, and particularly the Ad-RTS-immunomodulatory vector.

In yet another embodiment, a subject in need of treatment of a tumor is (a) administered with manipulated dendritic cells to express an immunomodulator, eg, an immunomodulator described herein, either constitutively or conditionally, and (b) the subject it is administered with one or more anti-cancer chemotherapeutic agents. In a preferred embodiment, engineered dendritic cells are engineered to express an Ad-immunomodulator vector, and particularly the Ad-RTS-immunomodulatory vector. One or more anti-cancer chemotherapeutic agents can be administered before the manipulated dendritic cells are administered, after the manipulated dendritic cells are administered, or concurrently with the administration of the manipulated dendritic cells. In a preferred embodiment, the anti-cancer chemotherapeutic agent is paclitaxel a derivative or analogue of paclitaxel, temozolomide, temozolomide, a derivative or analogue of temozolomide, sunitinib, a derivative or analogue of sunitinib, gemcitabine, or a derivative or analog of gemcitabine. .

In a further embodiment, a subject in need of treatment of a tumor is (a) administered with manipulated dendritic cells to express an immunomodulator, eg, an immunomodulator described herein either constitutively or conditionally, (b) a vector expressing an immunomodulator, for example, an immunomodulator described herein, either constitutively or conditionally, is injected intratumorally into the subject, and (c) the subject is administered with one or more anti-cancer chemotherapeutic agents. In a preferred embodiment, the dendritic cells are manipulated to express an Ad-immunomodulator vector and particularly the Ad-RTS-immunomodulator vector. In another preferred embodiment, the vector that is intratumorally injected into the subject is an Ad-immunomodulatory vector, and particularly the Ad-RTS-immunomodulatory vector. One or more anti-cancer chemotherapeutic agents can be administered before the manipulated dendritic cells and the vector expressing the immunomodulator are administered, after the manipulated dendritic cells and the vector expressing the immunomodulator are administered, or concurrently with the administration of the manipulated dendritic cells and the vector that expresses the immunomodulator. In a preferred embodiment, the anti-cancer chemotherapeutic agent is paclitaxel, a paclitaxel derivative or analog, temozolomide, temozolomide, a temozolomide derivative or analogue, sunitinib, a sunitinib derivative or analogue, gemcitabine, or a derivative or analog of gemcitabine. .

In any of the methods of the present invention, the disease or disorder may be a disease or disorder described in the present application. In one embodiment, the disease or disorder is a disease or disorder listed in Table 1 herein. In a further embodiment, the disease or disorder is a disease or disorder listed in Table 3 herein.

In any of the methods of the present invention, the cancer or tumor may be a disease or disorder described in the present application. In one embodiment, the cancer or tumor is a cancer or tumor listed in Table 1 herein. In a further embodiment, the cancer or tumor is a cancer or tumor listed in Table 3 herein.

It is possible to measure the effect of an immunomodulator and / or the expression of IL-12 on a population of cells by measuring the expression level or activity of the Thl / Tcl cytokine, IFN-gamma in a biological sample from a subject.

For the purposes of the invention, the invention provides a method for determining the efficacy of a therapeutic regimen based on TSC or immunologically engineered in vitro, in a patient with cancer, comprising: to. measure the level of expression or the level of activity or both of the interferon-gamma (IFN-gamma) in a first biological sample obtained from a human patient before administration of the cells in vitro for example, immune cells or TSC, with which generates a level of control; b. intratumorally administer to the patient the cells manipulated in vitro; c. administering to the patient an effective amount of the activation ligand; d. measuring the level of expression or activity level or both of IFN-gamma in a second biological sample obtained from the patient at a time after the administration of the activation ligand, whereby data are generated for a test level; Y e. compare the control level to the IFN-gamma test level, where the data showing an increase in the level of expression, activity or both of IFN-gamma at the test level relative to the control level, indicate that the Therapeutic treatment is effective in the patient. The invention may also further comprise the additional steps of F. take a biopsy and count the tumor infiltration lymphocytes (TIL) and / or g. Observe the regression of the tumor in response to treatment.

The term "subject" means an insect, plant or animal intact. It is also anticipated that the ligands will work equally well when the subject is a fungus or yeast. Animals for use with the invention include, but are not limited to, vertebrates for example, mammals such as humans, rodents, monkeys, and other animals, with humans or mice being most preferred. Other animals include veterinary animals such as dogs, cats, horses, cattle, sheep, goats, pigs, and the like.

The invention provides a method for increasing the expression of the immunomodulator, for example, TNF-alpha, by introducing into the vector, for example, an adenoviral vector deficient in replication, one or more regulatory sequences and optionally, a nucleic acid coding for a signal peptide, wherein the vector conditionally expresses the immunomodulator. As used herein, the term "protein expression" includes without limitation transcription, post-transcription, translation and / or post-translation. Also included in the invention is a method for increasing the expression of RA or proteins of an immunomodulator, for example, TNF-alpha, which comprises generating a vector that conditionally expresses TNF-alpha, wherein the vector further comprises one or more regulatory sequences connected to the polynucleotide sequence coding for TNF-alpha, and adding an activating ligand, thereby inducing expression of the immunomodulator, wherein the one or more regulatory sequences and / or signal peptide improves the expression of TNF- alpha. Several regulatory regions of the invention including, but not limited to, 5 '(5'UTR), 3'UTR, untranslated region, or both have been described. In one mode, the 5'UTR is 5U2. 5U2 is a canon SERCA2 fusion intron 2 with a mutated putative consensus poly-A site, with the splice donor of exon 2 flanking the 5 'end and the splice acceptor of exon 3 flanking the 3' end followed for a portion of the 5'UTR portion of bovine casein. In another embodiment, the 3 'regulatory region is a polyadenylation signal of SV40 or hGH.

In certain embodiments, the method of the invention also relates to enhancing the secretion of TNF-alpha by generating a vector that conditionally expresses TNF-alpha, wherein the vector further comprises a signal peptide, thereby increasing the TNF-alpha secretion compared to a vector comprising the signal peptide gene native to TNF-alpha, eg, wild type signal peptide of TNF-alpha. In particular, the signal peptide used in the invention is optimized by codon. In a specific embodiment, the signal peptide is encoded by the wild type signal peptide gene of IL-2. In a further specific embodiment, the signal peptide is encoded by the codon-optimized IL-2 signal peptide gene.

Without wishing to be bound by theory, the invention is expected to support the use of gene therapy based on TSC immune cells engineered in vitro., administered intratumorally, in the clinical scenario focusing on the objective clinical response as a primary endpoint of study, and anti-tumor CD8 + T-cells cross-primed (that produce IFN-gamma) as a secondary point of study. The ability to activate and deactivate the expression of the immunomodulator and / or IL-12 in vivo adds an element of safety and therapeutic control to the treatment since both the synchronization and the level of protein expression can be controlled by the administration of ligand, and additionally since it is expected that immunomodulatory and / or IL-12 synchronization is critical to the therapeutic effectiveness of the method.

The invention additionally supports the therapeutic applications of cells engineered in vitro by conditionally expressed genes of interest as innovative approaches for the effective and efficient treatment of human diseases.

The present invention also provides methods for treating a tumor, for reducing the size of a tumor, or for preventing the formation of a tumor in a mammal in need of the same, in which a vector for expression of the tumor is administered intratumorally to tumor microenvironments. conditionally proteins that have the functions of one or more modulators that are not contained within a cell,. In this embodiment, the vector is administered to the tumor without packaging or packaging in a cell, such as an immune cell or a TSC. The present invention also provides methods for treating a disease in a mammal in need thereof, wherein the mammal is administered a vector to conditionally express proteins having the functions of one or more immunomodulators that are not contained within a cell. In this embodiment, the vector is administered to the tumor without packaging or packaging in a cell, such as an immune cell or a TSC.

In one embodiment, immune cells, TSCs, dendritic cells, or bone marrow dendritic cells are not administered intratumorally with the vector.

In another embodiment, a vector of the invention that is not contained within a cell is administered intratumorally concurrently with, before, or after intratumoral administration of immune cells, TSCs, dendritic cells, or dendritic cells. of bone marrow.

In one embodiment, the vector of the invention that is not contained within a cell is intratumorally administered to the same lesion as the immune or TSC cells are administered. In another embodiment, the vector of the invention that is not contained within a cell is intratumorally administered to a different lesion in which the immune or TSC cells are administered.

In one embodiment, the vector is administered to the same lesion in each administration cycle. In another embodiment, the vector that is administered is not administered to the same lesions in each administration cycle.

In one embodiment, the tumor is a tumor of any of the cancers listed herein, for example, in Tables 1 and 3. In another embodiment, the tumor is a melanoma tumor, a colorectal tumor, a pancreatic tumor, a tumor of breast, a lung tumor or a kidney tumor. In another modality, the tumor is a malignant melanoma. In another embodiment, the tumor is a malignant melanoma of Stage IIC or Stage IV.

In one embodiment, the intratumoral dose is at least about 1.0 x 109 viral particles per cycle of vector administration. In another embodiment, the intratumoral dose is at least about 1.0 x 10 10 viral particles per cycle of vector administration. In another embodiment, the intratumoral dose is from about 1.0 x 109 to about 1.0 x 1013 viral particles per cycle of vector administration. In another embodiment, the intratumoral dose is from about 1.0 x 10 10 to about 1.0 x 10 13 viral particles per cycle of vector administration. In another embodiment, the intratumoral dose is approximately 1.0 x 10 10, approximately 1.0 x 10 11, approximately 1.0 x 10 12 or approximately 1.0 x 10 13 viral particles per cycle of vector administration. In one embodiment, the vector is AD-RTS-IL-12.

In another embodiment, the present invention further provides methods for treating a liver disease in a mammal in need thereof, wherein the mammal is administered a vector to conditionally express proteins that are not contained within a cell.

In another embodiment, the present invention further provides methods for treating a lysosomal storage disease in a mammal in need thereof, in which the mammal is administered a vector to conditionally express proteins that are not contained within a cell.

In another embodiment, the present invention further provides methods for treating a disease in a non-human mammal in need thereof, in which the mammal is administered a vector to conditionally express proteins that are not contained within a cell.

The dose of activating ligand is from about 5 to 100 mg / day, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 , 85, 90, 95 or 100 mg / day. In one embodiment, the activating ligand is administered at least once per day. In one embodiment, the activating ligand is administered once per day for approximately 14 days.

In one embodiment, at least two doses of the vector (e.g., approximately lxlO11 and lxlO12) are used in combination with at least three different dose levels of the activating ligand (e.g., from about 5 mg / day to about 100 mg / day ).

One skilled in the art will be able to optimize the doses in order to provide the range of effective plasma levels of the vector, for various degrees of activation of the activating ligand.

In one embodiment, the dose of activating ligand administered to the subject is changed during the period of administration of the activating ligand within the intratumoral vector administration cycle. In another embodiment, the dose of activating ligand administered to the subject is decreased during the period of administration of the activating ligand within the intratumoral vector administration cycle. In another embodiment, the dose of activating ligand administered to the subject is increased (scale) during the period of administration of the activating ligand within the intratumoral vector administration cycle.

In one embodiment, the subject is treated with 2, 3, 4, 5, 6, 7, 8, 9 or 10 intratumoral vector administration cycles. In another embodiment, the subject is treated with 3-7 cycles of intratumoral vector administration. In another embodiment, the subject is treated with 4-6 cycles of intratumoral vector administration. In another embodiment, the subject is treated with 5 or 6 intratumoral vector administration cycles. In another embodiment, the subject is treated with 6 cycles of intratumoral vector administration.

In one embodiment, each intratumoral vector administration cycle is performed with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks of separation. In another form of modality, each intratumoral vector administration cycle is performed with 4 weeks of separation.

In one embodiment, the dose of the vector is changed in each subsequent cycle of intratumoral vector administration. In another embodiment, the dose of the vector is decreased in each subsequent cycle of intratumoral vector administration. In another embodiment, the dose of the vector is increased in each subsequent cycle of intratumoral vector administration.

In one embodiment, the vector and activator ligand doses, and the number and duration of the intratumoral vector administration cycles, the frequency of vector delivery and the frequency of administration of activating ligand are set forth in Table 8 in Example 11 of the present.

In one embodiment, the invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a vector of the invention that is not contained within a cell. Suitable carriers include, but are not limited to, saline, distilled water, sodium chloride solutions, and mixtures of sodium chloride and inorganic salts or their similar mixtures, solutions of materials such as mannitol, lactose, dextran and glucose, amino acid solutions such as glycine and arginine, mixtures of organic acid solutions or saline solutions and glucose solutions, aqueous and non-aqueous, sterile, isotonic injection solutions, which may contain antioxidants, chelating agents, buffers, bacteriostats, and solutes which render the formulation isotonic, and sterile aqueous and non-aqueous suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations can be presented in sealed single-dose or multi-dose containers, such as ampoules and flasks, and can be stored in a freeze-dried (lyophilized) condition that requires only the addition of the sterile liquid carrier, eg, water, for injections, immediately before use.

In the case of conflict between any teaching or suggestion of any reference cited herein and in the specification, the latter shall prevail, for purposes of the invention.

All patents, patent applications and publications cited herein are fully incorporated by reference in their entirety.

It is to be understood that the embodiments described above and the exemptions are not intended to be limiting in any respect to the scope of the invention, and that the claims presented herein are intended to encompass all modalities and exemptions either explicitly or otherwise. not presented here.

U.S. Application No. 12 / 247,738, entitled "Dendritic Cells Manipulated by Engineering and Uses for Cancer Treatment", filed on October 8, 2008, is incorporated by reference herein in its entirety. The request of the United States no. 12 / 241,018, entitled "Therapeutic Gene and Bioreactor Change Constructions for the Expression of Biotherapeutic Molecules and Uses of Them" presented on September 29, 2008, is also incorporated by reference herein in its entirety.

