MXPA00004293A - Hyperthermic inducible expression vectors for gene therapy and methods of use thereof - Google Patents

Abstract

Methods and compositions are provided for transgene expression in target cells. Expression constructs using an inducible amplification system to drive expression of a therapeutic gene or other gene of interest in mammalian host cells are provided, as well as methods therefor. Inducible expression of the transgenes at high levels under physiologic conditions results from induction by hyperthermic conditions relative to the basal temperature of the host cells.

Description

EXPRESSION VECTORS INDUCI BLES HI PERTERMICOS FOR GEN THERAPY AND METHODS OF USE THEREOF BACKGROUND OF THE INVENTION (< 5 1. Field of the invention The present invention relates generally to the field for gene therapy, More particularly, it concerns methods and compositions for increasing the expression of transgenes. of the Related Art It is now thought that gene therapy is broadly applicable to the treatment of a variety of cancers and a variety of other diseases Viral vectors are a method employed as a gene delivery system. A great variety of viral expression systems has been developed and it has been evaluated according to its ability to transfer genes to somatic cells. In particular, adenovirus or retrovirus-based vector systems have been extensively investigated for a decade. Recently, the associated adenoviral virus (AAV) has emerged as a potential alternative to the most commonly used retroviral and adenoviral vectors. Lipid vectors including cationic lipids and liposomes are also used to deliver plasmid DNA containing therapeutic genes. The therapeutic treatment of diseases and disorders by therapy of genes involves the transfer and stable or transient insertion of new genetic information into cells. The correction of a genetic defect by re-introducing the normal allele of a gene encoding the desired function has shown that this concept is clinically feasible (Rosenberg et al., New Eng. J. Med., 323: 570 (1990). ). In ("5 reality, preclinical and clinical studies covering a wide range of genetic disorders, are currently under way to solve basic questions dealing with the efficiency of gene transfer, regulation of gene expression and potential risks of the use of viral vectors. The majority of gene transfer assays clinical studies using viral vectors perform the transfer of ex vivo genes to target cells, which are then administered in vivo. Viral vectors can also be provided in vivo, but repeated administration can induce neutralizing antibodies. A major issue facing the potential clinical application of gene therapy is the question of how to express heterologous genes in clinically significant amounts in selected tissues of the subject. The regulatory elements of genes, such as promoters and enhancers, have specific activities of cell type and can be activated by certain induction factors via elements of response. The use of such regulatory elements as promoters to drive gene expression facilitates the controlled and restricted expression of heterologous genes in vector constructs. For example, heat shock promoters can be used to handle the expression of a heterologous gene following thermal shock.

US Patents Nos. 5,614, 381, 5,646,010 and WO 89/00603, refer to managing the expression of transgenes using thermal shock at temperatures greater than 42 ° C. These temperatures are not practicable in human therapy, since they can not be maintained for a sustained period without harm to the individual. Gene therapy could be used in combination with a variety of conventional cancer therapy treatments including cytotoxic drugs and radiation therapies. It has been shown that hyperthermia intensifies the killing effect of radiation cells in vitro (Harisiadis et al., Cancer, 41: 21 31 -2142 (1 978)), significantly enhances the tumor response in animal tumors in vivo and improves the result in randomized clinical trials. However, the main problem with the use of hyperthermia treatment is that the hyperthermia system can not adequately warm large and deep tumors. Thus, it would be useful to develop vectors that can be used at temperatures of 42 ° C and below, systemically or locally, to treat a patient, so that the expression of the therapeutic gene (s) is preferentially activated in regions of the body that have have been subjected to conditions that induce such expression.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods for effecting the inducible expression of polynucleotides in cells. In particular, the use of heat shock promoters is taught in methods for effecting inducible expression of polynucleotides in mammalian cells. The present invention overcomes deficiencies in the prior art by providing heat shock controlled vectors that can be used at temperatures of 42 ° C and below. These methods can be used to treat a patient via the inducible expression of a therapeutic gene. In one embodiment, the present invention provides a method for effecting the expression of transgenes in a mammalian cell comprising first providing an expression construct comprising both (i) an inducible promoter operably linked to a gene encoding a transactivating factor, and (ii) a second promoter operably linked to a selected polynucleotide. The second promoter is activated by the transactivating factor expressed by the same construct. The method then includes the step of introducing the expression construct into the cell. Finally, the cell is subjected to conditions that activate the inducible promoter and result in the expression of the selected polynucleotide. In a preferred embodiment of the invention, the inducible promoter is a heat shock promoter and the conditions that activate the heat shock promoter are hyperthermic conditions. The hyperthermic conditions can comprise a temperature between about the basal temperature and about 42 ° C. As used herein, the basal temperature of the cell is defined as the temperature at which the cell is normally in its natural state, for example, a cell in the skin of a mammal can be at temperatures as low as 33 ° C. ° C, while a cell in the liver of an organism can be as high as 39 ° C. In specific modalities, the application of hyperthermia involves raising the temperature of the cell from the basal temperature, very normally 37 ° C to (<5 about 42 ° C or less.Alternatively, hyperthermic conditions can vary from about 38 ° C to about 41 ° C, or from about 39 ° C to about 40 ° C. The heat shock promoter is derived optionally from a promoter selected from the group of promoters heat shock protein (HSP) HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25 and HSP28. The ubiquitin promoter can also be used as the inducible heat shock promoter in the expression construct. A minimum thermal shock promoter derived from HSP70 and comprising the first approximately 400 bp of promobor HSP70B can optionally be used in the invention. In an alternative embodiment, the inducible promoter comprises a hypoxia response element (HRE). This hypoxia response element may optionally contain at least one binding site for hypoxia-inducible factor-1 (Hl F-1). In one embodiment of the invention, the second promoter can be selected from the group consisting of a human immunodeficiency virus 1 (HIV-1) promoter and a human immunodeficiency virus 2 (HIV-2) promoter. In preferred embodiments, the transactivating factor can be a transcription activator (TAT).

The selected polynucleotide can encode a protein or a polypeptide. For example, the polynucleotide can encode any of the following proteins: antidote protein ornithine decarboxylase, p53, pl6, neu, interleukin-1 (IL1), interleukin (IL2), interleukin-4 (IL4), ("5 interleukin-7 (I L7), interleukin-12 (IL1 2), interleukin-1 5 (IL1 5), ligand FLT-3, granulocyte-macrophage stimulating factor (GM-CSF), granulocyte-colony stimulating factor ( G-CSF), gamma-interferon (IFN?), Alpha-interferon (IFNa), tumor necrosis factor (TNF), herpes simplex virus thymidine kinase (HSV-TK) I-CAM 1, human leukocyte antigen-B7 (HLA-10 B7), or meloproteinase tissue inhibitor (TI MP-3). In such embodiment, the selected polynucleotide is placed in a sense orientation with respect to the second promoter. Alternatively, the expression of the selected polynucleotide may involve transcription but not translation, and produces a ribozyme. In this embodiment the selected polynucleotide is also placed in a sense orientation with respect to the second promoter. In yet another alternative embodiment, the expression of the selected polynucleotide involves transcription but not translation, and results in an RNA molecule, which serves as an antisense nucleic acid. In such an embodiment, the selected polynucleotide may be the target gene, or a fragment thereof, which is placed in the expression construct in an antisense orientation with respect to said second promoter. The expression construct may further comprise a gene encoding a selectable marker, such as hygromycin resistance, neomycin resistance, puromycin resistance, zeocin, gpt, DHFR, green fluorescent protein or histadinol. Alternatively, the expression construct may further comprise (i) a second selected polynucleotide, which is operably linked to said second promoter, and (ii) an internal ribosome entry site positioned between said first and second selected polynucleotides. The cell can be a tumor cell, a cell located inside a tumor, or a cell located inside a mammal. The introduction of the construction of expression in the cell can occur in vitro or in vivo. In one embodiment, the introduction of the expression construct into the cell is mediated by a delivery vehicle selected from the group consisting of liposomes, retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, simplex fish viruses and vaccinia viruses. In another embodiment of the invention, there is provided a method for providing a subject with a therapeutically effective amount of a product of a selected gene. This method involves providing a first expression construct which comprises an inducible promoter operably linked to a gene encoding a transactivating factor and providing a second expression construct which comprises a second promoter operably linked to a selected polynucleotide, where the second promoter it is activated by the transactivating factor encoded by the first expression construct. The first and second expression constructs are introduced into the desired cell of said subject and that cell is subjected to conditions that activate the inducible promoter, so that the expression of the selected polynucleotide is induced. In a preferred embodiment the first and second expression constructs are present in the same vector. In addition, the inducible promoter is preferably a heat shock promoter and the activating conditions comprise a temperature below 42 ° C and above about the basal temperature. The introduction of one or both expression constructs can be performed either in vivo or ex vivo. The expression product of the selected polynucleotide may optionally be harmful to a pathogen in the subject, such as a virus, bacterium, fungus or parasite. Alternatively, the expression product of the selected polynucleotide can inhibit the growth of the subject's cell. In yet another alternative embodiment of the invention, the expression product of the selected polynucleotide replaces a protein deficient in the subject. Alternatively, the expression product of the selected polynucleotide can promote nerve regeneration. In additional embodiments, a method for treating cancer in a mammal is provided., such as a human, comprising the steps of (a) providing an expression construct comprising (i) an inducible promoter, preferably a heat shock promoter, which is operably linked to a gene encoding a transactivating factor; and (ii) a second promoter operably linked to a selected polynucleotide, wherein the second promoter is activated by the transactivating factor; (b) introducing said expression construct into a tumor cell; and (c) subjecting the tumor cell to conditions that activate the inducible promoter, such that the selected polynucleotide is expressed in sufficiently high amounts to inhibit the growth of the tumor cells. If the inducible promoter is a heat shock promoter, the activating conditions comprise a temperature below about 42 ° C and above about the basal temperature. This method further comprises treating said tumor cell with an established form of cancer therapy, which is selected from the group consisting of external beam radiation therapy, brachytherapy, chemotherapy and surgery. The cancer can optionally be selected from the group of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testis, ovary, vulva, cervix cancer. , skin, head and neck, esophagus, bone marrow and blood. In a particular embodiment of the invention, the selected polynucleotide is anti-gamma ornithine decarboxylase protein. After the cell is subjected to conditions that activate the inductible promoter of the expression construct in the tumor cell, the tumor is treated with the protective radio WR-33278 or WR-1065. Finally, the tumor cell is treated with radiation therapy. Methods for eliciting an immune response in a mammal, such as a human, are also provided by the present invention. The provoked immune response can constitute either an imm ne humoral response or a cellular immune response. In one embodiment, the method comprises (a) providing an expression construct comprising (i) an inducible promoter, preferably a heat shock promoter, which is operably linked to a gene encoding a transactivating factor; and (ii) a second promoter operably linked to a selected polynucleotide, wherein the second promoter is activated by the transactivating factor (b) introducing said expression construct into a cell in the mammal; and (c) subjecting the cell to conditions that activate the inducible promoter, such that the selected polynucleotide is expressed with sufficient intensity to elicit an immune response in the mammal. If the inducible promoter is a heat shock promoter, the activating conditions comprise a temperature below about 42 ° C and above about the basal temperature. In one embodiment, the immune response that is elicited is directed against the cell in the mammal, which contains the expression construct. The method may also optionally involve treating the cell with an established form of cancer therapy selected from the group consisting of chemotherapy, external-beam radiation therapy, brachytherapy, and surgery. In another embodiment, an expression construct comprising (a) a gene encoding a transactivating factor is provided; (b) an inducible promoter operably linked to the gene; (c) a selected polynucleotide; (d) a second promoter that is operably linked to the selected polynucleotide. The second promoter of the construction is activated by the transactivating factor. In a preferred embodiment, the inducible promoter is a heat shock promoter and the expression of the selected polynucleotide can be induced by hyperthermic conditions comprising a temperature below about 42 ° C and above about 37 ° C. In an alternative modality, the inducible promoter of the expression construct may comprise a hypoxia response element. The expression construction can also comprise a second. selected polynucleotide, which is also operably linked to the second promoter and is separated by the first polynucleotide selected by I RES. A cell comprising the expression construct is also provided. The provided expression construct can also be optionally selected in a method for altering the genetic material of a mammal. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided by way of illustration only, because various changes and modifications within the spirit and scope of the invention are will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention will be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG. 1 shows the basic vector used to quantify the thermal shock promoter activity. The plasmid contains a • 7 * 5 minimal promoter derived from the HSP70B promoter (StressGen). A reporter gene, such as Enhanced Green Fluorescence Protein (EGFP), ß-gal, or IL-2 is easily inserted into the multiple cloning site (MCS), so that it is expressed under the control of the minimal HSP70B promoter. . The plasmid also contains the resistance genes neomycin and ampicillin for selection capacity in mammalian cells, as well as the standard elements for growth in a bacterial system. Plasmid S8 comprises the plasmid shown with EGFP inserted in the multiple cloning site. FIG. 2 shows histograms of activated cell sorting with fluorescence (FACS) for DU-145 cells transfected stably with the S8 plasmid. The fluorescence increases from left to right. The upper histogram is of transfected DU-145 cells that have not been subjected to thermal shock. The lower histogram is * transfected DU-145 cells, which have been subjected to a shock thermal at 42 ° C for 1 hour. FIG. 3 shows FACS histograms for three different populations of S8-transfected MCF7 cells. The MCF7 cells, transfected with the S8 construct, were classified by FACS. The original population came from a pollonal line of selected cells.