Example 1 A study was undertaken to determine the dose of dendritic cells and the most effective cytokine that is capable of inducing tumor-specific immune responses, and the antitumor activity in a renal cancer Renca tumor cell model.

Two lines of tumor cells are used in this study: Renca and Renca-HA. The last cell line is made by transfecting Renca cells with the influenza virus hemagglutinin (HA). The advantage of the Renca-HA model is the ability to trace antigen-specific T cells, since both epitopes derived from specific CD8 and CD4 HAs are known and have been used.

Specific Objective - determine the induction of HA-specific immune responses after intratumoral administration of dendritic cells ..

The Renca-HA tumor is established subcutaneously in BALB / c mice. When the tumor becomes palpable, the dendritic cells are injected intratumorally. The administration of dendritic cells will be repeated twice at intervals of 7 days, for a total of 3 administrations.

The following groups of mice are used (each Group includes 3 mice): 1. Mice not treated; 2. Mice treated with 5xl05 dendritic cells transduced with the control plasmid; 3. Mice treated with 106 dendritic cells transduced with the control plasmid; 4. Mice treated with 5xl06 dendritic cells transduced with control plasmid; 5. The same as Groups 2-4 using dendritic cells transduced with IL-12; 6. The same as Groups 2-4 using dendritic cells transduced with IL-15; Y 7. The same as Groups 2-4 using dendritic cells transduced with IL-21.

To test the effect of the combination of the different cytokines, the mice are treated simultaneously with: 8. 5xl05 dendritic cells transduced with IL-12, and 5xl05 dendritic cells transduced with IL-15, 9. 5xl05 dendritic cells transduced with IL-12 and 5xl05 dendritic cells transduced with IL-21, and 10. 5xl05 dendritic cells transduced with IL-21 and 5xl05 dendritic cells transduced with IL-21.

Four, days after administration, the lymph nodes of the tumor-bearing mice are harvested, and the cells are stimulated either with the peptide matching MHC class I (to detect CD8 + T cell responses) or the peptide equated to MHC class II (to detect CD4 + T cell responses).

The following tests are used: 1. ELISPOT IFN-Y and IL-2; 2. T cell proliferation; 3. Detection of TNFa, IL-10, IL-4, and GM-CSF release by lymph node cells.

In addition, the NK activity of the lymph node cells is evaluated, using YAC cells as targets.

In parallel, the cells are stimulated with anti-CD3 / CD28 antibodies to evaluate the non-specific response of T cells.

The most effective dose of dendritic cells capable of inducing antigen-specific immune responses are determined.

Specific Objective 2 - evaluate the antitumor activity of dendritic cells transduced with cytokine genes.

Only those dendritic cells transduced with cytokine that demonstrated statistically significant induction of immune responses are used in subsequent experiments.

The treatment of mice possessing the Renca-HA tumor is carried out as described in specific objective 1. A dose of DCs transduced with cytokines showing specific activity in previous experiments is used. As a control, dendritic cells transduced with control adenovirus are used. To reach statistical significance, each group includes 10 mice.

Tumor growth is evaluated. The Renca-HA rumor contains an immunogenic epitope that is useful for immune monitoring and for the initial test of the antitumor effect. However, to verify the potential antitumor activity of the treatment, non-transfected tumor cells need to be used. Therefore, the experiments described above are repeated using the Renca tumor model.

Example 2 The safety, tolerance, transgene function, and immune effects of the intratumoral injections or of the autologous dendritic cells, transduced with adenovirus, manipulated to express hIL-12, and one or more of other immunomodulators under control will be evaluated. of the RTS, in subjects with stage III and IV melanoma through procedures such as those described below.

A study involving the study subjects with stage III and IV melanoma will be conducted in 4 groups (groups) of subjects, each subject receiving a simple intratumoral injection (within a melanoma tumor) of autologous dendritic cells transduced with adenovirus (reinserted within the same subject from which they came) (DCs) engineered to express human interleukin-12 (hIL-12), and one or more other immunomodulators, at a dose of 5 x 107 in combination with daily oral doses of the activating drug (activation ligand). The study will use injections of transduced dendritic cells ex vivo (after the cells are removed from the subjects) with the adenoviral vector for the inducible expression of human IL-12 and one or more other immunomodulators. The production of IL-12 and one or more of other immunomodulators is "turned on" (induced) from the DCs injected through activation of the RTS by the oral administration of the activating drug (RG-115932). Safety and tolerance will be assessed through physical exams (including ECOG's operating condition), vital signs measurements, serum chemistry, urinalysis, hematology, "side effects" events, and antibodies and cellular immune response towards the adenoviruses, RTS components, and the activating drug. To assess progress, a simple dose and the resting pharmacokinetics / ADME of the oral activating drug, and its major metabolites, the analysis of hIL-12 levels, other levels of immunomodulators and the cellular immune response (cells T) in biopsies of target tumors, draining lymph nodes, and peripheral circulation, as well as a serum cytokine profile.

For example, 16 subjects with stage III and IV melanoma are divided into four groups with groups 1 and 2 that contain three subjects, and groups 3 and 4 that contain 5 subjects. All subjects will receive a single intratumoral injection of 5xl07 autologous DCs transduced with the adenoviral vector encoding human IL-12, and one or more other immunomodulators under the control of RTS. For example, subjects are administered with an intratumoral injection of autologous DCs transduced with the adenoviral vector encoding human IL-12 under the control of RTS and an immunomodulator such as IL-15 or IL-21.

Subjects will receive a simple daily oral dose of the activating drug (group 1: 0.01 mg / kg, group 2: 0.1 mg / kg, group 3: 1.0 mg / kg or group 4: 3 mg / kg) beginning the first dose approximately 3 hours before the DC injection on day 1 and continuing for 13 consecutive days more. The additional injections of autologous dendritic cells, adenovirally transduced, in combination with 14 single daily oral doses (once) of activated drug can be administered to the eligible subjects who meet the criteria for retreatment. The safety, tolerance, and function of dendritic cells are evaluated for all subjects in each group for up to one month after injection of dendritic cells manipulated in vitro, before enrolling subjects to receive the next dose plus high of the activating drug. The safety assessment will continue in all subjects for 3 months after the initial injection of engineered dendritic cells, with the possibility of extending the follow-up period up to a total of 6 months to monitor the safety of the subject if the toxicity is observed or the subject receives one or more additional injections of the dendritic cells.

Such a study demonstrates the safety and tolerance of one or more single or multiple intratumoral injections of autologous dendritic cells, transduced adenovirally, in combination with an oral activating drug in subjects with melanoma. The study provides resting pharmacokinetics / ADME of the oral activating drug. The study demonstrates functionality of the RTS in the subject by measuring the expression of hIL-12 and the expression of one or more other immunomodulators of the autologous dendritic cells transduced adenovirally, in target tumors and / or in drained lymph nodes, in response to the activation of the RTS by the oral administration of the activating drug. In addition, the study demonstrates the immune effects of autologous dendritic cells, transduced adenovirally, in terms of the cellular immune response in the target tumor, the draining lymph nodes, and the peripheral circulation after oral administration of the activating drug.

Melanoma is selected as an exemplary cancer. Melanoma, particularly among solid tumors, has been shown to respond to immunotherapy procedures, and melanone tumors are easily accessible for intratumoral injection and biopsy. The subjects included in the study have non-resectable stage III or IV melanoma, which is at least 0.5 cm in diameter, any tumor thickness, any involvement number of lymph nodes, metastasis in transit, or distant metastasis.

Preparation of Adenovirus harboring the RheoSwitch Therapeutic System, hIL-12 and One or More Other Immunomodulators The recombinant DNA is transferred to dendritic cells (DC) by transduction of the adenoviral vector ex vivo. Recombinant DNA is used to express human IL-12 (p70) and one or more other immunomodulators from immature, intratumorally injected dendritic cells, which confer survival and stimulate DC maturation in the tumor environment, resulting in their migration subsequent to the draining lymph nodes. This leads to a shift towards differentiation of the helper T cells to the Thl type, and also the activation of the tumor-specific cytotoxic cells, by cross-priming with the tumor antigens.

The recombinant DNA used as the recombinant adenoviral vector allows the expression of human IL-12 and one or more other immunomodulators under the control of the RheoSwitch® therapeutic system (RTS). The RTS comprises a bicistronic message expressed from the human ubiquitin C promoter, and codes for two fusion proteins: Gal4-EcR and VP16-RXR. Gal4-EcR is a fusion between the DNA binding domain (amino acids 1-147) of yeast Gal4 and the DEF domains of the ecdysone receptor from the Choristoneura fumiferana insect. In another modality more, the RTS consists of a bicistronic message expressed from the human ubiquitin C promoter, and codes for two fusion proteins: Gal4-EcR and VP16-RXR. GaM-EcR is a fusion between the DNA binding domain (amino acids 1-147) of yeast Gal4 and the DEF domain of the ecdysone receptor from the insect Choristoneura fumiferana. VP16-RXR is a fusion between the activation domain of HSV-VP 16 transcription and the EF domains of a chimeric RXR derived from human and lobster sequences. These Gal4-EcR and VP16-RXR sequences are separated by an internal ribosome entry site (IRES) of the EMCV. These two fusion proteins are dimerized when Gal4-EcR binds to a small molecule drug (RG-115932) and activates the transcription of hIL-12 and one or more other immunomodulators from a promoter that responds to Gal4, which It contains six binding sites to Gal4 and one synthetic minimal promoter. The above-described RTS transcription unit is placed 3 'of the hIL-12 and one or more other transcription units of the immunomodulators. This cassette RTS-ML12-complete immunomodulator, is incorporated into the genome of adenovirus 5 at the site where the El region has been deleted. The adenoviral main chain also lacks the E3 gene. A map for the Ad-RTS-hIL-12 adenoviral vector is shown in FIG. 8 of document US 2009/0123441 Al.

The recombinant adenoviral vector used in this study contains the following exemplary regulatory elements, in addition to the viral vector sequences: the human ubiquitin C promoter, the internal ribosome entry site, derived from EMCV, an inducible promoter containing 6 copies of the binding site to Gal4, 3 copies of the SP-I binding sites, and a synthetic minimal promoter sequence, the SV40 polyadenylation sites, and a transcription termination sequence derived from the human alpha-globin gene. It must be understood that other regulatory elements could be used as alternatives.

An exemplary recombinant adenoviral vector Ad-RTS-hIL-12-immunomodulator (s) is produced in the following manner. The coding sequences for the receptor fusion proteins, VP16-RXR and Ga -EcR separated by the EMCV-IRES (internal ribosome entry site), are inserted into the adenoviral shuttle vector under the control of the ubiquitin C promoter. human (constitutive promoter). Subsequently, the coding sequences for the p40 and p35 subunits of hIL-12, separated by IRES, and one or more other immunomodulators, are placed under the control of a synthetic inducible promoter containing six copies of the Gal4 binding site, they are inserted with the 5 'of the ubiquitin C promoter and the receptor sequences. The shuttle vector contains serotype 5 adenovirus sequences from the left end to map unit 16 (mul6), from which the El sequences are deleted and replaced by RTS, IL-12 and one or more immunomodulatory sequences (RTS-hIL- 12). The shuttle vector possessing the RTS-hIL-12-immunodulator (s) is tested by transient transfection in HT-1080 cells for IL-12 dependent on the activating drug, and another expression of the immunomodulatory agent (s). The shuttle vector is then recombined with the adenoviral backbone by cotransfection within HEK 293 cells to obtain recombinant adenovirus Ad-RTS-hIL-12-immunomodulator (s). The adenoviral backbone contains deletions of mu sequence from 0 to 9.2 at the left end of the genome, and the E3 gene. The shuttle vector and the adenoviral main chain contain the overlap sequence from mu 9.2 to mu 16 that allows the recombination of them and the production of the recombinant adenoviral vector. Since the recombinant adenoviral vector is deficient in the El and E3 regions, the virus is deficient in replication in normal mammalian cells. However, the virus can replicate in HEK 293 cells harboring the El region of adenovirus 5, and therefore provide the El function in the trans position.

An exemplary recombinant adenoviral vector is produced in the following manner: the linearized shuttle vector which possesses the DNA elements for the inducible expression of IL-12 and one or more other immunomodulators, and the adenoviral backbone are cotransfected into HEK293 cells . The recombination between the overlap sequences on the shuttle vector and the viral backbone, results in the production of recombinant adenoviruses and is packaged into viral particles in HEK293 cells. HEK293 cells are developed in DMEM containing fetal bovine serum.

The virus used for the proposed study was purified by centrifugation in a CsCl density gradient. The recombinant adenovirus undergoes two rounds of plaque purification and the resulting seed pool is used to produce a master viral bank (MVB) by amplification in HEK293 cells from a fully characterized library of master plates. The MVB undergoes intensive cGMP / GLP release tests that include replication competent adenovirus (RCA), sterility, mycoplasma, adventitious viruses, retroviruses, HIVl / 2 human viruses, HTLV1 / 2, HAV, HBV, HCV, EBV, B19 , CMV, HHV-6, 7 and 8, bovine and porcine virus, complete vector sequencing and functional test by AD-induced expression of IL-12, and one or more other immunomodulators in human cell lines.

The MVB virus was used for the production of the purified virus in a cGMP facility, and may again undergo release tests that include identity, RCA, sterility, mycoplasma, adventitious viruses, proportion of viral particles to infectious units, DNA contamination of host cell, endotoxin and proteins, and functional test by expression of IL-12 induced by AD, and one or more other immunomodulators in human cell lines.

A suitable study for producing recombinant adenoviruses is also set forth in Anderson, R.D., Gene Therapy 7: 1034-1038 (2000).

A suitable method for recombinant adenoviruses is host cells are set forth in Komita, H. et al., Cancer Gene Therapy 16: 883-891 (2009).

Transduction of Autologous Dendritic Cells by the Adenovirus that contains the Transgen hIL-12 and One or More Other Immunomodulators and RheoSwitch® Therapeutic Systems (RTS).

Dendritic cells derived from human subjects are transduced ex vivo and injected into the tumor. The DCs will be characterized before viral transduction for viability, purity (typically> 80% of cells showing the phenotype (DC), sterility, mycoplasma and endotoxin.) After viral transduction, the cells are washed repeatedly to remove any The supernatant from the last wash will be tested for the residual virus content by PCR, since the DCs are transduced ex vivo by the adenoviral vector (non-integrating virus) and the lifespan of the DCs after the Intratumoral injection and subsequent migration to drained lymph nodes is short, it is not expected that viral DNA will be incorporated into any of the non-target cells.The protocol used for the adenoviral transduction of DCs is expected to produce 80- 90% transduction and is considered very efficient.

PBMC harvest due to leucocitaferesis: The subjects suffer a standard leucocitaferesis of 90 to 120 minutes in the Outpatient Apheresis Unit of UPCI. The leukocyte procedure involves removing blood from a vein in one arm; the passage of blood through a centrifuge (cell separator), where its components are separated and one or more components are removed; and the return of the remnant components to the subject's vein in the same or in the other arm. No more than 15% of the total blood volume of the subject is removed at any time since the blood is processed through the cell separating device. In the cell separator, the blood is separated into plasma, platelets, white cells and red blood cells. The white blood cells (WBC) are removed and all other components are returned to the circulation of the subject. Each attempt is made to use two peripheral IV lines for this procedure. If that is not possible, a central line may be necessary. The subject has to be informed by the doctor who suffers from leucocitaferesis, and is routinely selected for vital signs (including blood pressure) before the procedure.

Processing: After harvesting, the leucopackage is manually distributed to the CPL, and is immediately processed by centrifugal elutriation in ELUTRATM. This is a closed system validated for clinical use. The monocyte fraction is recovered, and after the recovery and viability of the cells is established, they are transferred to an Astrom cartridge for the 6-day culture in the presence of IL-4 and GM-CSF. All processing and washing procedures are performed under sterile conditions.

Sowing in Initial Plate: The leukocytes recovered from the simple leucopackage are counted in the presence of trypan blue dye to determine the number of viable cells. Monocytes are evaluated for purity by flow cytometry. Monocytes are resuspended from 5 to 10 x 10 6 cells / ml in serum-free and antibiotic-free CellGenix medium, containing 1,000 IU / ml of IL-4 and 1,000 IU / ml of GM-CSF for SOP-CPL-0166, and placed in an Aastrom cartridge. A minimum loading volume of 50 ml and a minimum number of cells are required for c sete inoculation.

Cultivation: The Aastrom cartridge is placed in the incubator in the eplicell system, an automated culture device, compatible with cG P, completely closed, for the generation of immature DC.

DC Immature Harvest: On day 6, the Aastrom cartridge is removed from the incubator and the immature DCs are harvested. Cells are recovered by centrifugation at 1500 rpm, washed in CellGenix medium, counted in the presence of a blue trypan dye and verified for morphological and phenotypic characteristics.

Viability: This is determined by performing the counts of cells in a hemocytometer in the presence of trypan blue. In general, more than 95% of the harvested cells are viable, for example, they exclude a trypan blue dye. If the viability is less than 70%, the immature DCs will be discarded.

Phenotyping: The cells generated in culture are counted by microscopic observation in a hemocytometer, and a preliminary differential count (DC vs. lymphocytes) is obtained, using the trypan blue dye. The confirmation of the differential account is made by flow cytometry, comparing DC vs. lymphocytes using the properties of high front and lateral dispersion of immature DC as the criteria for their identification. Immature DCs routinely contain more than 80% cells with dendritic cell morphology and have a DC phenotype.

Power Assay of IL-12p70: It has been established that mature DCs (mDCs) have the ability to produce IL-12p70 spontaneously after activation with CD40L with or without the addition of innate immunity signals (eg LPS). A standardized IL-12p70 production assay was recently established and is applicable to small samples or large batches of DC vaccines generated under a variety of conditions. The current potency assay consists of two distinct steps, the first involving coincubation of responding DCs with J588 lymphoma cells stably transfected with the human CD40 ligand gene as stimulators. The second step involves testing the supernatants from these co-cultures for the levels of IL-12p70 secreted by the DCs stimulated with J558 / CD40L +/- LPS in the Luminex system. The power test has an inter-assay CV of 18.5% (n = 30) and a wide dynamic range, which facilitates the evaluation of various DC products characterized by very different levels of IL-12p70 production. The normal range for the assay established using the DC products generated from the monocytes of 13 normal donors was 8-999 pg / ml, with a mean of 270 pg / ml.

Production and Release Criteria for Dendritic Cells Each batch of dendritic cells generated in vivo is tested for the presence of microbial contaminants (aerobic and anaerobic bacteria, fungi and mycoplasma), as well as endotoxin and are functionally characterized. All dendritic cells that will be injected into the subjects will be fresh and will not undergo cryopreservation.

DC Quality Assurance Test: The DC generated as described above are evaluated for sterility, viability, purity, potency and stability. The criteria for cell product release are established and rigorously followed.

Feasibility: The cells generated in culture are counted by microscopic observation on a hemocytometer, and a differential count (DC vs. lymphocytes) is obtained using a trypan blue dye. This account provides the percentage of viable cells in the tested culture. More than 70% cell viability by tripan blue exclusion and a minimum of 70% of cells expressing HLA-DR and CD86 as CD markers derived from monocytes, are required to pass the release criteria. Additional markers can be included for exploratory analysis such as CD83 and CCR7 to assess the maturation status of DC, and CD3 and CD19 to assess lymphocyte contamination.

Purity: Two-color flow cytometric analysis of cells stained with mAbs conjugated to FITC and PE, is used to determine that the DC population identified morphologically expresses the surface antigens defined by DC and lacks the antigens of line of monocytes and T and B cells. For the preparation of vaccines, the generated DCs must express HLA-DR and CD86 and must not express CD3, CD19, or CD14. To be considered as mDC, the cells must express CD83 + and CCR7 +.

Power: To define a power measurement for DC, its ability to produce IL-12p70 was determined as described above.

Sterility: DC are tested by bacterial (aerobic and anaerobic) and fungal cultures using the BD Bactec system (Becton Dickinson Co., Sparks, MD) at the Microbiology Laboratory of the University of Pittsburg Medical Center. The final results of the microbial cultures are available in 14 days. Before the release of DC for use in vaccines, a gram stain is performed and must be negative for the presence of microorganisms.

IMCPL tests for mycoplasma are performed using the Rapid Mycoplasma Tissue Culture Detection System (Gen-Probe, Inc. San Diego, CA), which is based on nucleic acid hybridization technology. The endotoxin test is performed using the Limulus Amoebocyte Lysate Pyrogen Plus assay (Bio Whittaker, Inc., Walkerville, MD). The endotoxin test is performed on the cell culture at the time of harvest and before the release of the final product. The acceptable endotoxin level is < 5EU / kg of body weight. The non-transduced and transduced dendritic cells will be cryopreserved for future analysis.

It is expected that all transduced cells will express the transgene. It is expected that more than 80% of the DCs will be transduced. The product will be biologically active since the active coding sequence is maintained in the transgene. The virally transduced DCs, injected into the tumor, are of immature DC phenotype and do not express IL-12 and one or more other immunomodulators until it is matured, and therefore at this stage, the expression of IL-12 and one or more than other modulators is mainly from the transgene. Since the expression of IL-12 and one or more other immunomodulatory transgenes is induced by the small molecule activating drug RG-115932 in a dose-dependent manner, the level of transgene expression in transduced DCs can be controlled , to the desired levels. A small portion of the transduced DCs prepared for administration to human subjects can be tested in vitro for drug-dependent induction, expression of IL-12 and one or more other immunomodulators. The expression of IL-12 and one or more of other immunomodulators can be evaluated by ELISA with a sensitivity of 4 ng / ml.

It is expected that induction in vitro and IL-12 and one or more of other immunomodulators from cells transduced by the vector used in the proposed study will produce approximately 500 ng of IL-12 and one or more of other immunomodulators by 10 6 cells. in 24 hours, determined by Elisa. In preclinical studies using the melanoma mouse model, intratumoral injection of 106 or more transduced DCs show efficacy. However, it is expected that the required intratumoral injection can show efficacy at levels below this amount, and therefore transducted 5xl07 DCs injections can be used as a starting point to determine if smaller or larger amounts are required.

For example, in vitro, human and mouse cell lines and primary cells transduced with the recombinant adenoviral vector which possesses the genes for IL-12 and one or more other immunomodulators, show induction of IL-12 expression in response to the activating drug in a dose-dependent manner. 6. 3 Formulation of the Activating Drug The activator drug used herein is formulated in any of the following formulations: (1) 100% Labrasol; (2) Labrasol flavored with Listerine (Latitude Pharmaceuticals Inc., USA) comprising (a) menthol, (b) thymol, (c) eucalyptol, (d) aspartame, (e) sodium saccharin, (f) citric acid, ( g) mint flavor, (h) cream flavor, (i) labrasol; (3) Mygliol 812 and phospholipon 90G (Latitude Pharmaceuticals Inc., USA); or (4) Mygliol 812, phospholipon 90G and tocopheryl-polyethylene glycol succinate Vitamin E (Latitude) Pharmaceuticals Inc., USA).

Distribution While a variety of specific concentrations and protocols can be imagined, an example for treating patients could include patients receiving one or more intratumoral injections of transduced autologous dendritic cells (AdDCs) at a concentration of 5 x 107 suspended in sterile saline, manipulated to express hIL-12 (human interleukin 12) and one or more other immunomodulators under the control of RTS, in combination with the oral activating drug (RG-115932).

Initial Treatment Day 1 Internal Patient Visit: On day 1, a baseline physical examination (including vital signs, weight and ECOG condition) is performed. Urine is collected and blood drawn for baseline serum chemistry, urinalysis and hematology (safety profile). Approximately 3 hours before intratumoral injection of dendritic cells manipulated in vitro, each subject is dosed with an activating drug (group 1 - 0.01 mg / kg, 0.3 mg / kg, 1.0 mg / kg, and 3 mg / kg) immediately after a meal Blood is drawn at the specified time intervals (predose, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 16, and 24 hours after the AD dose) on day 1 for evaluation of the pharmacokinetics of simple dose of the activating drug and its major metabolites. Each subject receives a simple intatumoral injection of the transduced, adenoviral autologous dendritic cells at a concentration of 5 x 10 7 cells, engineered to express hIL-12 and one or more other immunomodulators under the control of RTS. Subjects are carefully monitored for reactions at the local injection site and / or hypersensitivity reactions. Day 2 to day 14 of the view of the internal Patient: Days 2 to 14, each subject is dosed with the activating drug immediately after a meal. The vital signs and adverse events are collected daily on days 2 to 14. On day 4 + 24 hours, biopsies of the tumor and / or drainage of lymph nodes are removed from approximately 50% of the subjects for the measurement of hIL-12 and the cellular immune response. On day 8 the weight is measured. On day 8 ± 24, biopsies are removed from the tumor and / or drained from lymph nodes, from subjects who did not have a biopsy performed on day 4 for the measurement of hIL-12, and one or more other immunomodulators and the cellular immune response. Blood is drawn on day 4 ± 24 hours and on day 8 + 24 for the evaluation of the potential antibodies and the cellular immune response against the adenovirus and / or the RTS components. A serum cytokine profile is also obtained to determine whether the expression of other cytokines is affected by treatment with hIL-12 and one or more other immunomodulatory transgenes. On day 8, urine is collected and blood drawn for baseline serum chemistry, urinalysis, and hematology (safety profile). On day 8, blood is drawn at the specified time intervals (predose, 0.5, 1, 2, 4, 6, 8, 12, 16, and 24 hours after the AD dose) for the assessment of pharmacokinetics / ADME in resting state of the activating drug and its major metabolites.

Internal Patient visit on day 14: On day 14, each subject is dosed with the Activating Drug immediately after a meal. Each subject receives a physical examination (including vital signs), height, weight and ECOG condition). Urine is collected and blood is drawn for serum chemistry, urinalysis, and hematology (safety profile). Blood is drawn on day 14 ± 24 hours for the evaluation of the potential antibodies and the cellular immune response against the adenovirus and / or the RTS components. A serum cytokine profile is also obtained to determine if the expression of other cytokines is affected.

Blood is collected from subjects at specified visits from inpatients and outpatients to measure potential antibodies and the cellular immune response to the adenovirus and the RTS components. Blood is obtained for a baseline serum cytokine profile. The infectivity blocking assay of AdVeGFP is used to detect an antibody response to the adenoviral vector (Gambotto, Robins et al., 2004). The response of the antibody to the RTS components will be evaluated by Western blot analysis and / or ELISA using the serum from the patient and the RTS proteins produced from an expression vector. In addition, the complex cytokine test will be performed on serum by Luminex for IL-12, IFN-gamma, IP-IO, and other Thl / Th2 cytokines such as IL-2, TNF-alpha, IL-4, IL-5 , and IL-10. These antibody and cytokine assays will require approximately 10 ml of blood.

Potential Antibody and Cellular Immune Response to Adenovirus and / or RTS Components: Blood will be collected from subjects at specified inpatient and outpatient visits to evaluate the cellular immune response and the potential antibody response to the adenovirus and RTS components and the tumor antigens. The infectivity blocking assay of AdVeGFP will be used to detect an antibody response to the adenoviral vector (Nwanegbo, et al., 2004). The response of the antibody to the RTS components will be evaluated by Western blot analysis and / or ELISA using serum from the subjects and the RTS proteins produced by an expression vector. In addition, the multiplex cytokine test will be performed on serum by Luminex for IL-12, IFN-gamma, IP-10, and other Thl / Th2 cytokines such as IL-2, TNFa, IL-4, IL-5 and IL -10. These antibody and cytokine assays will require approximately 10 ml of blood.