This activated cell line population (ie, cells expressing EGFP) was separated from the non-activated population. After sorting, the positive population was grown and then reclassified to obtain a purer positive cell line. In this case, the polyclonal MCF7-S8-PS cells were classified twice, producing a highly positive population MCF7-S8-PS2. FIG. 4 shows the expression of EGFP in different cell lines tested by FACS. Cell lines were transfected with plasmid S8. The cells were then cloned or a polyclonal line was grown. In some cases, the cell lines were classified for EGFP expression by FACS. The total average fluorescence was quantified and graphed. FIG. 5 shows the expression of EGFP in stably transfected DU-145 cells, which had been classified twice (DU-S8-PS2) following the thermal shock. The DU-S8-PS2 cells were either heated to 40 ° C or 42 ° C and allowed to recover several times. The cells were then analyzed by FACS. FIG. 6 shows levels of EGFP expression in DU-145 cells stably transfected 16 hours after exposure to heat stress. A population of cells (DU-S8-PS2) was stably transfected with plasmid S8. Another population (DU-V9-PS2) was stably transfected with plasmid V9, a plasmid identical to S8, except that the EGFP of plasmid V9 is operably linked to a CMV promoter, instead of HSP70B (see FIG.7). The cells were heated at various temperatures and allowed to recover for 16 hours. Untransfected DU-145 cells were included as a control.

FIG. 7 shows a schematic diagram of plasmid V9, which contains a CMV promoter that is operably linked to the gene encoding the Enhanced Green Fluorescence Protein I (EGFP). FIG. 8 shows the basic vector design for a vector 5 containing a second promoter, which allows the amplification of the thermal shock response. The plasmid contains a multiple cloning site (MCS) operably linked to an HSP70B promoter, but also contains a therapeutic gene operably linked to a second promoter. The plasmid also contains the resistance gene neomycin, the ampicillin resistance gene and standard elements for growth in bacteria. In plasmid pC8, the second promoter is the long terminal repeat (LTR) of H IV-1 (LTR) and the therapeutic gene is I L2. In mp 1 2, tat is inserted into the MCS, the second promoter is the LTR of H IV-1 and the therapeutic gene is I L2. Another plasmid, p007, is same as pf 1 2, except that LTR of H IV-2 is used as the second promoter. FIG. 9 shows amplifier constructions containing the therapeutic gene I L-2 driven either by the HIV-1 or HIV-2 promoter. The amplifier part is controlled by either the CMV or HSP promoter 70 driving the expression of TAT. The plasmids also contain the gene for resistance to neomycin and elements for growth in bacteria. These constructs were used in the amplifying studies of Examples 2 and 3. FIG. 9A shows a plasmid designated X1 4 containing an expression cartridge CMV-TAT-H IV-1-I L2; FI G. 9B shows a plasmid designated Y 1 5 containing an expression cartridge CMV-TAT-H IV-2-I L2; FIG. 9C shows a plasmid designated pfl2 containing an expression cartridge HSP-TAT-HIV-1-I L2; and FIG. 9D shows a plasmid designated p007 containing an HASP-TATHIV-2-I L2 exprssion cartridge. (• FIG.1.0 shows the DNA sequence of the BamH 1-HindIII fragment of p173OR from StressGen Biotechnology Corp. This fragment contains the minimum HSP70B promoter fragment of approximately 0.4 kb used in constructions of the specific examples, Example 1 and 3, below.10 DESCRIPTION OF ILLUSTRATIVE MODALITIES 1. The present invention Gene therapy faces two main technical problems: as both regulate and intensify the expression of therapeutic genes in vivo. The present invention solves both issues by combining hyperthermia treatment with constructs of inducible expression. The inventors have demonstrated increases in the efficiency of inducible and specific gene expression. 20 The ability to express therapeutic gene (s) at very high levels and the ability to control expression levels are important targets in the development of gene therapy. The inventors have created new sets of expression vectors to solve these objectives. The inventors use an amplifier strategy to handle the expression of the or genes of interest. Amplifiers consist of the expression of human HSP70B promoter-driven proteins that are transcriptional activators of other promoters, which, in turn, drive reporter genes. These additional promoters and their operably linked reporter genes are *. '5 included, preferably, in the same vector with the HSP70B promoter element and the gene encoding the transactivating protein. In studies of transfection of mammalian cells using human IL-2 as the reporter gene, the inventors have shown that gene expression was dramatically increased using their amplifier constructions for all temperature conditions used, compared to reporter gene expression produced by the constitutive CMV promoter or by HSP70B alone (see specific example, Example 3, below). The constructs containing both the HSP70B promoter, upstream of the virus gene of Human immunodeficiency (HIV) such as the long terminal repeats of H IV1 or H IV2, upstream of the interleukin-2 gene (I L-2), exhibited promoter activity at 37 ° C, which was further amplified by heat shock . The co-transfection experiments indicated that the activities of the constructions of The expression of HSP promoter, HSP / HIV1 and HSP / H IV2 was 0.4, 6.9 and 83.3 times respectively, that of the construction of CMV promoter expression in mammalian cells. These data indicate that, although less active than the CMV promoter per se, this minimal heat shock promoter can be used in conjunction with a second promoter to noticeably amplify gene expression, while still retaining some dependence. Of temperature. Previous studies have examined the use of the heat shock promoter to handle the expression of transactivating proteins to conditionally express other promoters (Schweinfest et al, Gene, _ 71 (1): 207-21 0, 1 988; EPO 01 1 8393; WO 89/00603, U.S. Patent No. 5,614,381; US patent no. 5,646,010; EO 0 299 1 27). The inventions described herein differ from these prior approaches, for example, by the use of 1) different heat shock promoters, (Schweinfest et al., Uses Drosophila promoters), 2) different modes of delivery (the present inventors have both promoters incorporated in a simple construction - while others have used 'co-transfection'), 3) different temperatures for induction (the previous work used temperatures greater than 42CC, while the present invention advantageously operates at temperatures of 42 ° C and minors); and 4) use in context of gene therapy instead of industrial production. Additionally, the present inventors are able to use either promoters of H IV-1 or H IV-2 and the present invention shows a clear distinction in the expression levels resulting from these two promoters. In a preferred aspect of the present invention, methods are provided for effecting the expression of transgenes in a mammalian cell by using an inducible thermal shock element. The heat shock sequence is used to handle the expression of a transactivating gsne. Thus, when the expression construct is subjected to hyperthermia, the expression of the transactivating element is induced. The transactivating gene acts on a second promoter, which is activated to handle the expression of the therapeutic gene of interest. In a particular embodiment, a promoter derived from the HSP70 promoter is used. A particularly useful aspect of this promoter is that it has a low basal level of expression at ambient temperatures and is inducible. The present invention further provides methods for providing a subject with a therapeutically effective amount of a gene product and for inhibiting the growth of a cell or eliciting an immune response. The compositions and methods employed in order to fulfill the objectives of the present invention are discussed in more detail below. 2. Thermal shock response Thermal shock or stress response is a universal response that occurs in organisms that vary from plants to primates. It is a response that can be caused as a result not only of thermal shock, but also as a result of a variety of other stresses, including ischemia, anoxia, glucose deprivation, amio acid analogues and ionophore glucose, ethanol, metals from a series of transition, drugs, hormones and bacterial and viral infections. Additionally, there is evidence that over-expression of heat shock protein genes may be associated with enhanced proliferation and tumor cell stress (Finch et al., Cell Growth and Differentiation 3 ( 5): 269-278, 1992).

This response is characterized by the synthesis of a family of well-conserved proteins of varying molecular sizes that are induced and localized differently. These proteins are among the most phylogenetically conserved and are characterized according to their weights. Transcription activation of genes encoding stress proteins occurs in a matter of minutes in response to environmental and / or physiological trauma. This rapid response has been attributed to the lack of introns in the vast majority of heat shock proteins. This absence of introns allows the heat shock proteins to circumvent a block in introns processing that occurs at high temperature. Thus, the heat shock protein is transferred with very high efficiency, often at the expense of other proteins. Activation of the tension genes is mediated by the conversion of a pre-existing heat shock transcription factor (HSF) from an inactive to an active form. There is a large difference in the molecular weight of this DNA binding protein (e.g., 83 kDa in humans and 1 50 kDa in yeast). The thermal shock element is a upstream conserved regulatory sequence of HSP70, which binds to HSF. Although the main function of heat shock proteins is to facilitate the folding of proteins and prevent aggregation, it is evident that these proteins play some role in providing an organism with a protective mechanism against environmental aggression and aiding the subsequent recovery from trauma.

Like most eukaryotic sequence-specific transcription factors, HSF acts through a highly conserved response element found in multiple copies upstream of the heat shock gene. The thermal shock response element is composed of three contiguous inverted repeats of a 5-base pair sequence, whose consensus was defined as nGAAn and more recently was defined as AGAAn. The regulation of HSF comprises mainly a change in activity instead of an alteration in synthesis or stability. 3. Hyperthermia therapy Many clinical studies have shown the effectiveness of hyperthermia as an adjunctive treatment for malignancies, when used in combination with radiotherapy or chemotherapy (Hahn, GM, Hyperthermia and Cancer), 2nd ed., New York, Plenum, 1982; Scott et al., Int. J. Rad. Oc. Biol. Phys. 10 (11) 221 9-2123, 1884; Lindholm et al., Rec. Res. In Cancer Res. 07: 1 52-1 56, 1 988. The reason for the application of heat, indication and contraindications, is developed based on experimental evidence that desirable physiological responses can be produced through the use of heat and based on controlled clinical studies Lehman provides an extensive treatise for the therapeutic use of heat in other applications (Therapeutic Heat and Cold, Rehabilitation Medicine Library, published by Williams &Wilkins, 1990, incorporated by reference), the reader is referred in particular to chapter 9, which discusses the use of heat in the context of therapeutic interventions, both medical and surgical. "Hyperthermia" is intended to refer to a temperature condition that is higher than the ambient temperature of the subject to which the treatment is being administered. Hence, a hyperthermal temperature, as used herein, will normally vary from about 37 ° C to about 42 ° C. In preferred embodiments, the temperature will vary from about 38 ° C to about 42 ° C, in other embodiments, the temperature range will be from about 39 ° C to about 41 ° C, in other embodiments, the temperature will be about 40 ° C. With the devices currently available for the application of hyperthermia in auxiliary therapies, it is possible to maintain the temperature of hyperthermia treatment up to approximately 0.5 ° C for temperatures up to 42 ° C. From there, the therapeutic treatments of the present invention can be performed at 37.0 ° C, 37.2 ° C, 37.4 ° C, 37.6 ° C, 37.8 ° C, 38.2 ° C, 38.4 ° C, 38.6 ° C, 38.8 ° C , 39.2 ° C, 39.4 ° C, 39.6 ° C, 39.8 ° C, 40.2 ° C, 40.4 ° C, 40.6 ° C, 40.8 ° C, 41.2 ° C, 41.4 ° C, 41.6 ° C , 41.8 ° C or 42.0 ° C. Prior to the present invention, the efficacy of hyperthermia required that temperatures within a tumor (s) remain above about 43 ° C for 30 to 60 min, although the limit temperatures for safety considerations in normal tissues are below 42 ° C. Achieving uniform temperatures above 42 ° C in your home is very difficult and often is not possible.

The tissues in mammals can be heated using a variety of technologies including, ultrasound, electromagnetic techniques, including either propagated (for example, microwave), resistive (for example, radio frequency) or inductive (radiofrequency or magnetic (Hahn, GM, Hyperthermia and Cancer, 2nd ed. New York, Plenum, 1982; Lehman LB, Postgard Med., 88 (3): 240-243, 1990, both incorporated herein by reference.) In some simple applications, temperatures US Pat. No. 4,230, 129 to Le Veen, incorporated herein by reference, refers to a method for heating body tissue and monitoring changes in temperature in the body. Tumor in real time with the help of a scintillation detector.The method provides the coupling of radiofrequency (RF) energy to the patient's body to avoid any absorption significant heat in the greasy tissues. This is obtained by focusing the RF energy on the tumor with an orbital movement of the applicator, so that the energy is not being constantly applied to the same confined area within the patient's body. U.S. Patent No. 3,991, 770 for Le Veen, also incorporated herein by reference, shows a method for treating a tumor in a human by placing the part of the human body containing the tumor in a radiofrequency electromagnetic field to heat the tumor tissue and cause necrosis of the tumor without damaging the adjacent normal tissue.

In preferred embodiments of the present invention, hyperthermia is applied in combination with the gene therapy vectors described herein to achieve inducible gene expression at a particular tumor site. Additionally, the hyperthermia / gene therapy treatment regimens may be used in combination with other conventional therapies, such as, the chemotherapies and radiotherapies discussed below, to effectively treat cancer. Other methods for inducing hyperthermia in the art are also known. Methods and devices for the regional and / or systemic application of hyperthermia are well known to those of skill in the art and are described, for example, in US Pat. 5284, 144; 4,230, 1 29; 4, 1 86,729; 4, 346.71 6; 4,848, 362; 4.81 5.479; 4,632, 1 28, all incorporated herein by reference. 4. Engineering Expression Constructs In certain embodiments, the present invention involves the manipulation of genetic material to produce expression constructs encoding therapeutic genes. Such methods involve the use of an expression construct containing, for example, a heterologous DNA encoding a gene of interest and a means for its expression, replicating the vector in an appropriate helper cell, obtaining viral particles produced therefrom, and infecting cells. with the particles of recombinant virus. The gene will be a therapeutic gene, for example, to treat cancer cells, to express immunomodulatory genes to fight viral infections, or to replace a gene function as a result of a genetic defect. In the context of the gene therapy vector, the gene will be a heterologous DNA, which means that it includes DNA derived from a different source of the viral genome, which provides the backbone of the vector. Finally, the virus can act as a live viral vaccine and express an antigen of interest for the production of antibodies against it. The gene can be derived from a prokaryotic or eukaryotic source, such as a bacterium, a virus, a yeast, a parasite, a plant or an animal. The heterologous DNA can also be derived from more than one source, i.e., a multigene construct or a fusion protein. The heterologous DNA may also include a regulatory sequence, which may be derived from a source and the gene from a different source. a) Therapeutic genes The polynucleotide of the present invention can optionally be a therapeutic gene. Any of a wide variety of therapeutic genes are suitable for use in the vectors and methods described herein. The therapeutic genes, which are suitable for the application of the present invention to a particular disorder, medical condition or disease, will be discernable to one skilled in the art. In one embodiment of the invention, the selected polynucleotide is the gene encoding the antitry protein ornithine decarboxylase. Antithymic protein ornithine decarboxylase (ODC) is an important component of feedback regulation of intracellular polyamine deposit sizes (Hayashi et al., Trends in Biochemical Sciences 21 (1): 27-30, 1996, incorporated herein by reference). The levels of this protein are directly related to the levels of intracellular polyamines, which stimulate the translation of the antizyme message. The anti-gamma protein focuses on ornithine decarboxylase, the first and often the speed-limiting enzyme in the synthesis of polyamine, for degradation. This protein also suppresses polyamine uptake. Thus, the low levels of endogenous polyamines leads to low levels of antisense, which in turn maximize the synthesis of polyamine via ODC and polyamine uptake. Conversely, high levels of endogenous polyamines cause high levels of antisense protein, which in turn minimize the synthesis of polyamine via ODC and suppress the uptake of polyamyria. The radioprotective WR-33278 (N, N "- (dithiodi-2, 1-ethanediyl) bis-1,3-propanediamine) is a polyamine analog containing disulfide, which is taken up by cells using the polyamine transporter (Mitchell et al. al., Carcinogenesis, 16: 3063-3068, 1995, incorporated herein by reference.) This carrier is inhibited by antisense.The evidence from animal models indicates that this radioprotector is taken up by at least some normal tissues to a greater degree. that some tumors (Ito et al., International Journal of Radiation Oncology, Biology, Physics 28: 899-903, 1994) Agents such as WR-33278 have been used in clinical radiotherapy in attempts to protect normal tissue dose-limiting toxicity. ad, if n reduce the effectiveness of radiotherapy tumor control (Spencer and Goa, Drugs, 50 (6): 1 001-31, 1995, incorporated herein by reference.) The reason for the difference in WR uptake -33278 it may be that tumor cells proliferant is frequently contain higher levels of polyamines than non-proliferating cells in normal tissues. Thus, the tumors would express higher levels of antisense than * «5 normal tissues. The inventors have placed an antisense cDNA lacking the necessary sequences for polyamine-dependent regulation under the control of the human 70B heat shock promoter. The inventors have stably transfected DU-145 cells derived from cancer of human prostate with this construction and have selected clones that show the heat-inducible suppression of polyamine uptake (indicating the activity of heat inducible antisense). The therapeutic application of this gene therapy (HSP70B promoter regulation of antizyme expression) will be put to use in future clinical trials in men with localized prostate cancer. Patients are treated with this gene therapy, administered intratumorally, combined with systemic WR-33278 and localized radiation therapy. Normal dose-limiting tissues adjacent to these prostate tumors will not express antisense in response to hyperthermia and will take the radioprotective WR-33278, while the tumor tissue will not take the radioprotector because it will express antizyme in response to hyperthermia. This strategy will allow to provide higher doses of radiotherapy to the prostate, with the intention of improving the local control of prostate cancer. In an alternative mode, other products can be used The metabolites of the medicament cytoprotective etiol (also known as amifostine, WR-2721 or S-2- (3-aminopropylamino) ethyl phosphorortic acid) other than WR33278 in conjunction with the expression constructs described herein. For example, WR-1065 (2- (3-aminopropylamino) ethanethiol) can be used in its place as the V 5 radioprotector. There are many other genes that can be delivered using the vectors of the present invention. For example, it is contemplated that the vectors of the present invention can be used to transfer tumor suppressors, antisense oncogens and activators of promedicamento, such as, the gene HSV-TK (Rosenfeld et al., Annals of Surgery, 225: 609-618, 1997; Esandi et al., Gene Therapy, 4: 280-287, 1997), for the treatment of Cancer. Other genes that could be optionally used in the expression constructs of the present invention include p53, p16, p21, p27, C-CAM, HLA-B7 (Gleich, et al., Arch.

Otolaryngol Head Neck Surg, 124: 1097-104, 1998; Heo et al., Hum. Gene Ther. 9: 2031-8, 1998; Nabel et al., Proc. Nat. Acad. Sciences USA, 90: 15388-15393, 1996; Stopeck et al., Journal of Clinical Oncology, 15: 341-349, 1997), IL2 (O'Malley et al., Molecular Endocrinology, 11: 667- P 673, 1997, Otova et al., Folia Biológica, 43: 25-32, 1997), IL4 (Kling, Nature Biotechnology, 15: 316-317, 1997), IL7 (Toloza et al., Annals of Surgical Oncology, 4: 70-79, 1997; Sharma et al., Cancer Gene Therapy, 3: 303-313, 1996), IL12 (Hiscox and Jiang, In Vivo, 11: 125-137, 1997; Chen et al., Journal of Immunology, 159: 351-359, 1997), GM-CSF (Kreitman and Pastan, Blood, 90: 252-259 , 1997; Homick et al., Blood, 89: 4437-4447, 1997; et al., Haematologica, 82: 239-245, 1997), IFN? (Noguchi et al., Clinical Infectious Diseases, 24: 992-994, 1997; Kanemaru et al., European Archives of Oto-Rhino-Laryngology, 254: 158-162, 1997; Tanaka et al., Journal of Gastroenterology and Hepatology. , 11: 1155-1160, 1996; Imai et al., /.Ver, 17: 88-92, 1997), I-CAM1 and TNF (Corcione et al., Annals of the fc 5 New York Academy of Sciences, 815 : 364-366, 1997). (All articles cited in this paragraph are incorporated herein by reference.) P53 is currently recognized as a tumor suppressor gene (Montenarh, Crit. Rev. Oncogen, 3: 233-256, 1992). High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation and several viruses, including SV40. The p53 gene is a frequent target of mutation inactivation in a wide variety of human tumors and it is already documented that it is the gene that most frequently undergoes mutation in common human cancers. Suffers mutation above 50% of human NSCLC and in a broad spectrum of other tumors. P16INK4 belongs to a newly described class of CDK inhibitor proteins that also includes p16B, p21WAF1 'C1P1-SDM, and p27KIP1. The p16INK4 gene maps to 9p21, a chromosomal region frequently deleted in most types of tumors. The suppressions and homologous mutations of the p16INK4 gene are frequent in cell lines of human tumors. This evidence suggests that the p16INK4 gene is a tumor suppressor gene. However, this interpretation has been challenged by the observation that the frequency of gene alterations p16INK4, is much lower in primary tumors not cultivated than in lines of cultured cells. Restoration of the wild-type p16INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cells (Okamoto, et al., Proc. Nati. Acad. Sci. USA, 91: 1 1045-1 1049, 1994, Arap, et al., Cancer Res., 55: 1351 -1354, 1995, both incorporated in the ¿. • 5 present by reference). C-CAM is expressed virtually in all epithelial cells. C-CAM, with an apparent molecular weight of 105 kD, was originally isolated from the plasma membrane of rat hepatocyte by its reaction with specific antibodies that neutralize cell aggregation.

Recent studies indicate that, structurally, C-CAM belongs to the immunoglobulin (Ig) superfamily and its sequence is highly homologous to the carcinoembryonic antigen (CEA). It has been shown that the first Ig domain of C-CAM is critical for cell adhesion activity. 15 Cell adhesion molecules, or CAMs, are known to be involved in a complex network of molecular interactions that regulate organ development and cellular differentiation (Edelman, Annu, Rev. Biochem 54: 1 35- 169, 1885). . Recent data indicate that aberrant expression of CAMs may be involved in tumorigenesis of various neoplasms; for example, the diminished expression of E-cadherin, which is expressed predominantly in epithelial cells, is associated with the progression of several kinds of neoplasms. In addition, Giancotti and Ruoslahti, Cell, 60: 849-859, 1990, incorporated herein by reference, demonstrated that the increasing expression of a5β integrin by gene transfer can reduce the tumorigenicity of Chinese hamster ovary cells in vivo. It has now been shown that C-CAM suppresses tumor growth in vitro and in vivo. Other tumor suppressors may be employed in accordance with the present invention. For example, the selected polynucleotide may be any of the following genes: retinoblastoma (Rb); adenomatous adenomatous coli (APC) gene; colorectal carcinomas with suppression (DCC); neurofibromatosis 1 (NF-1); neurofibromatosis 2 (NF-2); Wilm tumor suppressor gene (WT-1); Multiple endocrine neoplasia type 1 (MEN-1); Multiple endocrine neoplasia type 2 (MEN-2); BRCA1; von Hippel-Lindau syndrome (VHL); colorectal cancer with mutation (MCC); p1 6; p21; p57; p27; and BRCA2. In an alternative embodiment of the invention, the methods and vectors of the present invention can be used to promote regeneration processes, such as nerve regeneration, by stimulating the production of growth factors or cytokines. In such an embodiment, the selected polynucleotide may be a neurotrophic factor. For example, the selected polynucleotide may encode ciliary neurotrophic factor (CNFT), brain-derived neurotrophic factor (BDNF), or cell-derived neurotrophic factor (GDN F) (Mitsumoto et al., Science, 265: 1 1 07 -1 1 1 0, 1 994 and Gash et al., Ann. Neurol., 44 (3 Suppl 1): S 1 21 -125, 1998, both incorporated herein by reference). Alternatively, the polynucleotide selected from the expression construct can optionally encode tyrosine hydroxylase, GTP cyclohexyllase 1, or L-amino acid aromatic decarboxylase (Kang, Mov. Disord., 1 3 Suppl 1: 59-72, 1 998, incorporated in the present by reference).

In yet another embodiment, the construction of therapeutic expression may express a growth factor, such as insulin-like growth factor-1 (IGF-I) (Webster, Mult. Scler., 3: 1 1 3-1 20, 1 997, incorporated herein by reference). (5 Examples of other diseases for which the present vectors are useful include but are not limited to diseases and hyperproliferative disorders, such as, rheumatoid arthritis or restenosis by therapeutic gene transfer, eg, gene encoding angiogenesis inhibitors or inhibitors. of cell cycle 10 b) Antisense constructions Oncogenes, such as ras, myc, neu, raf, erb, src, fms, jun, trk,? ret, gsp, hst, bel and abl are also suitable targets. However, for therapeutic benefit, these oncogenes would be expressed as a antisense nucleic acid, so that it inhibits the expression of the oncogene. The term "antisense nucleic acid" is intended to refer to complementary oligonucleotides for the sequences of RNA bases and DNA coding oncogene. The antisense nucleic acid, when expressed in a target cell, specifically binds to its nucleic acid It targets and interferes with transcription, RNA processing, transport and / or translation. Focusing double-strand DNA (ds) with polynucleotides leads to the formation of the triple helix; Focusing RNA will lead to the formation of the double helix. Antisense constructions can be designed to leave the promoter and other control regions, exons, neutrons or even exon-intron limits of a gene. Antisense RNA constructs, or DNA encoding such antisense RNAs, can be employed to inhibit the transcription or translation of genes or both within a host cell, either in vitro or in vivo, such as within a host animal, including a host animal. human subject. Nucleic acid sequences comprising "complementary nucleotides" are those that are capable of forming base pairs according to the complementary Watson-Crick standard rules. That is, the larger purines will form base pairs with the smaller pyrimidines to form only combinations of guanine paired with cytosine (G: C) and paired adenine with either thymine (A: T), in the case of DNA, or adenine paired with uracil (A: U) in the case of RNA. As used herein, the terms "complementary sequences" or "antisense" mean nucleic acid sequences that are substantially complementary to their full length and have very few base inequalities. For example, nucleic acid sequences of fifteen bases in length can be called complementary when they have a complementary nucleotide in thirteen or fourteen positions with only simple or double inequalities. Of course, nucleic acid sequences that are "completely complementary" will be nucleic acid sequences, which are fully complementary throughout their entire length and do not have base inequalities. Although all or part of the gene sequence can be used in the context of the antisense construct, statistically, any sequence of 17 long bases should occur only once in the human genome and, therefore, sufficient to specify a single objective sequence. Although shorter oligomers are easier to make and increase the accessibility in vivo, several other factors are involved in determining the specificity of the hybridization. Both the binding affinity and the sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs will be used. One can easily determine whether a given antisense nucleic acid is effective to target the corresponding host cell gene simply by testing the constructs in vitro to determine whether the function of the endogenous gene is affected or whether the expression of related genes having complementary sequences is affected. In certain embodiments, one may wish to employ antisense constructs, which include other elements, for example, those that include C-5 propynyl pyrimidines. It has been shown that oligonucleotides containing C-5 propyne analogs of uridine and cytidine bind to RNA with high affinity and are potent antisense inhibitors of gene expression (Wagner et al., Science, 260: 1 51 0- 1 51 3, 1 993, incorporated herein by reference). c) Ribozyme constructions As an alternative to focused antisense delivery, focused ribozymes can be used. The term "ribozyme" refers to an RNA-based enzyme capable of focusing and cutting particular base sequences on DNA or, more typically, RNA. In the present invention, ribozymes are introduced into the cell as an expression construct that encodes the desired ribozimal RNA. The targets of the ribozymes are very similar to those described for antisense nucleic acids. Many ribozymes are known to catalyze the hydrolysis of phosphodiester bonds under physiological conditions. The ribozymes of the present invention catalyze the specific sequence cut of a second nucleic acid molecule, preferably, an mRNA transcript, and optionally an mRNA transcript from an oncogene. In general, ribozymes bind to a target RNA through the target binding portion of the ribozyme, which flanks the enzymatic portion of the ribozyme. The enzymatic portion of the ribozyme cuts the target RNA. He The strategic cut of an objective RNA destroys its ability to encode protein directly or indirectly. After the enzymatic cleavage of the target has occurred, the ribozyme is released from the target and seeks another target, where the process is repeated. In a preferred embodiment of the invention, the ribozyme is a hammerhead ribozyme, a small molecule of RNA derived from plant viroids (Symons, Ann, Rev. Biochem 61: 641-671, 1992; Clouet-D'Orval and Uhlenbeck, RNA, 2: 483-491, 1 996; Haseloff and Gerlach, Nature 334: 585-591, 1988; Jeffries and Symons, Nucleic Acids Res. 1 7: 1 371 - 1 377, 1989; Uhlenbeck, Nature 328: 596-600, 1987; all incorporated in the present by reference).