Tests of the cellular immune response use approximately 50-60 ml of blood and the subsets of CD4 and CD8 cells will be separated from it. The separated T cells will be mixed with analogous DCs transduced with the AdV-empty vector, AdV-RTS, or the immunomodulatory vectors of AdV-RTS-hIL12 - in an ELISPOT assay for the production of IFN-gamma by the T cells activated by antigens. AdV and RTS derivatives, if any. Similar assays will be performed using the tumor cells as such and / or DCs expressing shared melanoma antigens, to evaluate the early immune response to the tumor. Additional trials may also be conducted as necessary.

Pregnancy Test- Women with the potential to be pregnant are given the urine pregnancy test at the screening visit and before the first inpatient visit of the retreat phase. The test is performed at least 72, 48, 24, or 12 hours before the administration of the Activating Drug during the initial treatment and all periods of retreatment. If the urine pregnancy test is positive, then the confirmation will be obtained with a serum pregnancy test. If the pregnancy is confirmed, the subject will not be allowed to enter the test or continue in the retreatment phase. The pregnancy test can be performed again as many times as necessary.

Concomitant medication qions: In the selection, and before the first inpatient visit of the retreatment phase, each subject is asked to provide a list of concurrent medications to determine any possible relationship to the adverse events that occur during the testing and follow-up phase.

Retreatment Criteria: If a subject has tolerated previous ADDC inoculation without adverse reactions that are limiting, and has not shown disease progression or symptomatic decline at the time of potential retreatment, they will be considered for retreatment. If in the opinion of the principal investigator, and the treating physician, there is a potential clinical benefit for one or more intratumoral injections of AdDCs in combination with the activating drug (maximum tolerated group 1 dose) for 14 consecutive days, retreatment will be offered to the subject, with the condition that the following criteria are met: 1. There should be no limiting toxicities; 2. The subject's disease is stable or shows clinical or subjective signs of improvement, and 3. There is no evidence of cellular immune response or antibody to the adenoviral components of the RheoSwitch® Therapeutic System.

Evaluation of the function of the transgene and immune effects: Biopsies by puncture or removal of the tumor and drainage of associated lymph nodes, will be collected during the selection (day -12 a day -7), day 4, day 8 and day 14 of the test and at month 1 of follow-up (see Table 3-5) for the in vivo evaluation of transgene expression of the expression of hIL-12 and one or more other immunomodulators, and the cellular immune response. Fine needle aspiration biopsies of the tumor and associated drained lymph nodes will be collected on day -12 to -7 and on day 14 of the treatment period for in vivo evaluation of the transgene expression of hIL-12 , and one or more other immunomodulators, and the cellular immune response. Biopsies will be evaluated using standard light microscopy and immunohistochemistry to evaluate the cellular infiltration of the T cells into the tumor and the draining lymph nodes. The biopsy sections will be read by a pathologist not aware of the background of the subject of the study. To distinguish between expression of endogenous IL-12 induced by DCs in the tumor and drained lymph nodes, RT-PCR on RNA will be used with appropriately designed primers. Blood will be drawn for a serum cytokine profile in the selection, on day 4, on day 8 and on day 14 of the test, at month 1 of follow-up and on day -12 to -7, on day 8 and the 14th day of the retreatment period (see Tables 3-5). A serum cytokine profile will be obtained to determine if the expression of the other cytokines is affected by the treatment with the hIL-12 transgene. The Multiplex cytokine test will be performed in the serum by Luminex for IL-12, IFN-gamma, IP-10, and other Thl / Th2 cytokines such as IL-2, TNFa, IL-4, IL-5 and IL-10. . These antibody and cytokine assays will require approximately 10 ml of blood.

Simple dose and pharmacokinetics in resting state of the activating drug: Blood will be drawn at the specified time intervals (predose, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 16, and 24 hours after the morning dose) ) on day 1 of the test for the evaluation of the single-dose pharmacokinetics and on day 8 of the test for the measurement of the resting pharmacokinetics / ADME of the activating drug, and its major metabolites. The plasma will be evaluated by HPLC to obtain the following resting pharmacokinetic endpoints of the activating drug and major metabolites: Cmax (maximum observed plasma concentration), Tmax (time to maximum observed plasma concentration), Ctrough (minimum observed plasma concentration) computed as the average of the concentrations at 0 and 24 hours), C24h (plasma concentration at 24 hours), AUC24h (area under the curve of plasmatic concentration-time from time 0 to 24 hours), Ke (rate of elimination Apparent), and T112 (apparent half-life).

It should be understood that the above-described embodiments and exemplifications are not intended to be limiting in any respect to the scope of the invention, and that the scope of the claims presented herein are intended to encompass all modalities and exemplifications, whether explicitly or not presented here.

Example 3 A matrix of rationally selected modular gene components can be rapidly assembled into DNA expression constructs through the application of a combinatorial transgene technology such as ULTRAVECTORMR. To demonstrate that the assembled gene components that individually affect the transcription process, post-transcription, translation and posttranslation, can jointly impact the level of gene expression, RheoSwitch technology is used, optionally, in combination with the artificial 5'UTR, several signals of 3 'Reg + poly (A) (SV40 and hGH) , signal peptides (TNF-alpha and IL-2), and codon optimization schemes (+/-) to modulate transcription, increase a cell's ability to produce and secrete TNF-alpha. Figure 11 illustrates the modular elements used, and graphically represents each modular element flanked by unique restriction sites to provide a method for the precise assembly of modular combinations.

Modular assembly was carried out in the context of a fundamental DNA structure designed to accept synthetic genes. An example of a fundamental ULTRAVECTORMR structure engineered for adenoviral packaging is depicted in Figure 12. The combinatorial modular design combines well with the adenoviral distribution of a therapeutic product since it allows the use of compact regulatory sequences that may be shorter than those found in nature.

In vitro assembly of modular combinations Table 7. A matrix that produces 11 test vectors and mounted DNA Transient vectors were transfected into the HEK293T cell line to assess which modular combinations result in increased TNF-alpha performance. To induce the expression of TNF-alpha, control or vehicle or ligand was administered to the cells. The supernatant was collected and TNF-alpha levels were measured by ELISA. To adjust a line or base approaching the wild type, vector 43318 contains the 5'UTR of TNF-alpha wild-type conformed by UV, the signal peptide, the coding sequence, and the SV40pA 3'Reg. Individually, the modular changes of Opton codon optimization (1, 2) of the mature protein coding sequence or TNF-alpha signal peptide result in increasing increases in protein secretion (vectors 43319, 43320). The additional modular substitution of the 5U2 5'UTR for the 5'UTR of TNF-wt further raises the secretion levels (vectors 43322, 43323). Greater secretion of TNF-alpha is achieved when the wild-type 5'UTR, signal peptide, and coding sequence modules are replaced with the respective modules of 5U2, IL2, and TNFOptUV (vector 43329).

To demonstrate that the increased secretion of TNF-alpha is not dependent on the cell type, the 11 experimental vectors were transfected into CH0-K1 cells and 2 control vectors were added (see figure 27). Vector 43534 (figure 26) and vector 43533 (figure 25)) contain wild-type TNF-alpha modules to serve as controls. Vector 43534 is composed of the TNF-alpha wild-type sequence without the ULTRAVECTORR mounting pins being present. Notably, the presence of ULTRAVECTORMR mounting pins does not adversely affect the production and secretion of THF-alpha. In this data set it was also shown that 3'Reg wild-type TNF-alpha substitution with either polyA modules containing SV40e or hGH results in an increase in TNF-alpha secretion. Maximum yield of TNF-alpha was achieved with the combination of 5U2, IL2 signal peptide, TNFOptUV, and hGHpA. The CHO-K1 cell data exhibits the same tendency of increasing growth with each 5U2 module substitution in the 5'UTR, IL-2 signal peptide, and TNFOptUV coding sequence. Interestingly, the magnitude of increase is slightly different in the two cell types. This illustrates that while modules perform similarly in each cell type, physiological differences in tissue type or specific cell can influence the magnitude of the modular substitution effect. The increase of the combinatorial matrix to include more modules in each category may allow the identification of a superior combination depending on the tissue or cell type tested. Examples of additional modules that can be included in a larger matrix are included as SEQ ID NOs: 41-46.

Example 4. Evaluation of therapeutic candidate in an animal model To demonstrate the effectiveness of optimized inducible TNF-alpha constructs for treating cancer, for example, prostate cancer or head and neck cancer, a model of the disease in mouse head and neck cancer can be employed. It has been shown that individual gene suppression of Smad4 produces a spontaneous model of squamous cell carcinoma of the head and neck, human, malignant (HNSCC). (PMID: 19841536) In the absence of this mouse strain, naked human-derived HNSCC tumor cells can be implanted in nude mice. In the post-tumor setting, optimized TNF-alpha constructs can be introduced into the tumor with adenovirus. Variable doses of ligand can be administered to the mouse to regulate the level of optimized TNF-alpha produced. The tumor burden and the assessed tumor necrosis will be measured to identify potential therapeutic candidates of the optimized TNF-alpha constructs.

Example 5. Example of therapeutic modality A TNF-alpha transgene engineered to a patient is administered by intratumoral injection of a non-replicating adenovirus DNA vector. This gene program codes for mature cytokine optimized by codon in mammal, fused with a signal peptide optimized by codon for IL-2. In turn, the cds transgene (IL-2 SP + TNF-alpha) is flanked by 5'UTR of TNF-alpha wild type and the signal SV40 3'Reg + poly (A), and its expression is controlled by RheoSwitch technology by administering a carefully "programmed" dose of the activating ligand (i.e., the DNA incorporated in the vector WN-43320, see figure 15). Preliminary data show that this combination of DNA elements produces the highest possible induction of secreted TNF-alpha while deepening "strict" and "no-drip" control of its expression. That is, the non-induced basal level of expression remains low in this transgene configuration, and will probably be lower to exert out-of-target effects on the patient.

In an alternative embodiment of the invention, the engineered TNF-alpha transgene is administered by adenovirus to a patient in a modular DNA configuration similar to vector WN-43329 (see Figure 24), which exhibits both high basal expression and increased transgenic expression. This DNA employs the 5'U2 artificially engineered and the poly (A) signal of hGH, as well as full optimization by codon in the mammalian and IL-2 signal peptide. Systemic toxicity control is achieved in the patient by using a low MOI of adenovirus in the intratumoral injection. To a lesser degree, the level of expression and temporal control is also modulated through the activating ligand RheoSwitch. Additional control of the distribution of this gene product can be achieved through the incorporation of tissue-specific mRNA response elements that can prohibit out-of-target expression in vital organs, or through artificially engineered adenoviral capsid engineering. for improved tropism.

In a further embodiment of the invention, the engineered TNF-alpha trangne is similar to DNA in WN-43328 (Figure 23), which is hypothesized to confer high stability to artificial AR m through the 5'U2 element . However, this construct does not make use of an IL-2 signal peptide for enhanced secretion, and retains the wild type sequence of the DNA encoding the N'-term of TNF-alpha. For unknown reasons, the high-level secretion of artificial TNF-alpha can be shown to be detrimental to the patient's consequences despite the context, while the natural mechanism of "cytokine release" by metalloproteinases can limit the toxicity to the patient, by confining the factor to its tumor environment. If the natural detachment of exogenous TNF-alpha still demonstrates out-of-target effects, its ectodomain stem can be truncated by mutation to prohibit solubilization by native proteases, and the factor will enhance activity through cell-cell contacts as a transmembrane protein. type II de facto. Alternatively, a transgene similar to the construct described by vector WN-43328 can encode a constitutively expressed TNF-alpha with a mutated stem ectodomain containing a cleavage site for an exogenous protease. This exogenous protease will in turn be under a promoter element controlled by RheoSwitch technology, it will only be expressed in the presence of the activating ligand. In this way, cleavage and in vivo solubility (but not surface expression) of TNF-alpha will be controlled through modular transgenic elements.

Example 6. Antitumor efficacy of Ad-RTS-IL-12 The antitumor effect of Ad-RTS-IL-12 has been evaluated in a series of murine tumor models of melanoma, colorectal, pancreatic, breast, lung and renal cancer. An example dose response experiment is shown below. C57B1 / 6 immuno-competent female mice (6-8 weeks of age) were inoculated subcutaneously with B16F0 murine melanoma cancer cells. Eleven days after inoculation with tumor cells, when macroscopic tumor nodules were evident (tumor volumes averaged approximately 40 mm 3), the mice were separated into groups of 5 animals each. There were 9 groups, which included: treated with saline control; treated with ligand activator; Ad-RTS-mIL12 (leelO vp) alone and 6 groups were treated with different doses of Ad-RTS-mIL-12 vector (le7, le8, le9, 5e9, lelo, 5el0 viral particles), plus ligand activator. Mice in the + ligand groups were provided with 100 mg / kg of activating ligand in rodent diet meal of 18% global protein 2018 Teklad (Harían Laboratories) (1000 mg of ligand / kg of food) one day before the administration of the vector. The mice of the control groups received the rodent diet meal of 18% Global 2018 Teklad protein. An individual administration of Ad-RTS-mIL12 was injected in 100 μl of PBS into the tumor on day 12. Tumor volume and body weights were measured every 2-3 days using calibrators and a weight scale, and the animals were followed until the control tumors reached 2000 mm3. The data was uploaded in the Study Log study software.

As shown in Figure 31, a substantial antitumor effect was observed in the 12 doses of Ad-IL-RTS above read8 vp (range 73-99%). The lowest dose of Ad-RTS-IL-12 tested, read7 vp, does not show antitumor effect. In the absence of activating ligand, high-dose Ad-RTS-IL-12 at leelO vp showed no effect, illustrating the combination requirement of both Ad-RTS-IL-12 and activating ligand. The ligand treatment by itself showed no effect. Therefore, this study illustrates the potent antitumor effects mediated by Ad-RTS-IL-12 in combination with the activating ligand.

Body weight analyzes are presented in Figure 32. Animals treated at the highest dose level (5eel0 vp) of Ad-RTS-IL-12 showed momentary weight loss on day 19, but recovered by day 26. Animals in the treatment with other groups showed no clear dose response relationship and only minor changes in weight gain were observed.