In other embodiments, the ribozyme can be a group I intron, a hairpin ribozyme, VS RNA, a ribozyme of Delta virus hepatitis or a RNase P-RNA ribozyme (in association with an RNA leader sequence). Examples of fork motifs are described by Hampel < ? 5 et al. , Nucleic Acids Res. 18: 299, 1990 and Hampel and Tritz, Biochemistry 28: 4929, 1989; an example of the hepatitis delta virus motif is described by Perrotta and Been, Biochemistry 31: 16, 1992; an example of the RNAseP motif (associated with an external leader sequence) is described by Yuan et al. , patent no. 5,624,824; a ribozyme motif of VS RNA or Neurospora is described in Saville and Collins, Proc. Nati Acad. Sci. USA 88: 8826-8830, 1 991, Collins and Olive, Biochemistry 32: 2795-2799, 1 993; the intron of group I is described in Cech et al. , US patent no. 5, 354,855. The aforementioned reasons should not be considered as limiting with respect to the present invention and those experts in the The technique will recognize that ribozymes that can be used herein comprise a specific substrate binding site, which is complementary to an objective mRNA. Such ribozymes also comprise an enzymatic portion, which imparts RNA cutting activity to the molecule. The enzymatic portion resides within or surrounds, the binding site of substrate. d) Selectable markers In certain embodiments of the invention, the therapeutic vectors of the present invention contain nucleic acid constructs whose Expression can be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell allowing easy identification of cells containing the expression construct. Usually, the inclusion of a drug selection marker helps in the cloning and in the (5) selection of transformants For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers Alternatively enzymes such as thymidine kinase (tk) can be used. herpes simplex, and immunological markers can also be used. believes that the selectable marker employed is important, as long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Additional examples of selectable markers are well known to some skill in the art and include reporters, such as, EGFP, β-gal and chloramphenicol acetyltransferase (CAT). e) Multiple constructs and IRES In certain embodiments of the invention, the use of elements of internal ribosome binding sites (I RES) is used to create messages of multigenes or policistronics. The I RES elements are able to bypass the 5'-methylated Cap dependent translation ribosome tracking model and start the translation at internal sites. I RES elements of two members of the picanovirus family (polio and encephalomyocarditis) (Pelletier and Sonenberg, Nature, 334: 320-325, 1988), as well as an IRES of a mammalian message (Macejak and Sarnow, Nature, 353: 90-94, '1 991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an I RES, creating polycistronic messages. Under the ^ r 5 IRES element, each open reading frame is accessible to ribosomes for efficient translation. In this way, multiple genes can be efficiently expressed using a simple promoter / enhancer to transcribe a simple message. Any heterologous open reading frame can be linked to 10 I RES elements. This includes genes for secreted proteins, multiple subunit proteins encoded by independent genes, membrane or intracellular bound proteins and selectable markers. f) Control Regions 15 In order that the expression construct affects the expression of a transcript encoding a therapeutic gene, the polynucleotide encoding the therapeutic gene will be under the control of transcription of a promoter and a polyadenylation signal. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell host, or introduced synthetic machinery, which is required to initiate the specific transcription of a gene. A polyadenylation signal refers to a DNA sequence recognized by the synthetic machinery of the host cell, or introduced synthetic machinery, which is required to direct the addition of a series of nucleotides at the end of the transcription of M RNA for processing and appropriate transcription of the transcript out of the nucleus into the cytoplasm for translation. The phrase "under transcription control" means that the promoter is in the correct location relative to the polynucleotide to control the initiation of RNA polymerase and the expression of the polynucleotide. < ? The term "promoter" will be used herein to refer to a group of transcription control modules that are clustered around the initiation site for RNA polymerase I I. Much of what is thought about how promoters are organized is derived from analyzes of several viral promoters, including those for HSV thymidine kinase (tk) and units of SV40 early transcription. These studies, augmented by more recent work, have shown that the promoters are composed of discrete functional modules, each consisting of approximately 7-20, bp of DNA, and containing one or more recognition sites for repressor or activating transcription proteins. 15 At least one module in each promoter functions to place the start site for RNA synthesis. The best known example of this is the almost TATA, but in some promoters that lack a TATA box, such as the promoter for late SV40 genes, a discrete element that overlaps the start site by itself helps to set the place of initiation. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the 30-1 10 bp region upstream of the start site, although it has recently been shown that a variety of promoters also contain functional elements downstream of the start site. The separation between promoter elements is often flexible, so that the promoter function is conserved when the elements are inverted or moved in relation to one another. In the tk promoter, the separation between promoter elements can be increased to 50 bp apart before the activity starts to decline. Depending on the promoter, it seems that individual elements can work either cooperatively or independently to activate transcription. Where a human cell is focused, it is preferable to place the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter could include either a human or viral promoter. A list of promoters is provided in Table 1.

TABLE 1 PROMOTER Immunoglobulin heavy chain Immunoglobulin light chain T cell receptor HLA Dq a and DQ ß ß-interferon interleukin-2 Interleukin receptor-2 M HC Class II 5 I HC Class II HLA-DRa ß-actin Creatine kinase muscle Prealbumin (Transthyretin) KZZ Elastase / Metallothionein Collagenase Albumin Gene a-Fetaprotein t-globin ß-globin c-fos c-HA-ras I nsulin Neural cell adhesion molecule (NCAM) or ^ -antitrypsin H2B (TH2B) Histone Collagen mouse or type I Glucose-regulated proteins (GRP96 and GRP78) Rat growth hormone Human serum amyloid A (SAA) Troponin I (TN I) Platelet-derived growth factor Duchenne muscle dystrophy Polyneoma Retrovirus Papilloma virus Hepatitis virus B% Human immunodeficiency virus Cytomegalovirus Simian leukemia virus of bón It is not believed that the particular promoter that is employed to control the expression of the therapeutic gene is critical, as long as it is capable of being activated by the gene product linked to the inducible promoter. In a preferred embodiment of the invention, the transactivating protein is tat, and the promoter that is operably linked to the therapeutic gene is LTR of HIV-1 or H IV-2. For example, a promoter element containing an AP-1 site would respond to the inducible expression of the c-jun or c-fos proteins. Another factor combination The transactivant / adept promoter would be known to one of skill in the art. The promoter that controls the expression of the gene encoding the transactivating factor must be an inducible promoter. An inducible promoter is a promoter, which is inactive or exhibits relatively low activity except in the presence of an inducing substance. Some examples of promoters that may be included as a part of the present invention include, but are not limited to, MT II, MMTV, collagenase, stromelysin, SV40, murine MX gene, α-2-macroglobulin, h-2kb gene class I of MHC, proliferin, tumor necrosis factor or thyroid stimulating hormone gene a. The associated inducers of these promoter elements are shown in Table 2. The Egr-1 promoter and multi-drug resistance gene (MDR1) promoter are also options for inducible promoters. In preferred embodiments, the inducible promoter is inducible heat shock and is derived from one of the following promoters: HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25, ubiquitin and HSP28. In another preferred embodiment, the inducible promoter comprises a hypoxia responsive element, such as, those responsive to HI F-1. It is understood that any inducible promoter can be used in the practice of the present invention and that all such promoters will fall within the spirit and scope of the claimed invention.

TABLE 2 In particularly preferred embodiments, the tat protein is used as the transactivating factor. The genome of H IV-1 and H IV-2 share a large number of similarities with simian immunodeficiency viruses (SIVS) and has been studied extensively. It has been discovered that in addition to the gag, env, pol genes that are common to all retroviruses, there is a variety of regulatory genes that are important in HIV transcription. The viral tat protein is one of these regulating factors and is characterized by its ability to greatly increase the activity of the promoter of H IV-1 and H IV-2 (Sodroski et al., J. Virol. 55 (3): 831 -835, 1989; Sodroski, et al., Science, 229 (4708); 74-77, 46 1990) and receptor-mediated transfection (Wu and Wu, J. Biol. Chem. 262: 4429-4432, 1987; Wu and Wu, Biochemistry, 27: 887-892, 1988). (The articles cited in this paragraph are incorporated herein by reference). Once the construct has been delivered into the cell, the nucleic acid encoding the therapeutic gene can be placed and expressed in different places. In certain modalities, the nucleic acid encoding the therapeutic gene can be stably integrated into the genome of the cell. This integration can be in the location and cognate orientation via homologous recombination (gene replacement) or can be integrated in a non-specific, random location (gene accretion). In still further embodiments, the nucleic acid can be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to allow indeterminate maintenance and replication, or in synchronization with, the host cell cycle. How the expression construct is delivered to a cell, and where in the cell the nucleic acid remains depends on the type of expression construction used. In a particular embodiment of the invention, the expression construct can be trapped in a liposome. Liposomes are vesicular structures characterized by a bilayer membrane of phospholipids and an internal aqueous medium. Multilamellar liposomes have multiple layers of lipids separated by aqueous medium. They form spontaneously when the phospholipids are suspended in a therapeutic excess of the present invention in a cell. Such transfer can employ viral or non-viral methods of gene transfer. This section provides a discussion of gene transfer methods and compositions.

A. Non-viral transfer In a preferred embodiment, the therapeutic constructs of the present invention, for example, various genetic constructs (ie, DNA) must be delivered into a cell. In certain preferred situations, the introduction of the expression construct into a cell is mediated by non-viral means. Several non-viral methods for the transfer of expression constructs in cultured mammalian cells are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52: 456-467, 1973; Chen and Okayama, Mol Cell. Biol., 7: 2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10: 689-695, 1990) DEAE-dextran (Gopal, Mol Cell Biol., 5: 1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol Cell Biol., 6: 716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81: 7161: 7165, 1984), direct microinjection (Harland and Weintraub, J. Cell Biol., 10 1: 1094-1099, 1985), liposomes loaded with DNA (Nicolau and Sene, Biochim, Biophys, Acta, 721: 185-190, 1982, Fraley et al., Proc. Nati, Acad. Sci. USA, 76: 3348-3352, 1979), cellular sonication (Fechheimer et al., Proc. Nati, Acad. Sci. USA 84: 8463-8467, 1987), bombardment of genes using high-speed microprojectiles (Yang et al., Proc. Nati. Acad. Sci. USA, 87: 9568-9572, of aqueous solution The lipid components undergo self-regulation before the formation of closed structures and trap water and solutes dissolved in between the lipid bilayers. The addition of DNA to cationic liposomes causes a topological transition from liposomes to ('> 5 condensed liquid-crystalline beads optically bi-refringent.These DNA-lipoid complexes are potential non-viral vectors for use in gene therapy.The delivery of liposome-mediated nucleic acid and expression of Strange DNA in vitro has been very successful. Using the ß-lactamase gene, Wong et al. , Gene, 1 0: 87-94, 1 980, the feasibility of liposome-mediated delivery and delivery of foreign DNA in cultured chicken embryo, HeLa and hepatoma cells was demonstrated. Nicolau et al. , (Methods Enzymol., 149: 1 57-176, 1987, incorporated herein by reference) achieved successful gene transfer mediated by liposomes in rats after intravenous injection. Several commercial approaches involving "lipofection" technology are also included. In certain embodiments of the invention, the liposome can form a complex with a haemagglutinating virus (HVJ). It has been shown that this facilitates fusion with the cell membrane and promotes the entry of encapsulated DNA cells into liposome. In other embodiments, the liposome can form a complex or be used in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In still further embodiments, the liposome can form a complex or be used in conjunction with both HVJ and HMG-1. Because such expression constructs have been successfully employed in the transfer and expression of nucleic acid in vitro and in vivo, they are applicable to the present invention. Other vector delivery systems that can be employed to deliver a nucleic acid encoding a therapeutic gene in cells are delivery vehicles mediated by receptor. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because the specific distribution of the cell type of several receptors, the delivery can be highly specific (Wu and Wu, Adv. Drug Delivery Rev., 1 2: 1 59-167, 1 993). Vehicles that target genes mediated by receptors generally consist of two components: a specific ligand of a cellular receptor and a DNA binding agent. Several ligands have been used for gene transfer mediated by the receptor. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and WU, J. Biol. Chem., 262: 4429-4432, 1987) and transferrin (Wagner et al., Proc. Nati. Acad. Sci. 87 (9 ): 341 0-3414, 1990). Recently, a synthetic enoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al., FASEB J., 7: 1 081 -1 091, 1 993).; Perales et al. , Proc. Nati Acad. Sci USA, 91: 4086-4090, 1 994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous cell carcinoma cells (Myers, EPO 0273085). In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. , Methods Enzymol. , 149: 1 57-176, 1987, used lactosyl-ceramide, a galactose terminal asialganglioside, incorporated in liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a therapeutic gene also > 5 can be delivered specifically to a cell type, such as, prostate, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, the human prostate-specific antigen (Watt et al., Proc. Nati. Acad. Sci. 83 (2): 31 66-31 70, 1986) can be used as the recipient for delivery mediated nucleic acid in prostate tissue. In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. The transfer of the construction can be done by any of the aforementioned methods, which can permeate physics or chemically the cell membrane. This is particularly applicable for in vitro transfer, however, it can also be applied for in vivo use. Dubensky et al. (Proc. Nati, Acad. Sci. USA, 81: 7529-7533, 1884), successfully injected polyomavirus DNA in the form of CaPO4 precipitates in the liver and spleen of adult and newborn mice, demonstrating active viral replication and acute infection. Benvenisty and Neshif, Proc. Nat. Acad. Sci. USA, 83: 9551 -9555, 1986, also demonstrated that direct intraperitoneal injection of precipitated CaPO4 plasmids results in the expression of the transfected genes. It is anticipated that the DNA encoding a CAM can also be transferred in a similar way in vivo and express CAM.

Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate microprojectiles coated with DNA at a high speed, allowing them to pierce cell membranes and enter cells without killing them (Klein et al., Nature, 327: 70-73, 1987, incorporated herein by reference). Several devices have been developed to accelerate small particles. One such device is based on a high voltage discharge to generate an electric current, which in turn provides the motor force (Yang et al., Proc. Nati. Acad. Sci. USA, 87: 9568-9572, 1 990). The microprojectiles used have consisted of biologically inert substances, such as tungsten or gold beads.

B. Viral vector-mediated transfer Another method for achieving gene transfer is via viral transduction using infectious viral particles as a delivery vehicle, for example, by transformation with an adenovirus vector of the present invention, as described in FIG. present later. Alternatively, the bovine or retroviral papilloma virus can be used, both allowing the permanent transformation of a host cell with one or several genes of interest. Thus, in one example, viral infection of cells is used in order to deliver therapeutically significant genes to a cell. Normally, the virus will simply be exposed to the appropriate host cell under physiological conditions that allow uptake of the virus. Although exemplified with adenovirus, the present methods can be advantageously employed with other viral vectors as discussed below. Such methods will be familiar to those of ordinary skill in the art. a) Adenovirus Adenovirus is particularly suitable for use as a gene transfer vector due to its medium-sized DNA genome, ease of manipulation, high titer, broad target cell range and high infectivity. The viral genome of scarcely 36 kB is joined by repeats of inverted terminals (ITR) of 1 00-200 base pairs (bp), in which cis-acting elements necessary for the replication and packaging of viral DNA are contained. The early (E) and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication. The E 1 region (E1 A and E 1 B) encodes proteins responsible for the transcription regulation of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell closure (Renan, 1990). The products of the late genes (L1, L2, L3, L4 and L5), including most of the viral capsid proteins, are expressed only after significant processing of a simple primary transcript induced by the late main promoter. (M LP). The MLP (located in 16.8 map units) is particularly efficient during the late phase of infection, and all m RNAs emitted from this promoter possess a tripartite 5 'leader sequence (TL), which makes them the preferred mRNAs for translation . In order to optimize the adenovirus for gene therapy, it is necessary to maximize the carrier capacity, so that long segments of DNA can be included. It is also very desirable to reduce the toxicity and immunological reaction associated with certain adenoviral products. The two objectives are, to a degree, confining because the elimination of adenoviral genes serves both ends. By practice of the present invention, it is possible to achieve both objectives by retaining the ability to manipulate the therapeutic constructs with relative ease. The large displacement of DNA is possible because the cis elements required for viral DNA replication are all located in the inverted terminal repeats (ITR) (100-200 bp) at either end of the linear viral genome. Plasmids containing ITR's can replicate in the presence of a non-defective adenovirus. Consequently, the inclusion of these elements in an adenoviral vector should allow replication. In addition, the packaging signal for viral encapsulation is located between 1 94-385 bp (0.5-1 .1 map units) at the left end of the viral genome. This signal mimics the site of protein recognition in bacteriophage DNA, where a specific sequence near the left end but outside the cohesive end sequence mediates the binding to proteins that are required for the insertion of DNA into the DNA. head structure. Ad-substitution vectors have shown that a 450 bp fragment (0-1.25 map units) at the left end of the viral genome could direct packaging in 293 cells. It has previously been shown that certain regions of the adenoviral genome can be incorporated into the genome of mammalian cells and the encoded genes expressed by it. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in adenoviral function encoded by the cell line. As well There have been reports of complementation of adenoviral vectors deficient in replication by "helper" vectors, eg, wild type viruses or conditionally defective mutants. Adenoviral vectors deficient in replication can be supplemented, in trans, by helper viruses. This observation alone does not allows the isolation of replication-deficient vectors, however, due to the presence of helper virus, necessary to provide replication functions, would contaminate any preparation. Thus, an additional element was necessary, which would add specificity to replication and / or packaging of the replication-deficient vector. That The element, as provided in the present invention, is derived from the adenovirus packaging function. It has been shown that a packaging signal for adenovirus exists at the left end of the conventional adenovirus map. Later studies showed that a mutant with a deletion in the E 1 A region (1 94-358 bp) of the genome grew poorly even in a cell line that complemented early function (E1 A). When a compensating adenoviral DNA (0-353 bp) was recombined at the right end of the mutant, the virus was packaged normally. Additional mutation analysis identified a position-dependent, repeated, short element at the far left of the Ad5 genome. It was found that one copy of the repeat was sufficient for efficient packing if it was present at either end of the genome, but not when moving into the DNA molecule Ad5. By using mutation versions of the packaging signal, it is possible to create helper viruses that are packaged with varying efficiencies. Normally, mutations are mutations or point deletions. When a helper virus with low efficiency packaging is grown in helper cells, the virus is packaged, albeit at reduced proportions compared to wild type viruses, thereby allowing the helper to propagate. When these helper viruses are grown in cells along with viruses containing wild-type packaging signals, however, wild-type packaging signals are preferentially recognized on the mutated versions. Given a limiting amount of packaging factor, the virus containing the wild type signals are selectively packaged when compared to helpers. If the preference is large enough, materials should be approximated to homogeneity. b) Retrovi rus Retroviruses are a group of simple filament RNA viruses, characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process for reverse transcription. The resulting DNA is then stably integrated into the 5 cellular chromosomes as a provirus, and directs the synthesis of viral proteins. The integration results in the retention of viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes - gag, pol and env - that encode capsid proteins, polymerase enzyme and envelope components, respectively. A The sequence found upstream of the gene gag, called?, Functions as a signal for packaging the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5 'and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration into the host cell genome. In order to construct a retroviral vector, a nucleic acid encoding a promoter is inserted into the viral genome at the site of certain viral sequences to produce a virus that is defective in replication. In order to produce virions, a cell line is constructed of packaging containing the genes gag, pol and env, but without the T and LTR components. When a recombinant plasmid containing a human cDNA, together with the sequences of LTR and? retroviral, is introduced in this cell line (by precipitation of calcium phosphate, for example), the sequence? allows the RNA transcript of the The recombinant plasmid is packaged in viral particles, which are then secreted into the culture media. The medium containing the recombinant retroviruses is collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. However, the integration and stable expression of many types of retroviruses require the division of host cells. Recently, an approach designed to allow specific targeting of retrovirus vectors was developed, based on the chemical modification of a retrovirus by the chemical addition of galactose residues to the viral envelope. This modification could allow the specific infection of cells, such as, hepatocytes via asialoglycoprotein receptors, if desired. A different approach was designed for focused recombinant retroviruses, in which biotinylated antibodies were used against a retroviral envelope protein and against a specific cellular receptor. The antibodies were coupled via the biotin components when using streptavidin (Roux et al., Proc. Nati, Acad. Sci. USA, 86: 9079-9083-1 989). Using antibodies against major histocompatibility complex antigens class I and class I I, infection of a variety of human cells that support these surface antigens with an ecotropic virus was demonstrated. c) Adeno-associated virus AAV uses a simple, linear filament DNA of approximately 4700 base pairs. The inverted terminal repeats flank the genome. Two genes are present within the genome, originating a variety of products from different genes. The first, the cap gene, produces three different virion (VP) proteins, uncoupled VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products are responsible for transactivating AAV transcription. The three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, p1 9 and p40. The transcription originates six transcripts, two initiated in each of the three promoters, with one of each pair being spliced. The splice site, derived from map units 42-46, is the same for each transcript. The four non-structural proteins are apparently derived from the longest of the transcripts, and three virion proteins arise from the smallest transcript. AAV is not associated with any pathological state in humans. Interestingly, for efficient replication, AAV requires "helper" functions of viruses, such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovi rs. The best characterized of the auxiliaries is adenovirus, and many 'early' functions for this virus have been shown to assist in the replication of AAV It is believed that the low level expression of AAV rep proteins supports the structural expression of AAV in control, and it is thought that helper virus infection removes this blockage.Termite repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid, such as p201, which contains a modified Aav genome (Sarnulski et al. al., J. Virol., 61 (1 0): 3096-31 01, 1987, incorporated herein by reference), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the B / 5 terminal repeats based on the published sequence of AAV. The technician can determine, by well-known methods, such as suppression analysis, the minimum sequence or part of the AAV ITRs, which is required to allow the function, i.e., the stable and site-specific integration. The technician can also determine which minor modifications of the sequence can be tolerated, as long as the capacity of the terminal repeats is maintained to direct the specific, stable site integration. AAV-based vectors have proven to be safe and effective vehicles for in vitro gene delivery, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo. The efficient transfer and expression of genes mediated by AAV in the lung has led to clinical trials for the treatment of fibrosis Cysticer (Cárter and Flotte, Ann. N. Y. Acad. Sci., 770: 79-90, 1 995; Flotte et al., Proc. Nati. Acad. Sci. USA, 90: 10613-10617, 1993). In a similar way, the prospects for the treatment of muscular dystrophy by delivery of the AAV-mediated gene from the dystrophin to skeletal muscle gene, from Parkinson's disease by delivery of tyrosine gene hydroxylase to the brain, of hemophilia B by delivery of Factor IX gene to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart, seem promising because it has recently been shown that transgene-mediated expression AAV in these organs is highly efficient (Fisher et al., J. Virol, 70: 520-532, 1996, Flotte et al., Proc. Nati, Acad. Sci. USA, 90: 10613-10617; 1993; Kaplitt et al., Nat. Genet., 8: 148-153, 1994; Kaplitt et al., Arm Thord. Surg., 62: 1669-1676, 1996; Koeberl et al., Proc. Nati. Acad. Sci. USA, 94: 1426-1431, 1997; McCown et al., Brain Res., 713: 99-107, 1996; Ping et al, Microcirculation, 3: 225-228, 1996; Xiao et al., J. Virol. , 70: 8098-8108, 1996). d) Other viral vectors Other viral vectors can be used as expression constructs in the present invention. Virus-derived vectors, such as vaccinia virus, can be used (Ridgeway, En: Vectors: A survey of molecular cloning vectors and their uses (Vectors: A study of molecular cloning vectors and their uses), Rodríguez RL, Denhardt DT. Ed., Stoneham: Butterworth, pp. 467-492, 1988; Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press, pp. 117-148, 1986; Coupar et al., Gene, 68: 1-10, 1988), canary pustule eruption virus, lentivirus and herpes virus. 6. Cell approaches The methods and vectors of the present invention can be used to target a wide variety of cells, organs and tissues within a mammal. In some embodiments, the expression constructs described herein are used to treat cancer. The cell that is focused can be either a tumor cell, a cell within a tumor or a cell near a tumor. The tumor can be optionally in the brain, lung, liver, spleen, kidney, bladder, lymph node, small intestine, pancreas, colon, stomach, chest, endometrium, prostate, testicle, vulva, cervix, ovary, skin, head and neck, esophagus, bone marrow or blood. One of ordinary skill in the art will be able to readily discern an appropriate therapeutic gene to be expressed in a given type of tumor. In alternative modalities, a medical condition other than cancer is being treated. For example, the present invention provides highly effective protein replacement therapy. In such a case, a specific type of cell, tissue or organ can be targeted by expression of a protein, which is underexpressed in the subject, especially if the activity of the protein is limited to that specific cell, tissue or organ type. . Again, somebody of ordinary skill in the art will be able to discern which cells are focused in the most appropriate way. The expression construct may be introduced into the cell of interest through an in vitro, ex vivo or in vivo method. A lot of therapy genes are currently performed ex vivo, because the transfection or transduction of an isolated cell is often more efficient. The choice of the introduction method will be dependent on the type of cell, tissue or organ being treated, as well as the particular delivery vehicle chosen. Someone of ordinary skill in the art can easily navigate such an election. Because the expression constructs of the present invention require induction to be active, in many cases the expression construct can be delivered to a larger part of the subject's body than only to the cell, tissue or organ in which it is ded. the expression. The exposure of the subject to the activating conditions, which induce the expression of the transferred expression constructs, may then be limited to achieve expression specificity. In many cases, this is preferred. For example, exposure of a subject to radiation therapy is preferably limited to only those necessary areas. The application of hyperthermia will generally also be limited in its range. In other modalities, activation conditions may be conditions inherent to the self-focused cell. For example, the hypoxic environment of a tumor will trigger expression when the expression construct has a inducible promoter containing a hypoxia response element. In such cases, the resulting expression will be, by its very nature, very localized, even if it is not the delivery of the expression construction. 7. Combination Therapy The expression constructs of the present invention can be advantageously combined with one or more traditional clinical therapies. One goal of current cancer research is to find ways to improve the effectiveness of chemo and radiotherapy. One way is to combine such traditional therapies with gene therapy.