Example 7. Efficiency of Ad-RTS-IL-12 in Lewis lung cancer model Immunocompetent mice C57b / 6 females 6 to 1 weeks of age were inoculated subcutaneously (s.c.) with murine Lewis lung carcinoma cells (CLL). Five days after inoculation of the cells, the mice were randomized and assigned to treatment and control groups (n = 5) for a total of four groups, without treatment (control), activator alone (RG-115932), Ad- RTS-mIL12 alone and Ad-RTS-mIL12 plus activator. The cohorts receiving the activator (L) were fed (rodent diet meal of 18% global protein 2018 Teklad (Harían Laboratories) mixed with activator (1000 mg / kg of food) ad libitum.The cohorts receiving treatment with Ad -RTS-mIL12 alone or without treatment continued to receive a regular diet Treatment was started when the tumor reached 28 ± 6 mm 3. Ad-RTS-mILl2 (lelO vp / 100ul in PBS) was given to mice through intratumoral injections (it) on day 6, 9 and 13 after inoculation with tumor cells The food with activator (L) was started to be given to the mice 24 hours before the administration of the vector, the tumor size and weight were monitored body of each mouse three times a week using gauges and a weigh scale until the end of the experiment.The experiment was terminated when the mouse tumor size exceeded> 1200 mm 3. The data was uploaded to the animal study software Study Log.

The post-treatment tumor volume is shown in Figure 33A. Mice that had Lewis lung tumor in the control and activator groups (L) exhibited approximately similar kinetics of tumor growth. Three doses of Ad-RTS-mIL12 alone led to intermediate tumor growth. Importantly, Ad-RTS-mIL12 with activator (L) produced marked inhibition of tumor growth (78%) in relation to the control group. These data suggest that Ad-RTS-mIL12 in the presence of activator inhibits the growth of Lewis lung tumor. Body weight was monitored as an indicator of toxicity. No major loss of body weight was found during the course of the experiment.

Example 8. Antitumor efficacy of Ad-RTS-IL-12 in a melanoma model Immunocompetent mice C57b / 6, females 6 to 8 weeks old were inoculated subcutaneously (s.c.) with murine melanoma cancer cells (B16F0). Ten days after inoculation with the cells, the mice were randomized to treatment and control groups (n = 5) for a total of nine groups: no treatment (control), only activator (L) (RG-115932) , and only Ad-RTS-mIL12, and only Ad-RTS-mIL12 with different doses of activator (50, 100, 250, 500 and 1000 mg / kg) of ligand. The cohorts that receive activator (L) were fed rodent diet meal of 18% global protein 2018 Teklad mixed with activator (1000 mg / kg of food) ad libitum. Cohorts receiving treatment with Ad-RTS-mIL12 alone or without treatment continued to receive a regular diet of rodent diet meal of 18% global protein 2018 Teklad. Treatment was started when the tumor reached 56 + 18 mm3. A single dose of Ad-RTS-mIL12 (lelO vp / l00ul in PBS) was given to mice via intratumoral injection (i.t.) on day 13 after inoculation with tumor cells. The food with activator (L) was given to the mice 24 hours before the injection of the vector. The size of the tumor and the body weight of each mouse were monitored three times a week using calipers and a weigh scale until the end of the experiment. The experiment was terminated when the tumor size exceeded > 2000 mm3. The data was uploaded to the animal study software Study Log.

Changes in tumor volume and body weight after treatment are shown in Figures 34A and 34B. Mice that have melanoma tumor in control groups and only activator (L) showed similar aggressive tumor growth. The kinetics of tumor growth indicated that the food with activator has no antitumor activity. A slight inhibition of tumor growth was observed (12%) on day 26, when the animals received a single dose of Ad-RTS-mIL12 (lelO vp) without ligand. Treatment with Ad-RTS-mIL12 plus activator (L) resulted in inhibition of tumor growth (73-98%) compared to control mice. A single dose of Ad-RTS-mIL12 with 50 mg / kg activator (L) produced significant tumor reduction in relation to control tumors. Notably, significant antitumor activity (90-98%) was evident as the dose of the food with activator was increased from 100-1000 mg / kg, compared to 50mg / kg of food with activator. These data clearly show that Ad-RTS-mIL12 is active in the melanoma model and exhibits a broad therapeutic window of dose of activating ligand. Body weight was monitored as an indicator of toxicity. On days 13 and 17, slight momentary changes in body weight (<5%) were found in the activator dose of 1000 mg / kg. No major loss of body weight was found during the rest of the experiment. No changes were found in body weight related to the dose response without activator. The treatment with AdRTS-mIL12 under different doses was well tolerated without any sign of toxicity.

Example 9. Antitumor efficacy of Ad-RTS-IL-12 in a colon cancer model Immunocompetent Balb / C mice 6 to 8 weeks old, females subcutaneously (s.c.) were inoculated with murine, stable, murine colon cancer cells expressing luciferase (CT26Luc). Ten days after inoculation with cells, the mice were randomly assigned to treatment and control groups (n = 5) for a total of three groups, without treatment (control), only activator (L) (RG-115932), and Ad-RTS-mIL12 plus activator. The cohorts that receive activator (L) were fed with rodent diet food 18% of global protein 2018 Teklad (Harían Laboratories) mixed with activator (1000 mg / kg of food) ad libitum. The cohorts that do not receive treatment continued to receive food from rodent diet 18% of Teklad 2018 global protein. Treatment was started when the tumor reached 40 + 17 mm3. Ad-RTS-mIL12 (lelO vp / 100ul in PBS) was given to mice via intratumoral injection (i.t.) on day 11 and 18 after inoculation with tumor cells. The food with activator (L) was given to the mice 24 hours before the injection of the vector. The size of the tumor and the body weight of each mouse were monitored three times a week using calipers and a weigh scale until the end of the experiment. The experiment is terminated when the mouse tumor size exceeds > 2000 mm3. The data was uploaded to the animal study software Study Log.

Changes in tumor volume and body weight after treatment are shown in Figures 35A and 35B. Mice that had colon carcinoma in the control groups and only activator (L) showed similar aggressive tumor growth. The kinetics of tumor growth indicated that food with activator does not inhibit tumor growth. Two doses of Ad-RTS-mIL12 plus activator (L) alone resulted in complete regression and inhibition of tumor growth (100%) compared to control mice. Notably, five of the five animals were completely tumor free as a result of treatment with Ad-RTS-mIL12. Mice that became tumor free following treatment with Ad-RTS-mIL12 were further stimulated with parental CT26Luc cells. Five Balb / c animals without previous treatment were also inoculated subcutaneously with CT26Luc as the control group. The control animals developed tumor nodules as expected. Importantly, the tumors did not develop in all animals restimulated at four weeks after restimulation. This study indicates that therapy with Ad-RTS-mIL12 developed strong antitumor immunity against the aggressive colon cancer model. Body weight was monitored as an indicator of toxicity. No major loss of body weight was found during the course of the experiment.

Example 10. Antitumor efficacy of Ad-RTS-IL-12 in a model of pancreatic cancer Immunocompetent C57B / 6 female mice 6 to 8 weeks old were inoculated subcutaneously (s.c.) with syngenic PA O2 pancreatic cancer (ATCC) cells. Six days after cell inoculation, mice were randomized into groups of five animals each in four untreated groups, single activator (RG-115932), only Ad-RTS-mIL12 and Ad-RTS-mIL12 plus activator. The cohorts receiving ligand activator were fed with rodent diet meal of 18% global protein 2018 Teklad (Harían Laboratories) mixed with activator (1000 mg / kg of food) ad libitum. Cohorts receiving treatment with Ad-RTS-mIL12 alone or without treatment continued to receive rodent diet meal of 18% global protein 2018 Teklad. Mice were treated with an individual intratumoral injection (i.t.) of Ad-RTS-mIL12 at a dose level of 10 vp / 100ul in pBS, on day 7 and on day 14 after implantation of tumor cells. Tumor size averaged rare STGT3 at the time of initiation of vector treatment.

The size of the tumor and the body weight of each mouse were monitored three times a week until the end of the experiment. The experiment was terminated when the mouse tumor size exceeded 600mm3. Since pancreatic tumors grow very slowly, it was defined as the termination of the experiment. Tumor growth in mice that do not receive treatment was normal.

In this tumor model, minor delay in tumor growth was seen in mice receiving treatment with either activator alone or only Ad-RTS-mIL12. In contrast, the tumor growth in all mice treated with Ad-RTS-mIL12 was drastically inhibited (97%) compared to that in mice. control that did not receive treatment. Body weight was measured throughout the experiment using gauges and a weigh scale as a measure of toxicity. The body weight of animals injected with Ad-RTS-mIL12 showed no significant decrease in body weight after administration, except for a momentary decrease in body weight (<5%) on day 12-13. In addition, no pathological behavior (lethargy, lyse fur, lameness, dehydration, stooped posture, etc.) was observed in any of the animals. The tumor regression was maintained until day 37, when the control animals were sacrificed. The data was uploaded to the animal study software Study Log.

The results are shown in figures 36A and 36B.

Example 11. Antitumor efficacy of Ad-RTS-IL-12 in a breast cancer model The purpose of this study was to evaluate the intratumoral treatment with Ad-RTS-mIL12 for its efficiency and toxicity in the murine breast cancer model.

BalbC female mice 6 to 8 weeks old were purchased from Charles River Laboratories or Harían (E.U.A). The experimental and animal care procedure was carried out according to the Intrexon institutional animal use and care guide.

Murine breast carcinoma cell lines (4T1) from ATCC (Manassas, VA) were purchased. 4T1 cells were cultured in the Roswell Park Memorial Institute (RPMI) 1640 (ATCC, Manassas, VA) medium supplemented with heat inactivated fetal calf serum (FCS) 10% v / v, L-glutamine 2-m ( Atlanta Biologicals, Inc., Lawrenceville, GA), 100 IU / ml penicillin G, and 100 ug / ml streptomycin.The cells were cultured at 37 ° C in 5% C02.All cell lines were routinely tested and They found that they are free of mycoplasma.

Immunocompetent BALB / c mice from 6 to 8 weeks old females were inoculated subcutaneously (s.c.) with syngeneic breast cancer cells (4T1), le5 cells / 50ul. Eight days after cell inoculation, the mice were randomized into groups of five animals each in four groups, without treatment, only activator, only Ad-RTS-mIL12 and Ad-RTS-mIL12 plus activator. Cohorts receiving ligand activator were fed rodent food mixed with activator (1000mg / kg) ad libitum. Cohorts receiving treatment with Ad-RTS-mIL12 alone or without treatment continued to receive a normal diet (Harán Laboratories, E.U.A.). The activating ligand is administered through a virtual diet created from Harlan Teklad (a division of virtual diet of Harian) formulated to 1000 mg of activating ligand at 1 kg of the same food that is administered to the control animals. Mice were treated with an individual intratumoral injection (i.t.) of Ad-RTS-mIL12 at a dose level of 10 vp / 100ul in pBS, on day 9, 12 and 14 after implantation of tumor cells. The mean volume of tumor size was 36 mm3 at the time of initiation of vector treatment. The tumor size and body weight of each mouse were monitored three times per week until the end of the experiment. The experiment was terminated when the mouse tumor size exceeded > 1000mm3.

Eight days after inoculation with 4T1 breast cancer cells, the mice were randomized and assigned to treatment and control groups (n = 5 / group) for a total of four groups, without treatment (control), only activator (L ), only Ad-RTS-mIL12 and Ad-RTS-mIL12 plus activator, as shown in the table below.

Treatment was started when the tumor reached the mean volume of 36 mm3. The post-treatment tumor volume is shown in Figure 39A. Mice that have tumor 4T1 in the groups with only control, with only Ad-RTS-IL-12 and only activating ligand (L) showed inhibition of tumor growth -20% and 35% respectively on day 26 (figure 39A). Importantly three doses of Ad-RTS-mIL12 with activating ligand led to marked inhibition of tumor growth (82%), in relation to the control group (p <; 0.005). These data suggest that Ad-RTS-mIL12 in the presence of activator exhibits potent antitumor activity in the breast cancer model (4T1). Body weight was monitored as an indicator of toxicity. No major loss of body weight or deaths was found during the course of the experiment (Figure 39B).

The results demonstrate that the direct intratumoral injection of Ad-RTS-mIL12 plus activator ligand is highly effective in inducing tumor regression and is safe in the breast cancer model. The antitumor activity was significant (p <0.005) in this model.

Example 12. Clinical Protocol for Administration of Ad-RTS-IL-12 Vector Free of Immune Cells The following is a clinical protocol that can be used to practice the invention in the form of administration of the Ad-RTS-IL-12 vector for the treatment of non-resectable malignant stage III C or IV melanoma.

The objectives of this Phase Ib clinical trial are to assess the safety and objective response, tumor response speed, and immunological and other biological activities of six treatment cycles of intratumor injections of Ad-RTS-IL-12 in combination with 14 oral doses. activator ligand daily The dose of Ad-RTS-IL-12 will be administered initially (first cycle) to 1 x 1011 viral particles (vp) together with a dose of 5 mg / day of activating ligand. The doses of both the viral particles and the activating ligand will then be scaled up for each repeated treatment cycle for each patient, according to a fixed schedule (Table 8), with the condition that the previous treatment cycle was tolerated by the patient. patient.