For example, the gene for herpes simplex thymidine kinase (HS-tk), when delivered to brain tumors by a retroviral vector system, successfully induces susceptibility to the antiviral agent ganciclovir. However, the effective use of gene therapy in combination with traditional cancer therapies have been obstructed by the need for clinically meaningful expression of the genes once they have been transferred to the target cell. To kill cells, inhibit cell growth, inhibit metastasis, decrease tumor size and reverse or reduce other In the manner of the malignant phenotype of the tumor cells, using the methods and compositions of the present invention, one would generally introduce an expression construct of the present invention into the "target" cell to induce expression by the application of hyperthermia or other conditions, which activate the inducible promoter. This therapy genes can be combined with compositions comprising other effective agents in the treatment of cancer. These compositions would be provided in a combined amount effective to kill or inhibit the proliferation of the cell. This process may involve introducing the expression construction and the agent or factors in the cell at the same time. This can be achieved by contacting the cell with a simple composition or pharmacological formulation that includes both agents, or by exposing the cell to two different compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent. Alternatively, gene therapy / hyperthermia treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In modalities where the other agent and expression construction are applied separately to the cell, one would generally ensure that a significant period does not expire between the time of each delivery, so that the agent and expression construction would still be able to exert an effect advantageously combined on the cell. In such cases, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other, and more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours. In some situations it is more desirable to extend the period for treatment significantly, however, where several days pass (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 4, 5, 6 , 7 or 8) between the respective administrations. It is also conceivable that more than one administration of any expression construct or the other agent is desired. Several combinations can be used, where the expression construction is "A" and the other agent is "B", as exemplified below: A / B / AB / A / BB / B / AA / A / BB / A / AA / B / BB / B / B / AB / B / A / BA / Á / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BB / B / B / AA / A / A / BB / A / A / AA / B / A / AA / A / B / AA / B / B / BB / A / B / BB / B / A / B Other combinations are contemplated. Again, to achieve cell death, both agents are delivered to a cell in an effective combined amount to kill the cell. Suitable agents or factors for use in a combination therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage, such as, radiation ?, X-rays, UV radiation, microwaves, electronic emissions and the like. In one embodiment of the invention, radiation therapy, which is combined with gene therapy, constitutes external beam radiation. External beam radiation treatment usually delivers high-energy radiation, such as high-energy x-ray beams. Alternatively, internal radiation or brachytherapy can be used in combination with gene therapy. Methods for delivering brachytherapy include intracavital or interstitial placement of radiation sources, instillation of colloidal solutions and parenteral or oral administration. The sealed sources are encapsulated in a metal, wire, tube, needle or the like. Unsealed radioactive sources are prepared in a suspension or solution. The encapsulated radioactive elements are placed in body cavities or inserted directly into tissues with suitable applicators. The applicator is usually placed in the cavity or body tissue surgically or using fluoroscopy. The applicators, usually plastic or metal tubes, can be sutured on or near the tumor to hold them in place. The radioactive isotope is then placed in the applicator ("posterior charge"). Radioactive implants are used in the treatment of cancer of the tongue, lip, chest, vagina, cervix, endometrium, rectum, bladder and brain. Encapsulated sources can also be left inside a patient as permanent implants. "Sowing" with small beads of radioactive material is an approach used for the treatment of localized prostate cancer and localized but inoperable lung cancer. A variety of chemical compounds, also described as "chemotherapeutic agents" functions to induce DNA damage, with the intention of using all of them in the combined treatment methods described herein. Chemotherapeutic agents contemplated for use include, for example, adriamycin, 5-fluorouracil (5FU), etoposide (VP-1 6), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. The invention also encompasses the use of a combination of one or more DNA damage agents, either current or radiation-based compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide. For example, to treat cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct, and induce the expression of the gene by the application of hyperthermia. This can be achieved by applying hyperthermia locally at the tumor site or systemically to the individual. This treatment may be in combination with irradiation of the tumor with radiation such as X-rays, UV light, gamma rays or even microwaves. Alternatively, the tumor cells may come into contact with (5) the agent when administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound, such as adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C or more preferably, cisplatin. prepared and used as a combined therapeutic composition, or together, when combined with a therapeutic expression construct, as described above. It is anticipated that agents that directly cross-link nucleic acids, specifically DNA, will facilitate DNA damage leading to an antineoplastic, synergistic combination with the constructions of expression of the present invention. Agents, such as cisplatin, and other DNA alkylating agents can be used. Cisplatin is not absorbed orally and therefore must be delivered via intravenous, subcutaneous, intratumoral or intraperitoneal injection. Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, veraparnil, podophyllotoxin, and similes. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through injections of intravenous bolus at doses ranging from 25-75 mg / m2 in 21-day intervals for adriamycin, up to 1,00 mg / m2 for etoposide intravenously or doubling the intravenous dose orally. Agents that break the synthesis and fidelity of precursors and nucleic acid subunits also lead to DNA damage. As such, a variety of nucleic acid precursors have been developed. Agents that have undergone extensive testing and are readily available are particularly useful. As such, agents such as 5-fluorouracil (5-FU), are used preferentially by neoplastic tissue, making this agent particularly useful for focusing neoplastic cells. Although it is quite toxic, 5-FU is applicable in a wide range of carriers, including topical administration, however, intravenous administration is commonly being used with doses ranging from 450-1,000 mg / m2 / day. Other factors that can cause DNA damage and have been used widely, include those commonly known as "rays", X-rays and / or the targeted delivery of radioisotopes to tumor cells. Other forms of factors that damage DNA are also contemplated, such as microwave and UV irradiation. Very probably all of these factors effect a wide range of DNA damage, in DNA precursors, in the replication and repair of DNA and in the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses from 50 to 200 roentgens for prolonged periods (3 to 4 weeks), at sim ples of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted and the uptake by the neoplastic cells. The expert is addressed to "Remignton's Pharmaceutical Sciences" (Remington Pharmaceutical Sciences), 5th edition, chapter 33, in P 5 particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for the administration will determine, in any case, the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet standards of sterility, pyrogenicity, general safety and purity, as required by the FDA Office of biological standards. The regional delivery of the therapeutic expression constructs of the present invention to cancer patients is a preferred method for delivering a therapeutically effective gene to counteract the clinical disease being treated. Similarly, chemo- or radiotherapy can be directed to a particular affected region of the subject's body. Alternatively, the systemic delivery of expression construct and / or the agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred. In addition to combinations of gene therapies with chemo- and radiotherapies, it is also contemplated that the combination of multiple gene therapies will be advantageous. For example, the approach of mutations p53 and p1 6 at the same time can produce an improved anti-cancer treatment. Any other gene related to tumor conceivably can be focused in this way, for example, p21, Rb, APC, DCC, NF-1, NF-2, BRCA2, p1 6, FHIT, WT-1, MEN-I, MEN-I I, BRCA1, VHL , FCC, MCC, ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bel and abl. 8. Pharmaceutical compositions and routes of administration It is contemplated that the therapeutic compositions of the present invention may be administered, in vitro, ex vivo or in vivo. Thus, it will be desirable to prepare the complex as a pharmaceutical composition suitable for the intended application. Generally, this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurity that could be harmful to humans or animals. One will also generally want to employ appropriate salts and buffers to make the compound stable and allow complex uptake by the target cells. The compositions of the present invention comprise an effective amount of the expression construct or a viral vector or liposome carrying the expression construct, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions can also be referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or otherwise unfavorable reaction when administered to an animal or a human, as appropriate. As used herein, the "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, antibacterial and antifungal agent, absorption and isotonic retardation agent, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except for any conventional medium or agent that is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Active complementary ingredients F '% »5 can also be incorporated into the compositions. Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, polyethylene glycols liquids and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The therapeutic compositions of the present invention may include classical pharmaceutical preparations for use in regimens therapeutic, including its administration to humans. The administration of therapeutic compounds according to the present invention will be via any common route, as long as the tissue or target cell is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical administration. Alternatively, the administration will be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions including carriers, buffers or other excipients. For application against tumors, direct intratumoral injection is contemplated, n injection of a bed of resected tumor, regional (for example, lymphatic) or systemic administration. It may also be desired to perform continuous perfusion for hours or days via a catheter to a disease site, for example, a tumor or tumor site. The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions, either as suspensions or liquid solutions; solid forms suitable for solution in, or suspension in, liquid can also be prepared prior to injection. These preparations can also be emulsified. A normal composition for this purpose comprises a pharmaceutically acceptable carrier. For example, the composition may contain about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers including aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like can be used. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include nutrient and fluid fills. The preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and the exact concentration of the various components of the composition are adjusaccording to well-known parameters.

Additional formulations that are suitable for oral administration are also contemplated. Oral formulations include normal excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations or powders. When the route is topical, the form may be a cream, ointment, balm or spray. An effective amount of the therapeutic agent is determined based on the intended objective, for example, (i) inhibition of tumor cell proliferation, (ii) elimination or death of tumor cells, or (iii) gene transfer for expression short or long term of a therapeutic gene. The term "unit dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen. The amount to be administered, both according to the number of treatments and dose of unit, depends on the subject to be treated, the state of the subject and the desired result. The present inventors contemplate the multiple gene therapeutic regimens. In one embodiment, a vector encoding a therapeutic gene is used to treat patients with cancer. Normal amounts of a viral vector used in cancer gene therapy is 1 06-1 01 5 PFU / dose (eg, 1 06, 1 07, 1 08, 1 09, 1 010, 1 01 1, 1012, 1 01 3, 1 014 and 1 01 5), where the dose is divided into several injections at different sites within a solid tumor. The treatment regimen also involves several cycles of administration of the gene transfer vector over a period of 3-10 weeks. The administration of the vector for longer periods from months to years may be necessary for a continuous therapeutic benefit. In another embodiment of the present invention, a viral vector encoding a therapeutic gene can be used to vaccinate humans or other mammals. Normally, an amount of virus effective to produce the desired effect, in this case vaccination, would be administered to a human or other mammal so that long-term expression of the transgene is achieved and a host immune response develops. It is contemplated that a series of injections, e.g., a primary injection followed by two booster injections, would be sufficient to induce a long-term immune response. A normal dose would be from 1 06 to 1 01 5 PFU / injection, depending on the desired result. Low doses of antigen generally induce a strong cell-mediated response; while high doses of antigen generally induce an immune response mediated by antibody. The precise amounts of the therapeutic composition also depend on the practitioner's judgment and are pecular for each individual.

Examples The following specific examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims. «5 EXAMPLE 1 The heat shock promoter can induce the expression of a reporter gene Vector constructions. To study the capacity of the promoter HSP70 to induce gene expression, was inserted either a promoter The minimum term shock (HS) or a minimal CMV promoter upstream of a reporter gene in a plasmid containing selectable markers of neomycin and ampicillin. The basic design of a plasmid (M5) containing a multiple cloning site operable to a promoter derived from HSP70 is shown in Figure 1. M5 was constructed by replacing the CMV promoter in pcDNA3.0 (Invitrogen, I nc. ) with a minimal HSP70B promoter (SEQ ID NO: 1, Figure 10), a 0.4 kb fragment (Hind ll l-Bam H 1) of the human heat shock protein promoter 70B (HSP70B), obtained from StressGen , Inc. The activity of the minimal HS and CMV promoters was determined by transfecting human cancer cells, MCF7 human breast carcinoma cells and human prostate carcinoma cells DU 145, with the S8 plasmid. Plasmid S8, derived from the M5 vector of Figure 1, contains the minimal HSP70B promoter operably linked to the gene encoding the Enhanced Green Fl uorescence Protein (EGFP). S8 was constructed by inserting the EGFP gene of pEGFP-1 (Clonetech, Inc.) into the multiple cloning site (MCS) of M5. Cell culture and transfection. Cells derived from human prostate cancer DU-145 and cells derived from human breast cancer MCF-7 were transfected with the vector S8 described above. To isolate cells stably transfected with S8, the cultures were transfected using standard calcium phosphate precipitation methods. The cells containing the integrated plasmids were selected for their ability to proliferate in the presence of neomycin. The thermal shock was administered by completely submerging culture flasks in a controlled temperature water bath (± 0.1 ° C).