The objectives of this phase Ib study are as follows: 1. To assess the safety and tolerance of repeated courses of treatment of intratumoral injections of ADRTS-IL-12 in an intra-patient dose scale in combination with scale-dose activating ligand in patients with malignant stage III C or IV non-resectable melanoma. 2. Obtain indications of efficiency when using diagnostic CT scans (Evaluation Criteria Response in Solid Tumors (RECIST 1.1), PET scans and photographs (as applicable). 3. To evaluate the functionality of the RheoSwitch ™ Therapeutic System (RTSMR) in patients when evaluating the immunological effects of AD-RTS-IL-12 in combination with activating ligand, in terms of cellular immune response (particularly gene expression of IL-12 and other cytokines, frequency of cytotoxic T lymphocytes and Tregs) and other biological activities (for example, apoptosis and immune cell infiltration) in target tumors injected, lymphatic drainage nodules comprised in tumor (if accessible) and in the peripheral circulation, and correlate changes in the immunological and other biological parameters with previous dose of activating ligand and with the tumor response. 4. To evaluate the degree of uptake of AD-RTS-IL-12 in tumor cells and in dendritic cells and macrophages in the tumor to determine which cells capture the virus if the degree of uptake is dose dependent of AD-RTS-IL- 12 To determine the inflammatory response and immune response (cellular, such as cytotoxic lymphocytes and Tregs, and the induction of cytokines) in the tumor, drain lymph nodes comprised in tumor (if accessible) and in the peripheral circulation. Changes in immunological and other biological parameters will correlate with the dose of AD-RTS-IL-12 and the activating ligand and with the tumor response. 5. To assess the pharmacokinetic profile during steady state in each cycle on days 8 - 9 in a subset of patients. 6. Evaluate QT / QTc intervals in ECG obtained by Holter monitoring, in patients who will undergo PK evaluation.

Indication: Malignant melanoma stage III C (in transit), Stage IV (Mine, Mlb or Mlc (LDH <2xULN) not excised with at least 4 accessible lesions.

Study Design: Multicenter, single-arm, open-label evaluation of phase b safety, tolerance, tumor response (RECIST 1.1), and immunological and other biological effects, of six cycles of treatment, each lasting for 28 days, each with an intra-tumoral injection of Ad-RTS-IL-12 in combination with 14 daily oral doses of the activating ligand. The dose of both AD-RTS-IL-12 and the activating ligand will be scaled according to Figure 1 and Table 1 for all patients who tolerated the preceding treatment cycle.

Study Population: Men and women of all races, > 18 years of age, with stage III C or IV non-resectable malignant melanoma with ECOG performance status of 0-1, who have a minimum of 4 accessible lesions (longer diameter <3 cm, shorter diameter> lcm) or lymph nodes comprised in palpable tumor (longest diameter <5 cm; shorter> 1.5 cm) for intra-tumoral lesions or biopsies.

Sample size: A minimum of 12 and a maximum of 28 patients with stage III C or IV melanoma will be introduced in this study.

All patients in this protocol will be introduced into an individual arm, with an intra-patient dose scale of AD-RTS-IL-12 and activating ligand with each repeated cycle of treatment according to Figure 1 and Table 1, with the condition that the previous treatment cycle was well tolerated.

Test Product: During each cycle, the patient will be treated with a combination of oral activating ligand and an intra-tumor injection of a gene therapy (Ad-RTS-IL-12) engineered to express inducible hIL-12 in a dose-dependent response to the activating ligand. AD-RTS-IL-12 will be prepared at a central processing site and then frozen and sent to the appropriate clinical site. All patients will receive intra-tumoral injections (one per cycle for up to six cycles, with 4 weeks apart) of AD-RTS-IL-12 (approximately 1.0 x 1011 and 1.0 x 1012 total viral particles per injection). Patients will also receive an individual daily oral dose of activating ligand for 14 consecutive days during each cycle. The dose of AD-RTS-IL-12 and / or the activating ligand will be scaled intra-patient at the beginning of cycles 2 to 6 (see Table 8), in all patients who tolerated the previous treatment cycle. AD-RTS-IL-12 will be injected in a different lesion in each cycle, and if the number of lesions is limited, the injections will be made in sequential rotation. One of the minimum of four accessible lesions will not be injected since this lesion will be used to evaluate the systemic effect of AD-RTS-IL-12. The dosage of the patients will be staggered at least 24 hours apart. Each intra-tumoral injection will be presented once during a cycle, approximately 3 hours (+ 30 minutes) after the first dose of the activating ligand.

Dosage: Ligand Activator: lower dose / day: 5 mg intermediate dose / day: 20 mg; highest dose / day: 100 mg. The activating ligand will be administered during the first 14 days of each cycle.

Ad-RTS-TL-12: dose: approximately 1.0 x 1011 or 1.0 x 1012 particles / tumor per injection suspended in a total volume of 0.5 ml of sterile solution, with the injection volume distributed throughout the lesion, especially in the area of the tumor margin.

Table 8: Dosing Schedule Treatment with the next higher dose level will not weigh until the safety of tolerability of the preceding treatments has been confirmed. If BAT is defined, an additional scale will not be presented.

Administration Route: Ligand Activator: a solution in a soft gelatin capsule taken orally within 30 minutes of a meal; AD-RTS-IL-12: It will be injected on the first day of each cycle in an accessible tumor lesion or drain lymph nodes comprised in tumor (palpable) when necessary.

Method of Patient Assignment: All patients will receive treatment according to Table 8 and will be introduced into an arm. Safety and tolerance will be rigorously assessed for all patients, during and after each treatment cycle. The dose scale can only take place if the treatment of the previous cycle was tolerated.

Trial Duration: This study will last for each patient for approximately 28 weeks after the test.

After a period of up to 23 days for exam evaluation (Days -30 to -7), patients will be approved for participation in the study. On Days -6 to -2, the baseline biopsy will be performed and on Day 0, the baseline assessment of cardiac function using Holter monitoring will be done on patients who evaluated for PK. On Day 1 of each cycle, the approved patient will begin to receive the experimental treatments (an intra-tumoral injection of AD-RTS-IL-12 and an oral dose of the activating ligand). The treatment with ligand activator in each cycle will continue for a total of 14 days, followed by 14 days of debugging and observation for safety. The study treatment consists of 6 cycles, each lasting a total of 28 days including 14 days of follow-up. A post-treatment follow-up evaluation will be performed 6 weeks after the last injection (4 weeks after the last dose of activating ligand). Viral DNA in blood will be determined. If viral DNA is present at 6 weeks after the last injection, additional viral DNA titers will be continued. However, if two consecutive negative Q-PCR results for each source are shown, no further tests will be necessary.

Primary Endpoints: Safety and tolerance will be assessed by physical exams (including ECOG performance status), QT / QTc interval in ECG (in patients with PK), vital signs, serum chemistry, urinalysis, hematology, and by patient reports of any adverse event. Objective response and response speed, as assessed by CT scans.

Secondary Endpoints: Stable-state pharmacokinetics of the activating ligand, in a subset of eight patients (four per dose level of AD-RTS-IL-12). b. Degree of immune inflammatory response (cellular, such as cytotoxic lymphocytes and Tregs, and induction of cytokines) in the tumor, in drain lymph nodes comprised of tumor (if accessible) and in the peripheral circulation, as a result of treatment. c. Correlate changes in immunological and other biological parameters with AD-RTS-IL-12 and doses of activating ligand and tumor response. d. Efficiency also valued by explorations of PET and photographs. and. Long-term follow-up will be presented for up to 5 years. Patients will be contacted annually by the investigator.

Inclusion criteria: to. Men or women of tosas the races? 18 years old; b. Stage III C (in transit) or Stage IV non-resectable melanoma (Mine, Mlb, Mlc with LDH < 2x ULN), arising from cutaneous primary melanoma, sublingual mucosa of any tumor thickness or from any unknown primary site; c. A minimum of 4 accessible non-visceral lesions (longer diameter <3 cm; shorter> 1 cm) or lymph nodes comprised in palpable tumors (longer diameter <5 cm; shorter> 1.5 cm) for intra-injection -tumoral or biopsies. At least one injury will not be injected). d. ECOG performance status of 0 or 1; and. Patients without visible brain metastasis as assessed by improved MEI screening in contrast at the time of the examination or at the time of 30 days before entry into the study; F. Function of organs and adequate hematological base line, valued by laboratory values in the space of 30 days before treatment with study treatments and before the repeated treatment cycles and the dose scale of activating ligand as follows: hemoglobin = 10 g / L, granulocytes > 2500 / mm3, lymphocytes > 1000 / mm3, platelets > 100,000 / mm3, serum creatinine < 1.5 x ULN, AST, ALT, alkaline phosphatase < 2.5 x ULN, LDH < 2 x ULN, bilirubin in serum < 1.5 x ULN, absolute neutrophils > 500 / mm3; g. An expected survival of at least approximately 6 months in the opinion of the researcher (as it is valued mainly in the performance state); h. Women must be post-menopausal or surgically sterile or have effective practical contraception; Men who are not surgically sterile and whose partners are not menopausal or surgically sterile should be practiced effective contraception; i. Normal coagulation parameters as measured by PT / PTT; j. Voluntary signed informed consent approved by IRB.

Exclusion criteria: to. Viral, bacterial or fungal active and acute infections that require specific therapy; b. HIV infection due to issues regarding the ability to mount an effective immune response; c. Active autoimmune disease requiring steroids (> 10 mg of prednisolone or comparable) or other immunosuppressive therapy; d. Patients with detectable brain metastasis at the time of examination (or within 30 days before study entry), as assessed by contrast enhanced MRI scans; and. Patients with injuries > 3cm (LD) or lymph nodes comprised in tumor, palpable > 5 cm (LD); f. Patients with a hemoglobin of < 10 g / L; g. Presence of Stage IV visceral metastases or other distant metastases if LDH > 2 x ULN; h. Patients who have been previously treated with AD-RTS-IL-12 or activating ligand; i. Patients who have been previously treated with intratumoral gene therapy. j. Patients of organ halografts; k. Another concurrent clinically active malignant disease, with the exception of other skin cancers; 1. Less than 30 days (before the first dose of study medication) have elapsed since the completion of previous chemotherapy, hormone therapy, radiation therapy, immunotherapy, or any first-line therapy; m. Clinically significant cerebrovascular disease; n. History of concurrent severe heart failure (New York Heart Association Class III or IV) or coronary artery disease; or. Acute medical conditions such as ischemic heart or lung disease that may be considered an unacceptable anesthetic or operative risk; p. History of current bleeding or coagulation disorders; q. Concurrent immunosuppressive therapy such as corticosteroids (> 10mg prednisolone or comparable) and cyclosporin A; r. Concurrent research treatments, or treatment with any research treatment in the space of the last 30 days (before the first dose of the study medication); s. Concurrent medications that are metabolized by the CYP450 3A4 route; t. Women who are breastfeeding or are pregnant; or. Patients who have a history of hypersensitivity that can be related to any component of the product, for example, benzoic acid that can be related to the activating ligand, which contains two benzene rings; v. Any medical or psychiatric condition that, in the opinion of the investigator, will reduce in an acceptable manner the safety or administration of the proposed treatment, or prevent obtaining voluntary informed consent.

Statistical Methods: The objective response (CR + PR) will be based on changes in the size of the injected and non-injected tumor lesions as well as lymphatic nodules comprised in tumor, palpable by CT scans [using Response Criteria in Solid Tumors (RECIST 1.1)] .

PET scans and / or photographs will be used to evaluate changes in metabolic activity or size (skin lesions), respectively.

The primary analysis of OS and ORR will include a confidence interval, and will be performed when the sample size reaches 12, 16, 20, 24 patients, and at 6 weeks after the last patient's treatment.

The measures of demographic, immunological and biological activity, as well as safety parameters that include proportions of adverse events and laboratory values, will be analyzed descriptively at the end of the follow-up. The results will be summarized in tables, graphs and patient list by patient.

Descriptive statistics, including mean, median, standard deviation and histogram, will be used to summarize continuous measurements. Frequency accounts will be used for categorical variables, including objective tumor response. Immunological and biological activities will be correlated with the antitumor effect. These statistics will be provided by stratum (size of tumor lesions: lcm of longest diameter [LD],> 1 cm LD, DLN size included: <3cm LD,> 3cm LD, location of lesions: visceral, not visceral; Injection state of lesions: injected, not injected). The statistics will be made at the end of each treatment cycle and together. For the complete analysis, observations will be combined across the strata but not through the cycles.

Compliance: The trials were conducted in compliance with the current Good Clinical Practice (cGCP).

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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (102)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A vector for conditionally expressing proteins having the functions of one or more therapeutic proteins comprising a polynucleotide encoding a gene change, characterized in that the polynucleotide comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide that encodes one or more proteins that function as a therapeutic protein operably linked to a promoter that is activated by the ligand-dependent transcription factor.

2. The vector according to claim 1, characterized in that the therapeutic protein is selected from the group consisting of erythropoietin, ghrelin, osteoprotegerin, RANKL, RANKL decoy, TNF-OI antagonist, an IL-1 antagonist, G-CSF, - GM-CSF, IFN-OI, IFN- ?, angiostatin, endostatin, TNF-OI, PP1DCY-LSRLOC, β-glucuronidase, IL-12, α-galactosidase A, Arylulfatase A, α-glucosidase, β-glucosidase, glucocerebrosidase , CLN6 protein, Juvenile associated with CL 3, N-sulfoglucosamine-sulfohirolase (SGSH), aN-acetylglucosaminidase, acetyl-CoA-glucosaminide acetyltransferase, N-acetylglucosamine-6-sulphatase, aL-iduronidase, arylsulfatase B, acid sphingomyelinase, yuduronate- sulfatase, and ceruloplasmin.

3. The vector in accordance with the claim 1, characterized in that the therapeutic protein is one or more modulators selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10R DN or a subunit thereof, IL-15, 1L-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, IFN-alpha 1, IFN-alpha 2, IL-15-R-alpha, CCL3 (MIP-la), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP- 3b), CXCL9 (M1G), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6Cinema), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, Flt3L, IFN -beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1 RNAi, an antisense oligonucleotide of PD-L1, TGFbRII DN, ICOS-L, S100, CD40L, p53, survivin, fusion p53-survivin, MAGE3, PSA and PSMA.