A positive clone, clone 4, and a polyclonal line were selected I with geneticin from the transfection of MCF7 cells. A polyclonal line was selected with geneticin from the transfection of cells DU-145. (In each case, the cells were selected with geneticin for 2 weeks.) The selected lines were then analyzed and classified by FACS. Isolation of positive cell lines. Cells expressing high levels of EGFP in response to heat shock were selected using both conventional serial dilution methods and fluorescence activated cell sorting methods (FACS). EGFP expression was quantified using flow cytometry. The Enhanced Green Fluorescence Protein (EGFP) is excited at a run of 490, allowing it to be seen under a fluorescence microscope or analyzed by FACS. Cells expressing EFGP were classified from cells that do not express EFGP when using the FACS method. This was done with cell lines selected with geneticin. The reason why this is required is that in a polyclonal cell line there are populations that have the S8 plasmid integrated in a < • 5 way that interferes with the expression of the reporting gene. By sorting the cells, these populations can be removed, leaving the population purely positive for further analysis. As seen in Figure 2, the average fluorescence of EFGP in DU-145 cells stably transfected with the shock promoter Minimum thermal conduction of EFGP (S8) and growing at 37 ° C was approximately 10 units of relative fluorescence. Four hours after exposure to 42 ° C thermal shock for one hour, the average relative fluorescence was 7-9 times higher. Relative gene expression is quantified subsequently by measuring changes in the relative fluorescence in stably transfected cells. The FACS classification of MCF7 cells transfected with plasmid S8 is illustrated in Figure 3. Kinetic studies. Survival studies were conducted on heat exposure to evaluate optimum temperatures / times at Which MCF7 cells could be heated without causing massive cell death. At 40 ° C and 42 ° C up to one hour, cell death was found to be negligible with less than 3% cell death. At 44 ° C for a time of only 30 minutes, almost 50% of the cells had died.

Using the above optimal survival times, initial kinetic studies were performed. Heating transfected MCF7 cells for 1 hour at 40 ° C and 42 ° C produced more EGFP than heating for only 30 minutes when tested by FACS. The optimal recovery time for the cells after heating was 4 hours. Any additional recovery time did not increase EGFP levels. For heat treatments made at 44 ° C for 30 min, the recovery time was longer, being 8 hours the maximum. Expression of EGFP at 37 ° C-44 ° C in several cell lines. The following heating / recovery times were used as identified in the kinetic studies to test the inducibility of EFGP driven by the HSP70-derived promoter in all lines of transfected cells of the inventors: 40 ° C - 1 h of treatment with heat, 4 h of recovery 42 ° C - 1 h of heat treatment, 4 h of recovery 44 ° C - 30 min of heat treatment, 8 h of recovery Using these temperatures / times, the following cell lines were tested for EGFP expression: MCF7: breast carcinoma parental cell line. Du145: parental cell line of prostate carcinoma. MCF7-S8-P: polyclonal line of MCF7 cells transfected with the S8 plasmid. MC F7-S8-PS 1: MCF7-S8-P cells which were classified by EG FP expression by FACS once.

MCF7-S8-PS2: MCF7-S8-PS 1 cells that were reclassified by EGFP expression by FACS. MCF7-S8-4: clone 4 of the transfection MCF7 S8. MCF7-S8-4S1: MCF7-S8-4 cells sorted once by EGFP expression. Du145-S8-P: polyclonal line of Du 145 cells transfected with the S8 plasmid.

The expression seen from the transfected lines of EFP driven by the promoter derived from HSP70 is shown in Figure 4. As the temperature increases, the relative amount of EGFP also increases. These data show that the heat shock promoter of the inventors really responds to heat. However, at 37 ° C there was still EGFP expression. Expression of EGFP in DU-145 cells transfected stably after thermal shock. The induction of endogenous heat shock promoters is transient and temperature dependent. When the DU-145 cells, stably transfected with the minimal HS promoter driving the expression of EGFP (S8) and selected twice by FACS (DU-S8-PS2 cells), were subjected to thermal shock several times and at various temperatures, the expression The reporter gene was dependent on temperature and the expression was transient with maximum values at 1 5-24 hours after the inductor voltage (Figure 5). These results indicate that the promoter is activated transiently under the conditions used herein and that EGFP is unstable, because the fluorescence decreases after 1 5-24 hours in these cells. The activity of the minimum heat shock promoter increases transiently by approximately 3 times after a thermal shock of 40 ° C for either 1 or 2 hours. The promoter activity increases 13 and 25 times after a thermal shock of 42 ° C either for 1 or 2 hours, respectively. Comparison of EGFP expression under thermal shock control and CMV promoters. The data presented in Figure 6 show that the minimal heat shock promoter activity in DU-S8-PS2 cells is dependent on the temperature over the range of 37-43 ° C. In contrast, DU-145 cells stably transfected with V9, a vector in which the CMV promoter drives EGFP expression (Figure 7), expresses almost 50% higher levels of promoter activity than those same cells transfected with the promoter. of minimum thermal shock and induced with a thermal shock of 43 ° C. The activity of the CMV promoter is not essentially affected by the temperature in these cells. The temperature dependence of the minimal HS promoter is not specific for Du-145 cells.

EXAMPLE 2 The expression of I L-2 can be amplified by the use of an HIV promoter and tat in a building. Initial amplifier studies. Studies involving new constructions capable of amplifying a therapeutic gene expression were carried out. To demonstrate the principle of the idea of amplifier, several constructions were produced. The constructs contain a constitutive promoter, the CMV promoter, instead of an inducible heat shock promoter. These constructs are plasmids L-27, X14, RR1 3, Y1 5 and SS 1 0. Table 3, below, shows the promoters / genes present in each plasmid and the amount of I L-2 produced. Four of the plasmids were obtained from a plasmid containing two multiple cloning sites. In these four plasmids, the CMV promoter was inserted upstream of either the gene tat or a multiple cloning site (MCS) and either of the long terminal repeats of H IV1 or H IV2 (LTRs) was inserted upstream of the gene of mouse interleukin-2 (I L-2). Plasmids X14 and Y1 5 are shown schematically in Figure 9A and 9B. Plasmid L-27 served as a reference. I L-2 was measured from tissue culture supernatants by ELISA using the IL-2 EASIA kit (Medgenix Diagnostics, Fleurus, Belgium). The sensitivity of the set is estimated at 0.1 I U I L-2 / ml. In this study, the cells of the SW480 were transfected with the liquid Dosper (see the transfection protocol of Example 3, below).

TABLE 3 Name of plasmid Promoter / gene Amount of IL-2 in I.U.

Lipid only Dosper 0.48 L-27 CMV / IL-2 15.63 RR13 HIV1 / IL-2 17.56 CMV / multiple cloning site X14 HIV1 / IL-2 173.7 CMV / TAT SS10 HIV2 / IL-2 12.83 CMV / cloning site / multiple Y15 HIV2 / IL-2 440.55 CMV / TAT It can be seen from this study that complete amplification constructs are capable of increased expression on the CMV promoter. In addition, the production of the transactivating factor, TAT, is required for this increased production.

EXAMPLE 3 Heat-inducible amplifiers. Vector construction. To determine if a second promoter could be used to increase the activity of the minimal HS promoter, MCF-7 cells were transiently transfected with a series of vectors, including pC8, pf 1 2 and p007 (Figures 8 and 9). Using a plasmid containing two multiple cloning sites, the minimal heat shock promoter was inserted upstream of either the gene tat or a multiple cloning site (MCS) and any of the repeats of γ. i. 5 long terminals of HIV1 or HIV2 (LTRs) was inserted upstream of the mouse interleukin-2 (I L-2) gene. The plasmids also each carried selectable markers of neomycin and ampicillin. Plasmid f 1 1 was first created by inserting an EcoRI fragment of 0.5 kb, containing the coding region of interleukin-2 (I L-2) (see GenBank access no. 577834), of the C5 plasmid in the EcoRI site of the M5 vector (see specific example, Example 1, above). Plasmid C8 was constructed by inserting a 1 kb BamHI fragment, containing the 0.4 kb HSP70B fragment upstream of an MCS of plasmid B4527 (see Tsang, et al., Biotechniques 20: 51-52, 1 996 and Tsang. et al., Biotechniques 22:68, 1997, both incroporated by reference), in the BamH1 site of plasmid DN P-1 (Tsang et al., 1 996, and Tsang et al., 1 997), which contains LTR of H IV1 upstream of the coding region of I L-2. The vector f 12 (Figure 9) was then constructed by inserting a Notl fragment of 0.4 kb, containing the region of coding for the HIV gene, on the Notl site of C8. An intermediate vector D10 was constructed by inserting the 1 kb BamHl fragment containing the minimal HSP70B promoter into plasmid M NP-7 (Tsang et al., 1996, and Tsang et al., 1 997), which contains the LTR of H IV2 upstream of the coding region of I L-2. Plasmid 007 (Fig. 9) was created by inserting the Not I fragment of 0.4 kb, coding the gene tat, into the Not I site of D 10. Transfection protocol. The transfection was performed according to the published procedure (Stopeck, et al., Cancer Gene Therapy, é "# 5 5: 1 19-126, 1998) .The MCF-7 cells were plated either in plates of 6 or 12 The next day, the cells were washed with Hanks buffered saline and replaced with 1 ml of transfection solution.The transfection solution was a 4: 1 l mass ratio to either DNA of Dosper (1, 3-di-oleoyloxy-2- (6-carboxymethyl) propylamide, from Boehringer Mannheim) or Dmrie C (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide, from Gibco BRL) either with 1 .25 μg or 2.5 μg of plasmid DNA in serum-free OptiMEM (from Gibco BRL) Fetal bovine serum (FBS) was immediately added to each well to a final concentration of 10% (vol / vol). determined as a better lipid than Dosper. The cells were incubated for 24 hours before heating and 24 hours after heating before quantitation of I L-2. Studies of heat-induced amplification. In a set of experiments, the cells transfected with the plasmids pC8, mp 12 or p007 were assayed by I L-2 expression activity. I L-2 was measured from tissue culture supernatants by ELISA using the I L-2 EASIA set (Medgenix Diagnostics, Fleurus, Belgium). The sensitivity of the set is estimated at 0. 1 I U I L-2 / ml. The data from this set of experiments are shown in Table 4, below. This Table shows the expression levels of I L-2 in MCF7 cells, which were transfected with Dosper, heated 24 hours later, and then assayed by ELISA 40 hours after transfection. All plasmids L-27 (a plasmid used for reference expressing I L-2 driven by the CMV promoter), 007, f1 2 and C8 were tested.

TABLE 4 I. U. of IL-2 Temperature: 37 ° C 39 ° C 41 ° C 42 ° C 44 ° C Duration of continuous continuous shock 1 h 1 h 0.5 h thermal: Lipid only 2.03 0.50 0.41 0.53 0.53 L-27 14.2 9.88 5.95 9.88 7.80 007 336.76 318.49 334.02 373.74 389.27 F12 8.40 6.88 49.93 60.02 88.13 C8 9.19 8.03 1 1 .74 8.73 16.37 From this study it can be seen that pf 12 responds to heating and produces larger quantities of the thermal shock amplifier construction of I L-2 than does pC8 or pL-27. At 37 ° C, pf12 produces 5 times more I L-2 than its control driven by CMV, L-27. When the cells were subjected to thermal shock at 39 ° C overnight, mp 1 2 produced 7 times more I L-2 than the controls driven by CMV at 37 ° C. A heat shock treatment of 1 hour at 41 ° C or 42 ° C increased the expression of the amplifying constructions by as much as 26 times, compared to the control vector driven by CMV at 37 ° C. (However, plasmid 007 at 37 ° C is already almost near its maximum activity and does not increase expression levels mostly with heat.) The activity of pf 12 is also at a high level at 37 ° C. These results showed that the amplifying strategy can increase the levels of gene expression at temperatures between about 37 ° C and about 42 ° C. In a different set of experiments, variations in transfection efficiencies were considered to respond to co-transfection with a control plasmid, in which the CMV promoter was upstream of β-galactosidase. The general protocol for these experiments was to transfect cells 24 hours after the subculture, thermally shock cultures for an additional 24 hours thereafter, change the culture medium and then collect medium for measurement of IL-2 levels 24 hours later. As seen in Table 5, below, the activity of the CMV promoter was affected only minimally after the thermal shock. The activity of the minimal heat shock promoter was very low in cells maintained at 37 ° C and was induced about 20 times by thermal shock at 42 ° C. As seen in the stably transfected cells, the thermal shock promoting activity was only about half that of the CMV promoter.

TABLE 5 Expression of interleukin -2 (IL2) * Promoter Vector 37 ° C 42 ° C ** Times (42/37) Relative *** L27 CMV-IL2 82.6 93.4 1.1 1.0 C8 HSP-MCS 84.7 70.6 0.8 1.9 HIV1-IL2 f11 HSP-IL2 2.3 54.0 23.7 0.4 (1) f12 HSP-TAT 107.6 347.4 3.2 6.9 (17) - HIV1-IL2 007 HSP-TAT 747.5 1642.9 2.2 83.3 (208) HIV2-IL2 * values in IU of IL2 produced per mg of cellular protein in 24 hours ** heat shock was for 1 hour based on values of 42 ° C and co-transfection with CMV-β-gal The HIV1 promoter, in the absence of tat expression, was similar to the CMV promoter and was almost independent of thermal shock. However, when the minimum heat shock promoter was used to express tat, the expression of the reporter gene was dramatically increased after 42 ° C of thermal shock. In cells transiently transfected with heat shock promoter / iaf and HIV1 / IL-2, the production of IL-2 was similar to that for the heat shock promoter / MCS and HIV1 / IL-2 in cells maintained at 37 ° C. This activity was increased about 3 times and at levels almost 7 times higher than the activity of the CMV promoter by itself, after 42 ° C HS.