4. The vector according to any of claims 1 to 3, characterized in that it is selected from the group consisting of plasmid, adenovirus, retrovirus, adeno-associated virus, pox virus, baculovirus, vaccinia virus, herpes simplex virus, Epstein-Barr, adenovirus, geminivirus, caulimovirus, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

5. The vector according to any of claims 1 to 4, characterized in that it is an adenoviral vector.

6. The vector according to any one of claims 1 to 5, characterized in that it further comprises a polynucleotide that codes for a protein having the function of IL-12.

7. The vector according to claim 6, characterized in that the polynucleotide coding for one or more proteins having the functions of the immunomodulator and the polynucleotide coding for the proteins having the function of IL-12 are under the control of a regulated promoter. of gene change.

8. The vector according to any of claims 1 to 7, characterized in that the gene change is a gene change based on ecdysone receptor (EcR).

9. The vector according to any of claims 1 to 8, characterized in that the polynucleotide encoding a gene change comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by the first transcription factor sequence and the second transcription factor sequence interact to form a protein complex that functions as a ligand-dependent transcription factor.

10. The vector according to any of claims 1 to 9, characterized in that the polynucleotide encoding a gene change comprises a first sequence of transcription factor and a second sequence of transcription factor under the control of a promoter, wherein the proteins encoded by the first transcription factor sequence and the second transcription factor sequence interact to form a protein complex that functions as a ligand-dependent transcription factor.

11. The vector according to claim 10, characterized in that the first transcription factor sequence and the second transcription factor sequence are connected by an internal ribosomal entry site of EMCV (IRES).

12. The vector according to any one of claims 1 to 11, characterized in that the polynucleotide encoding the one or more proteins having the functions of the therapeutic protein encode the human proteins.

13. The vector according to any of claims 5 to 12, characterized in that the polynucleotide encoding the protein having the function of IL-12 encodes human IL-12.

14. The vector according to any of claims 3 to 13, characterized in that the immunomodulator is TNF-alpha.

15. The vector according to claim 14, characterized in that the immunomodulator is human TNF-alpha.

16. The vector according to any of claims 3 to 15, characterized in that the immunomodulator comprises a sequence of amino acids at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 37 (human TNF-alpha).

17. The vector according to any of claims 1 to 16, characterized in that the polynucleotide coding for one or more proteins having the function of a therapeutic protein is optimized by codon.

18. The vector according to claim 17, characterized in that the polynucleotide encoding one or more proteins having the function of a therapeutic protein further comprises a nucleic acid sequence encoding a signal peptide.

19. The vector in accordance with the claim 18, characterized in that the nucleic acid sequence coding for the signal peptide is optimized by codon.

20. The vector in accordance with the claim 19, characterized in that the signal peptide is selected from the group consisting of: TNFOptUV and IL-2optUV.

21. The vector according to any of claims 18 to 20, characterized in that the signal peptide induces an enhanced secretion of TNF-alpha protein in comparison to the peptide sequence encoded by the TNF-alpha wild-type signal peptide gene.

22. The vector according to any of claims 1 to 21, characterized in that it also comprises the 5 'untranslated region (UTR).

23. The vector according to claim 22, characterized in that the 5 'UTR is derived from wild type TNF or 5U2.

24. The vector according to claim 22 or claim 23, characterized in that the 5 'UTR induces an improved level of mRNA coding for TNF-alpha, expression of TNF-alpha protein, or both.

25. The vector according to any of claims 1 to 24, characterized in that it also comprises the 3 'regulatory sequence.

26. The vector according to claim 25, characterized in that the 3 'regulatory region is a polyadenylation signal derived from SV40e or human growth hormone (hGH).

27. The vector in accordance with the claim 25 or claim 26, characterized in that the 3 'regulatory region induces an improved level of AR m that codes for TNF-alpha, TNF-alpha protein expression, or both.

28. The vector according to claim 27, characterized in that it comprises a 5'UTR derived from 5U2, an IL-2 signal peptide optimized by codon (lL-2optUV), TNF-alpha encoded by a nucleic acid sequence optimized by codon , and regulatory region 31 derived from hGH.

29. The vector according to any of claims 1 to 28, characterized in that it comprises a nucleic acid sequence selected from the group consisting of vector 43318, vector 43319, vector 43320, vector 43321, vector 43322, vector 43322, vector 43323, vector 43324 , vector 43325, vector 43326, vector 43327, and vector 43329.

30. The vector according to any one of claims 1 to 29, characterized in that the vector for conditionally expressing the therapeutic protein is injected directly into or near a tumor.

31. The vector according to any of claims 1 to 30, characterized in that the vector is not contained within a cell before in in vivo administration.

32. A method for producing a population of immune cells or therapeutic support cells (TSCs) that express proteins that have the function of one or more therapeutic proteins, characterized in that it comprises modifying the immune cells with the vector according to any of claims 1 to 31

33. The method according to claim 32, characterized in that the cells are human dendritic cells.

34. The method according to claim 33, characterized in that the dendritic cells are dendritic cells of bone marrow.

35. A population of immune cells or TSCs expressing proteins that have the function of one or more therapeutic proteins, characterized in that it comprises the vector according to any of claims 1 to 34.

36. The population of immune cells or TSC according to claim 35, characterized in that the cells are human dendritic cells.

37. An immune cell engineered in vitro by engineering or a TSC, characterized in that it comprises the vector according to any of claims 1 to 31.

38. The immune cell engineered in vitro or TSC according to claim 37, characterized in that the immune cell or TSC is a human dendritic cell.

39. A pharmaceutical composition, characterized in that it comprises the vector according to any of claims 1 to 31, the population of immune cells or TSC according to any of claims 35 to 36, the immune cells managed in vitro by engineering or TSC in accordance with any of claims 37 to 38, or any combination thereof.

40. The composition, characterized in that it comprises two or more of the vector according to any of claims 1 to 31, two or more populations of immune cells or TSCs according to any of claims 35 to 36, two or more of the immune cells managed in vitro by engineering or TSC according to any of claims 37 to 38, or any combination thereof.

41. The composition characterized in that it is in accordance with claim 39 or claim 40, and a pharmaceutically acceptable carrier.

42. The composition according to any of claims 39 to 41, characterized in that it also comprises a buffering agent.

43. The conformity composition 42, characterized in that the buffering agent is TRIS.

44. The composition according to any of claims 39 to 43, characterized in that it also comprises glycerin.

45. The composition according to any of claims 39 to 44, characterized in that the composition is suitable for oral, intravitreal, intratumoral, intraperitoneal, or subcutaneous administration.

46. The composition according to any of claims 39 to 45, characterized in that the population of cells comprises at least 104 cells.

47. The composition according to any of claims 39 to 46, characterized in that the population of cells comprises at least 107 cells.

48. The composition according to any of claims 39 to 47, characterized in that it reduces the tumor size or prevents the formation of tumors when the composition and the activating ligand are administered concurrently or in any order to a mammal in need of the same.

49. The composition according to any of claims 39 to 48, characterized in that it treats a tumor when the composition and an activating ligand are administered concurrently or in any order to a mammal in need of the same.

50. Use of (1) the vector according to any one of claims 31, the population according to any of claims 35 to 36, the immune cells engineered in vitro or TSC according to any of claims 35 to 38 , or the composition according to any one of claims 39 to 49 and (2) a therapeutically effective amount of one or more activating ligands in the manufacture of a medicament for treating a disease or disorder in a mammal in need of the same.

51. The use according to claim 50, wherein the disease or disorder is a tumor in the mammal.

52. The use according to claim 50 or claim 51, wherein the treatment is reducing the size of a tumor or preventing a tumor in the mammal.

53. Use of (1) the vector according to any of claims 1 to 31, the population according to any of claims 35 to 36, the immune cells engineered in vitro by engineering or TSC according to any of claims 35 to 38, or the pharmaceutical composition according to any of claims 39 to 49, and (2) a therapeutically effective amount of one or more activating ligands in the manufacture of a medicament for reducing, eliminating, or controlling systemic toxicity of TNF-alpha. in a mammal in need of the same, wherein the ligand is administered concurrently with, before, or after administration of the vector or composition and wherein synchronization of the administration of ligand activator reduces, eliminates, or controls systemic toxicity .

54. The use according to any of claims 50 to 53, wherein the administration of the activating ligand is stopped when the mammal demonstrates a side effect.

55. The use according to any one of claims 50 to 54, wherein the ligand is administered less than one hour before or after the vector, the population, the immune cells engineered in vi tro by engineering or TSC, or the composition.

56. The use according to any of claims 50 to 55, wherein the ligand is administered less than 24 hours after the vector or composition.

57. The use according to any of claims 50 to 55, wherein the ligand is administered less than 48 hours after the vector or composition.

58. The use according to any of claims 50 to 57, wherein the vector is not contained within a cell and is administered intratumorally to tumor environments in the mammal.

59. The use according to claim 58, wherein an immune cell or TSC is not intratumorally administered with the vector.

60. The use according to any of claims 50 to 59, wherein the tumor is a benign tumor.

61. The use according to any of claims 50 to 59, wherein the tumor is a malignant tumor.

62. The use according to any of claims 50 to 59, wherein the tumor is a melanoma.

63. The use according to any of claims 50 to 59, wherein the tumor is a malignant melanoma skin cancer.

64. The use according to any of claims 50 to 63, wherein the ligand is a diacylhydrazine.

65. The use according to any of claims 50 to 64, wherein the ligand is selected from RG-115819, RG-115932, and RG-115830.

66. The use according to any of claims 50 to 63, wherein the ligand is an amidoketone or oxadiazoline.

67. The use according to any of claims 50 to 66, wherein the ligand is administered less than one hour before or after the vector, immune cell population or TSC, immune cells engineered in vitro, or composition.

68. The use according to any of claims 50 to 66, wherein the ligand is administered less than 24 hours after the vector, immune cell population or TSC, immune cells engineered in vitro, or composition.

69. The use according to any of claims 50 to 66, wherein the ligand is administered less than 48 hours after the vector, immune cell population or TSC, immune cells engineered in vitro, or composition.

70. A method for determining the efficiency of the vector according to any of the claims 1 to 31, the population of TSC immune cells according to any of claims 35 to 36, immune cells engineered in vitro according to any of claims 35 to 38, or the composition according to any of the claims 39 to 49, based on a therapeutic regimen in a patient, characterized in that it comprises: (a) measuring the level of expression or activity level or both of interferon-gamma (IFN-γ) in a first biological sample obtained from the patient in need of the same before administration of the vector, TSC immune cell population , immune cells managed in vitro by engineering, or composition, thereby generating a level of control; (b) administering to a patient in need of the same vector according to any of claims 1 to 31, the population of TSC immune cells according to any of claims 35 to 36, the immune cells engineered in vitro by according to any of claims 35 to 38, or the composition according to any of claims 39 to 49; (c) administering to the patient in need thereof an amount of an activating ligand; (d) measure the level of expression or activity level or both of IFN-? in a second biological sample obtained from the patient in need of the same after administration of the vector, TSC immune cell population, immune cells engineered in vitro and ligand activator, thereby generating a level of testing; Y (e) compare the control level or the IFN-α test level, where an increase in the test level of expression, activity or both of IFN-? in relation to the level of control it indicates that the therapeutic regimen is effective in the patient in need of the same.

71. A kit, characterized in that it comprises (a) the vector according to any of claims 1 to 31, the population of TSC immune cells according to any of claims 35 to 36, the immune cells engineered in vi tro by engineering according to any of claims 35 to 38, or the composition according to any of claims 39 to 47, and (b) a ligand that activates the gene change.

72. The kit according to claim 71, characterized in that the ligand is RG-115819, RG-115830 or RG-115932.

73. A method for increasing the expression of TNF-alpha mRNA or TNF-alpha protein expression, characterized in that it comprises (1) generating the vector according to any of claims 1 to 31, wherein the immunomodulator is TNF-alpha and wherein the polynucleotide encoding one or more proteins having the function of an immunomodulator further comprises one or more regulatory sequences, and (2) adding an activating ligand, wherein the one or more regulatory sequences comprise the expression of TNF-alpha .

74. Use of (a) a vector for conditionally expressing proteins, wherein the vector comprising a polynucleotide encoding a gene change, and (b) a therapeutically effective amount of one or more activating ligands, in the manufacture of a medicament to treat a disease or disorder in a mammal in need of the same, wherein the polynucleotide encoding a gene change comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide that codes for one or more proteins operably linked to a promoter that is activated by the ligand-dependent transcription factor, wherein the vector is not contained within a cell; Y wherein the ligand, when administered, induces the expression of the one or more proteins in the mammal and to treat the disease.

75. The use in accordance with the claim 74, wherein the disease selects from the group consisting of chronic kidney disease, osteoarthritis, oncology, viral upper respiratory infection, feline plasma cell stomatitis, feline eosinophilic granulomas, feline leukemia virus infection, canine distemper infection, fungal infections systemic diseases, cardiomyopathy, mucopolysaccharidosis VII, and infectious disease.

76. The use in accordance with the claim 75, wherein the infectious disease is selected from the group consisting of bovine respiratory disease, porcine respiratory disease, avian influenza, avian infectious bronchitis, bovine spongiform encephalopathy, canine leishmaniasis, chronic atrophy disease, classical swine fever, Echinococcus, enzootic pneumonia, FIP, foot and mouth disease, Jaagsiekte, Maedi-Visna, Mastitis in animals, Microsporum canis, Orf (animal disease), Peste des petits ruminants, smallpox disease, beak and feather disease Psittacides, rabies, Mediterranean fever (Brucellosis) or Bang disease or undulant fever, malt fever, contagious abortion, epizootic abortion, food poisoning with Salmonella, enteric paratyphosis, bacillary dysentery, Pseudotuberculosis, plague, pestilent fever, Tuberculosis, vibrio, rodeo disease, Weil's disease (Leptospirosis) or canicola fever, hemorrhagic jaundice (icterohemorrhage due to Leptospira), fever dairy worker (L. hardjo), cyclical fever, tick-borne cyclic fever, spirocetal fever, vagabond fever, hunger fever, Lyme arthritis, Bannworth's syndrome (lyme disease), tick-borne meningopolineuritis, chronic migraine erythema, Vibriosis, Colibacteriosis, colitoxemia , white diarrhea, swine intestinal edema, enteric paratyphosis, staphylococcal food toxicosis, staphylococcal gastroenteritis, Canine Crown Virus (CCV) or canine parvovirus enteritis, feline infectious peritonitis virus, transmissible gastroenteritis virus (TGE), Hagerman Redmouth disease ( ERMD), infectious hematopoietic necrosis (IHN), pleuropneumonia due to Actinobacil (Haemofilo) porcine, Hansen's disease, streptotrichosis, sheep fungal dermatitis, Pseudomormo, Whitmore's disease, Francis's disease, deer fly fever, rabbit fever, O 'Hara disease, streptobacillary fever, Haverhill fever, erythema arthritis epidemic, sodoku, shipping or transport fever, hemorrhagic septicemia, ornithosis, Cotorus fever, Chlamydiosis, American blastomycosis, Chicago disease, Gilchrist's disease, cat scratch fever, Benign lymphocytic disease, benign non-bacterial lymphadenitis, Bacillary angiomatosis, Peliosis Hepatis Bacilar, consultation fever, Balkan influenza, Balkan flu, slaughter fever, tick-borne fever, pneumoricketiasis, American tick typhus, tick-borne typhoid fever, Vesicular Ricettsiasis, Kew Gardens spotted fever, flea-transmitted typhoid fever, endemic typhoid fever, urban typhoid, ringworm, dermatophytosis, ringworm, trichophytosis, microsporosis, Jokei proritus, Athlete's foot, Sporothrix schenckii, dimorphic fungus, cryptococcosis and histoplasmosis, Smallpox simian Epidermal Benign BEMP, Herpesvirus simian disease simian B, lethargic Encephalitis Type C, yellow fever, black vomit, hantavirus pulmonary syndrome, Haemorrhagic fever Korean, nephropathy Epidemic, Epidemic Hemorrhagic Fever, Nephritis, hemorrhagic osonephritis, lymphocytic choriomeningitis, Venezuelan equine encephalitis, California encephalitis / La crosse encephalitis, African Hemorrhagic Fever, Green or Vervet Monkey Disease, Hydrophobia, Smooth, infectious hepatitis, epidemic hepatitis, epidemic jaundice, rubella, Morbilli, Cervirus and Equine, Avian Plague, Newcastle Disease, Piroplasmosis, Toxoplasmosis, African Sleep Disease, Gambian Trypanosomiasis, Rhodesian Trypanosomiasis, Chagas Disease, Chagas-Mazza Disease, South American Trypanosomiasis, Histolytic Entamoeba, Balantidial Dysentery, Cryptosporidiosis, Giardiasis, Leishmaniasis cutaneous: ulcer chiclero, espundia, pianbols, uta, and buba (in the Americas); oriental ulcer, Aleppo's boil (in the Old Continent); Ulcer of Baghdad, Ulcer of Delhi, ulcer of Bauru, Visceral leishmaniasis; kala-azar, Microsporidiosis, Anischiasis, Trichinosis, Angiostrongilosis, eosinophil meningitis or meningoencephalitis (A. cantonensis), abdominal angiostrongilosis (A. costaricensis), Uncinariasis, Necatoriasis, Hookworm disease, Capillariasis, Brugiasis, Toxocariasis, Oesophagostomiasis, Strongyloidiasis, Trichostrongylosis, Ascariasis, Difilobrosis, Sparganosis, Hydatidosis, Hydatid disease, Echinococcus granulosis, cystic hydatid disease, Oritary worm infection, Schistosoma, Burkitt's lymphoma caused by EBV, Rous sarcoma caused by Rous retrovirus, Kaposi's sarcoma caused by herpes virus type 8, leukemia of adult T cells caused by HTLV-I retrovirus, and hairy cell leukemia caused by HTLV-II.

77. The use according to claim 75, wherein the infectious disease is selected from the group consisting of bovine respiratory disease, porcine respiratory disease, and Avian influenza.

78. The use according to claim 75, wherein the oncology is selected from the group consisting of osteosarcoma, leukemia, and lymphoma.

79. The use according to any of claims 74 to 78, wherein the one or more proteins is selected from the group consisting of erythropoietin, ghrelin, osteoprotegerin, RANKL, RA KL decoy, TNF-a antagonist, an IL antagonist. -1, G-CSF, GM-CSF, IFN-a, IFN- ?, angiostatin, endostatin, TNF-α, PP1DCY-LSRL0C, β-glucuronidase, and IL-12.

80. The use according to any of claims 74 to 79, wherein the one or more proteins is selected from the group consisting of IL-1, IL-2, IL-12, IL-3, IL-4, IL-5. , IL-7, IL-8, IL-9, IL-10R DN or a subunit thereof, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM- CSF, IFN-alpha, IFN-gamma, IFN-alpha 1, IFN-alpha 2, IL-15 -R-alpha, CCL3 (MIP-la), CCL5 (RA TES), .CCL7 (MCP3), XCL1 (lymphotactin ) (CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6Cinema), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, Flt3L, IFN-beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1 RNAi, an antisense oligonucleotide of PD-L1, TGFbRII DN, ICOS-L, S100, CD40L, p53, survivin, fusion of p53-survivin, MAGE3, PSA and PSMA.

81. Use of (a) a vector for conditionally expressing proteins, wherein the vector comprising a polynucleotide encoding a gene change, and (b) a therapeutically effective amount of one or more activating ligands, in the manufacture of a medicament for treat a lysosomal storage disorder in a mammal in need of the same, wherein the polynucleotide encoding a gene change comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a transcription factor dependent on a transcription factor. ligand, and (2) a polynucleotide that codes for one or more proteins operably linked to a promoter that is activated by the ligand-dependent transcription factor, wherein the vector is not contained within a cell prior to administration in vivo, - and wherein the ligand, when administered, induces expression of the one or more proteins in the mammal and treat the lysosomal storage disorder.

82. The use in accordance with the claim 81, wherein the lysosomal storage disorder is selected from the group of Pompe disease / glycogen storage disease type II, Gaucher disease (Type I, Type II, Type III), Fabry disease, Mucopolisaccaridosis II (Hunter syndrome) ), Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), Mucopolysaccharidosis I, Metachronic leukodystrophy, Neuronal ceroid lipofuscinosis or CLN6 disease (Late childhood Atypical CLN5 disease, late onset, early juvenile, late childhood variant of the Filandan variant, late childhood CLN2 / TPP1 disease / Jansky / Buelschowsky disease, NCL / CLN4 disease of onset adults / of Kufs, late childhood variant CLN8 disease / Northern epilepsy, CLNI / PPT infant / Santavuori-Haltia disease, Beta-mannosidosis), NCL / CLN3 Juvenile / Batten-Spielmeyer-Vogt disease, Sanfilippo syndrome type A, Sanfilippo syndrome type B, Sanfilippo syndrome type C, Sanfilippo syndrome type D, MPSI Hurler syndrome, Niemann-Pick disease (Type A, Type B, Type C, Type D), Deficiency of Activators / Gangliosidosis of GM2, Alpha-Mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon's disease, Farber's disease, Fucosidosis , Galactosialidosis (Goldberg syndrome), GM1 (Infantile, late childhood / juvenile, Adult / Chronic) gangliosidosis, I / Mucolipidosis II cell disease, Childhood Free Sialic Acid Storage Disease / ISSD, Youth Hexosaminidase A Deficiency, Krabbe (Early Childhood, Late Start), Mucopolisacharidosis Disorders (Pseudo-Hurler / Mucolipidosis IIIA Polydystrophy, Scheie Syndrome, MPS Syndrome I Hurler-Scheie, Morquio Type A / MPS IV A, Morquio Type B / MPS IVB, Deficiency of MPS IX Hyaluronidase, Sly Syndrome (MPS VII), Mucolipidosis I / Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV), multiple deficiency of sulfatase, Picnodisostosis, Sandhoff disease / adult onset / GM2 gangliosidosis, Sandhoff disease / GM2 gangliosidosis, Infant, Sandhoff disease / GM2 gangliosidosis, Juvenile, Schindler's disease, Salla's disease, Sialic acid storage disease Infant, Tay-Sachs / GM2 gangliosidosis, Wolman's disease, Asparilglucosaminuria, and prosaposin.

83. The use according to claim 81 or claim 82, wherein the lysosomal storage disorder is selected from the group consisting of Pompe disease / glycogen storage disease type II, Gaucher disease (Type I, Type II, Type III), Fabry disease, Mucopolysaccharidosis II (Hunter syndrome), Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), Mucopolysaccharidosis I, and Metachromatic leukodystrophy.

84. The use according to any of claims 81 to 83, wherein the one or more proteins is selected from the group consisting of a-galactosidase A, arylsulfatase A, a-glucosidase, b-glucosidase, glucocerebrosidase, protein CLN6, associated juvenile with CLN3, N-sulfoglucosamine-sulfohirolase (SGSH), a-Nacetylglucosaminidase, acetyl-CoA-glucosaminide-acetyltransferase, N-acetylglucosaraine-6-sulphatase, aL-iduronidase, arylsulfatase B, acid sphingoraleinase, and iuduronate-sulphatase.

85. Use of (a) a vector for conditionally expressing proteins, wherein the vector comprising a polynucleotide encoding a gene change, and (b) a therapeutically effective amount of one or more activating ligands, in the manufacture of a medicament to treat a liver disease in a mammal in need of the same, wherein the polynucleotide encoding a gene change comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide that codes for one or more proteins operably linked to a promoter that is activated by the ligand-dependent transcription factor, wherein the vector is not contained within a cell prior to in vivo administration; Y wherein the ligand, when administered, induces the expression of the one or more proteins in the mammal and treats the liver disease.

86. The method according to claim 85, characterized in that the liver disease is Hepatitis B.

87. The method according to claim 85, characterized in that the liver disease is Hepatitis C.

88. The method according to any of claims 85 to 87, characterized in that the protein is by IFN-OI.

89. The method according to any of claims 85 to 87, characterized in that the protein is ceruloplasmin.

90. Use of (a) a vector for conditionally expressing proteins, wherein the vector comprising a polynucleotide encoding a gene change, and (b) a therapeutically effective amount of one or more activating ligands, in the manufacture of a medicament to treat an eye disease in a mammal in need of the same, wherein the polynucleotide encoding a gene change comprises (1) at least one transcription factor sequence that is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide that codes for one or more proteins operably linked to a promoter that is activated by the ligand-dependent transcription factor, wherein the vector is not contained within a cell prior to in vivo administration; Y wherein the ligand, when administered, induces the expression of the one or more proteins in the mammal and treats ocular disease.

91. The use according to claim 90, wherein the ocular disease is selected from the group consisting of: glaucoma, Open Angle Glaucoma, Angle Closed Glaucoma, Aniric Glaucoma, Congenital Glaucoma, Juvenile Glaucoma, Glaucoma Induced by Lenses, Glaucoma Neovascular, Post-Traumatic Glaucoma, Steroid-induced Glaucoma, Glaucoma of Sturge-Weber Syndrome, and Glaucoma Induced by Uveitis, Diabetic Retinopathy, Macular Degeneration, Macular Degeneration, Choroidal Neovascularization, Vascular Leakage and / or Retinal Edema, Bacterial Conjunctivitis, Fungal Conjunctivitis, Viral Conjunctivitis, Uveitis, Keratotic Precipitates, Edema macular, inflammatory response after intraocular lens implantation, uveitis syndrome, retinal vasculitis, sarcoidosis, Eales disease, acute retinal necrosis, Vogt Koyanaki Harada syndrome, ocular toxoplasmosis, radiation retinopathy, proliferative vitreoretinopathy, endophthalmitis, ocular glaucoma, neuropathy ischemic optics, thyroid-associated orbitopathy, orbital pseudotumor, pigment expression syndrome (pigmentary glaucoma), scleritis, choroidopathies of episcleritis, retinopathies (eg, cystoid macular edema, central serous choroidopathy and presumed ocular histoplasmosis syndrome, vascular disease etinal, retinal artery occlusions, retinal vein occlusions, retinopathy of prematurity, retinitis pigmentosa, vitreous familial exudative retionpathy (FEVR), idiopathic polypoidal choroidal vasculopathy, epiretinal macular membranes and cataracts.

92. The use according to claim 90 or claim 91, wherein the vector is administered intravitreally.

93. The use according to any of claims 74 to 92, wherein the mammal is a human or a non-human animal.

94. The use according to any of claims 74 to 93, wherein the vector is selected from the group consisting of plasmid, adenovirus, retrovirus, adeno-associated virus, pox virus, baculovirus, vaccinia virus, herpes simplex virus, Epstein-Barr virus, adenovirus, geminivirus, caulimovirus, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

95. The use according to any of claims 74 to 94, wherein the vector is an adenoviral vector.

96. The use according to any of claims 74 to 95, wherein the gene change is a gene change based on EcR.

97. The use according to any of claims 74 to 96, wherein the polynucleotide encoding a gene change comprises a first transcription factor sequence under the control of a promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by the first transcription factor sequence and the second transcription factor sequence interact to form a protein complex that functions as a ligand-dependent transcription factor.

98. The use according to any of claims 74 to 97, wherein the polynucleotide encoding a gene change comprises a first transcription factor sequence and a second transcription factor sequence under the control of a promoter, wherein the proteins encoded by the first sequence or transcription factor and the second sequence of transcription factor interact to form a protein complex that functions as a ligand-dependent transcription factor.

99. The use according to claim 98, wherein the first transcription factor sequence and the second sequence of the transcription factor are connected by an internal ribosomal entry site of EMCV (IRES).

100. The use according to any of claims 74 to 99, wherein the ligand is a diacylhydrazine.

101. The use according to any of claims 74 to 99, wherein the ligand is selected from RG-115819, RG-115932, and RG-115830.

102. The use according to any of claims 74 to 99, wherein the ligand is an amidoketone or oxadiazoline.