The transfected cells of H IV2 / I L-2 and HS / .af promoter showed a substantial reporter gene expression in cells maintained at both 37 ° C and after thermal shock at 42 ° C. The relative activity of promoter, measured by the production of I L-2 was about 80 times greater than that of the CMV promoter alone. The temperature regulation was reduced with the reporter gene expression approximately 2 times higher after heat shock of 42 ° C, compared with the same activity in cells maintained at 37 ° C. The temperature dependence of the reporter gene expression was not influenced by the presence of a second promoter. As shown in Table 6, below, the reporter gene expression, in cells transiently transfected with the plasmid containing minimal heat shock promoter / faf and H IV2 / IL-2, was increased in a temperature-dependent manner between 37 and 44 ° C. These results are qualitatively similar to those seen in FIGS. 4 and 6 for cells stably transfected with only the minimal thermal shock promoter.

TABLE 6 Expression of I L-2 (IU / ml) * Promoter Vector 37 ° C 39 ° C 40 ° C 41 ° C 42 ° C 44 ° C C8 HSP-MCS 7 ~ 2 973 670 4 ~ 8 5 ~ 3 770 HIV1 -IL2 fl2 HSP-TAT 40.6 - - - 133.1 HIV1 -I L2 007 HSP-TAT 224 222 230 250 375 470 HIV2-I L2 Cancer cells Chest MCF7 were transiently transfected with vectors as shown; with thermal shock for 1 h, 24 hours later; the media was collected and I L2 was measured 24 hours after the thermal shock.

EXAMPLE 4 Animal Studies Mouse models of human cancer, with the histological characteristics and metastatic potential that resemble tumors seen in humans, can be treated with the therapeutic compositions of the present invention. In one embodiment of the present invention , SCI D mice are injected with human tumor cells stably transfected with reporter constructions, in which the HSP70B promoter is driving TAT expression and in which the H IV-1 or H IV-2 promoter is driving either the Expression of I L-2 or EG FP After the tumors have grown to an appropriate measurable size of for example, 1 cm in diameter, the tumors are heated using ultrasound at temperatures up to about 42 ° C. is quantified at various times after warming either by removing the tumor, making tissue cuts and measuring the fluorescence of EGFP or measuring tissue levels. or of IL2 tumor using ELISA. Using another embodiment of the present invention, human tumor cells are injected into SCI D mice. The tumors are grown to an appropriate measurable size and injected with DNA-lipid complexes. Tumors are heated using ultrasound and gene expression measured at moments after heating. The efficacy of these treatments is indicated by a decrease in tumor size, a decrease in metastatic activity, a decrease in cell proliferation or an arrest in tumor growth, as a result of the administration of the therapeutic compositions of the tumor. present invention. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed, should not be unduly limited to such specific embodiments. In fact, various modifications of the modes described for carrying out the invention, which are obvious to those skilled in the art, are intended to be within the scope of the following claims.

LIST OF SEQUENCES < 110 > Tsang, Tom Gerner, Eugene W. Harris, David T. Hersh, Evan < 120 > Hyperthermic inducible expression vectors for gene therapy and methods of using same < 130 > 15907-0016 < 140 > < 141 > < 150 > US 60 / 064,088 < 151 > 1997-11-03 < 160 > 1 / < 170 > Patentln Ver.2.0 < 210 > 1 < 211 > 469 < 212 > DNA < 213 > Homo sapiens < 400 > 1 ggatcctcca cagccccggg gagacctcgc ccctaaagtt gccgctt tg cagctctgcc 60 acaaccgcgc gccctcagag ggagctasaa ccagccggca gtttctttca ccttccccgc 120 gcagccctga gtcagaggcg ggctggcctt gcaagtagcc ccccagccrt cttcggtctc 180 acggaccgat ccgcccgaac cttctcccgg ggtcagcgcc gcgctgcgcc gcccggctga 240 ctcagcccgg gcgggcgggc gggaggct cgactgggcg ggaaggtgcg ggaaggttcg 300 cggcggcggg gtcggggagg tgcaaaasge tgaaaagccc gtggacggag ctgagcagat 360 ccggccgggc tggcggcaga gaaaccgcag ggagagcctc actgctgagc gcccctcgac 420 gcgggcggca gcagcctccg tggcctccag catccgacaa gaagcttac 469

Claims (10)

1 . A method for effecting the expression of a selected polynucleotide in a mammalian cell, comprising: (a) providing an expression construct, said expression construct (i) comprising an inducible promoter operably linked to a gene encoding an transactivating factor; and (ii) a second promoter operably linked to said selected polynucleotide, wherein said second promoter is activated by said transactivating factor; 10 (b) introducing said expression construct into said cell; and (c) subjecting said cell to conditions that activate said inductible promoter; wherein said conditions result in the expression of said selected polynucleotide.

2. The method of claim 1, wherein said inducible promoter is a heat shock promoter and the conditions that activate said inducible promoter are hyperthermic conditions.

3. The method of claim 2, wherein said hyperthermic conditions comprise a temperature between about

20 basal temperature for said cell and approximately 42 ° C. The method of claim 3, wherein said hyperthermic conditions comprise a temperature between about 37 ° C and about 42 ° C.

5. The method of claim 4, wherein said hyperthermic conditions comprise a temperature between about 38 ° C and about 41 ° C.

The method of claim 5, wherein said hyperthermic conditions comprise a temperature between about 39 ° C and about 40 ° C.

The method of claim 2, wherein said heat shock promoter is derived from a promoter selected from the group consisting of the promoters HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25, ubiquitin and HSP28.

8. The method of claim 1, wherein said inducible promoter comprises a hypoxia responsive element. The method of claim 1, wherein said second promoter is selected from the group consisting of an H IV-1 promoter and an IV-2 H promoter, and said transactivating factor is tat. The method of claim 1, wherein the expression of said selected polynucleotide results in the production of a polypeptide, a protein, a ribozyme or an antisense nucleic acid. eleven . The method of claim 1, wherein said selected polynucleotide encodes a protein selected from the group consisting of ornithine decarboxylase antisense protein, p53, p16, neu, I L1, I L2, I L4, I L7, IL 1 2, I L1 5, ligand FLT-3, GM-CSF, G-CSF, I FN ?, I FNa, TN F, HSV-TK, I-CAM 1, HLA-B7, and TI M P-3.

The method of claim 1, wherein said expression construct further comprises a gene encoding a selectable marker. The method of claim 1, wherein said construction of

(5) further comprises (i) a second polynucleotide selected operably linked to said second promoter, and (i) an internal rhombosome entry site positioned between said first and second selected polynucleotides 14. The method of claim 1 , wherein said cell is a tumor cell 15. The method of claim 1, wherein the introduction of said expression construct into said cell is mediated by a delivery vehicle of the group consisting of liposomes, retroviruses, adenovirus, adeno-associated virus, lentivirus, herpes simplex virus and vaccinia virus 16. The method of claim 1, wherein the introduction of said expression construct into said cell occurs in vitro. 1, wherein the introduction of said expression construct into said cell occurs in vivo 18. A method for providing a subject with a therapeutic amount. A method of providing a first expression construct, said expression construct comprising an inducible promoter operably linked to a gene encoding a transactivating factor;

(b) providing a second expression construct, said second expression construct comprising a second promoter operably linked to said selected polynucleotide, wherein said second promoter is activated by said transactivating factor; (c) introducing said first and second expression constructs into a cell of said subject; and (d) subjecting said cell to conditions that activate said inducible promoter, wherein the expression of said selected polynucleotide is induced by said conditions.

9. The method of claim 18, wherein said inducible promoter is a heat shock promoter and the conditions that activate said inducible promoter comprise a temperature between about the basal temperature and about 42 ° C. The method of claim 1, wherein said first and second expression constructs are in the same vector. twenty-one . The method of claim 20, wherein the introduction of said expression constructs into said cell occurs ex vivo. 22. The method of claim 18, wherein the introduction of said expression constructs into said cell occurs in vivo. The method of claim 18, wherein the expression product of said selected polynucleotide is harmful to a pathogen in said subject, wherein said pathogen is selected from the group consisting of viruses, bacteria, fungi, and parasites. 24. The method of claim 1 8, wherein the product of expression of said selected polynucleotide inhibits the growth of said cell.

25. The method of claim 18, wherein the expression product of said selected polynucleotide replaces a protein deficient in said subject. 26. The method of claim 18, wherein the expression product of said selected polynucleotide promotes nerve regeneration.

27. A method for treating cancer in a mammal, comprising the steps of: (a) providing an expression construct, said expression construct (i) comprising an operably inducible promoter

10 linked to a gene encoding a transactivating factor; and (i) a second promoter operably linked to a selected polynucleotide, wherein said second promoter is activated by said transactivating factor;

(b) introducing said expression construct into a tumor cell; and (c) subjecting said tumor cell to conditions that activate said inducible promoter, wherein said conditions result in the expression of said selected polynucleotide and the expression product of the selected polynucleotide is expressed in an amount effective to inhibit the growth of said tumor cell. The method of claim 27, wherein said inducible promoter is a heat shock promoter and the conditions that activate said inducible promoter are hyperthermic conditions, comprising a temperature between about the basal temperature and about 42 ° C. 29. The method of claim 27, further comprising treating said tumor cell with at least one established form of cancer therapy, which is selected from the group consisting of external beam radiation therapy, brachytherapy, chemotherapy, and surgery. 30. The method of claim 27, further comprising: (d) treating said tumor cell with the radioprotector WR-33278 or WR-1065

He 5 after submitting to hyperthermic conditions; and (e) in a final step, treating said tumor cell with radiation therapy, wherein said polynucleotide encodes ornithine decarboxylase anti-protein protein. 31 The method of claim 27, wherein said mammal is a human. The method of claim 27, wherein said cancer is selected from the group consisting of cancer of the brain, lung, liver, bladder, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, chest , endometrium, prostate,

15 testicle, ovary, skin, vulva, cervix, head and neck, esophagus, bone marrow and blood. 33. A method for eliciting an immune response in a mammal comprising: P (a) providing an expression construct, said expression construct (i) comprising an inducible promoter operably linked to a gene encoding a transactivating factor; and (ii) a second promoter operably linked to a selected polynucleotide, wherein said second promoter is activated by said transactivating factor; (b) introducing said expression construct into a cell in the mammal; and (c) subjecting said cell to conditions that activate said inducible promoter, wherein said conditions result in the expression of said selected polynucleotide and the expression product of the selected polynucleotide is expressed in an amount effective to elicit an immune response in said mammal. , said immune response being selected from the group consisting of a humoral immune response and a cellular immune response. 34. The method of claim 33, wherein the inducible promoter is a heat shock promoter and the conditions that activate said inducible promoter are hyperthermic conditions comprising a temperature between about basal temperature and about 42 ° C. 35. The method of claim 33, wherein the immune response is directed against said cell. 36. The method of claim 35, further comprising treating said cell with an established form of cancer therapy selected from the group consisting of chemotherapy, external beam radiation therapy, brachytherapy, and surgery. 37. The method of claim 33, wherein said mammal is a human. 38. A method for altering the genetic material of a mammal, comprising: (a) providing an expression construct, said expression construct (i) comprising an inducible promoter operably linked to a gene encoding a transactivating factor; and (ii) a second promoter operably linked to said selected polynucleotide, wherein said second promoter is activated by said transactivating factor; and (b) introducing said expression construct into a cell of said mammal. 39. An expression construct comprising: (a) a gene encoding a transactivating factor; (b) an inducible promoter operably linked to said gene; (c) a selected polynucleotide; and (d) a second promoter operably linked to said selected polynucleotide, said second promoter being activated by said transactivating factor. 40. The expression construct of claim 39, wherein said inducible promoter is a heat shock promoter and expression of said selected polynucleotide is induced by hyperthermic conditions, said hyperthermic conditions comprising a temperature between about 37 ° C and about 42 ° C. . 41 The expression construct of claim 40, wherein said heat shock promoter is derived from a promoter selected from the group consisting of promoters HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25, ubiquitin and HSP28. 42. The expression construct of claim 39, wherein said inducible promoter comprises a hypoxia response element.

43. The expression construct of claim 39, wherein said second promoter is selected from the group consisting of HIV-1 promoter and HIV-2 promoter and said transactivating factor is selected from the group consisting of tat. < ? 44. The expression construct of claim 39, wherein the expression of said selected polynucleotide results in the production of a polypeptide, protein, ribozyme or antisense molecule. 45. The expression construct of claim 39, wherein said expression construct further comprises (i) a second

10 selected polynucleotide operably linked to said second promoter; and (ii) an inner ribosome entry site positioned between said first and second selected polynucleotides. 46. A cell comprising the expression construct of claim 39. 15

r

SUMMARY

Methods and compositions for transgenic expression in target cells are provided. Expression constructions are provided using an inducible amplification system to induce the expression of a therapeutic gene or other gene of interest in mammalian host cells, as well as methods. The inducible expression of transgenes at high levels under physiological conditions results from the induction by hyperthermic conditions relative to the basal temperature of the host cells.