MXPA00008367A - Self-regulated apoptosis of inflammatory cells by gene therapy - Google Patents

Abstract

This invention relates to chimeric nucleic acids and to the therapeutic induction of apoptosis in activated inflammatory cells, or cells at a site of inflammation, by introducing into those cells the chimeric nucleic acid. The chimeric nucleic acid having at least one TNF&agr;promoter enhancer attached to a functional copy of a TNF&agr;promoter and further attached to at least one copy of an apoptosis-inducing gene, which is further attached to a 3'UTR. The apoptosis-inducing gene is Granzyme B. The invention also relates to methods of making and using self-regulated apoptosis chimeric nucleic acids and pharmaceutical compositions containing them for treating inflammatory diseases.

Description

SELF-REGULATED APOPTOSIS OF INFLAMMATORY CELLS THROUGH GENE THERAPY Related Request Data This application claims the priority of the US application. serial number 60 / 039,266 in process, filed on February 2, 1997.

TECHNICAL SECTOR OF THE INVENTION The present invention relates to the fields of molecular biology and immunology. More specifically, this invention relates to the induction of apoptosis in inflammatory cells by introducing into these cells a gene that induces apoptosis (programmed death of the cell or non-necrotic death of the cell) in these cells.

Background of the Invention In many inflammatory states, cytokines such as IL-1β, IL-10, GM-CSF and TNFα are produced excessively as a result of mass aggregation and accumulation of inflammatory cells (Brennan FM et al., Bptish Medical Bulletin 1995, 51/2 / 368-38).

The upregulation and / or dysregulation of cytokines in inflamed tissue can be directly or indirectly REF: 122667 responsible for the exacerbation of chronic inflammatory diseases. For example, the most severe pathology in rheumatoid arthritis (RA) is exhibited at the local site of inflammation (ie, the smovial joints). Therefore, it is likely that the cytokines produced in the smoothed joints of RA patients play an important role in the disease process. Of these cytokines, it is thought that IL-1β and TNFα are responsible for the destructive cartilage destruction and bone erosion that characterizes RA (Dayer JM et al., J. Exp. Med., 1985, 162, 1208-12-15; Go in M. Et al., Nature, 1983, 306, 378-380). The presence of excessive amounts of IL-lß and TNFa in the smovial joints has been shown to accelerate the development of collagen-induced arthritis in rodents (Brennan FM, et al., Clin.Expt. Immunol., 199, 97/1, 1 -3). Apoptosis is a fundamental physiological process for the development of the embryo and the conservation of tissue homeostasis. (Raff, M.C. Nature, 1992, 356, 397; Vaux, D.L. et al., Cell, 1994, 76, 777). The instability in this critical natural process is externalized in a variety of neoplastic, neurodegenerative and autoimmune diseases (Thompson, C.B., Science, 1995, 267, 1456). Biochemical attributes, which involve the cascade of signal transduction, are relatively complex and not fully understood. A variety of stimuli, including activation of specific receptors such as TNFR1 or Fas, trigger the evolutionarily conserved execution machinery that involves several signaling components that are orchestrated to cause cell death (Ashkensazi, A. Y Dixit, VM, Science , 1998, 181, 1305). Granzyme B is a sepá protease, which is mainly found in cytostatic T lymphocyte granules and natural exterminating cells. Granzyme B plays an important role in the Induction of apoptotic changes in target cells by cytotoxic cell mediated killing (Huesel JW et al., Cell, 76, 977-987, 1994; Shi, L. et al., J. Exp. Med. 175, 1521-1529 , 1992), in part catalyzing the excision and activation of several caspases (? Alvesen, G.S. and Dixit, V.M., Cell, 91, 443-446, 1997), as well as by independent caspase pathways (Andrade, F. et al., Immumty 8, 451-460, 1998). Structurally, Granzyme B is produced in the form of a polypeptide that contains a peptide conductor separated by a dipeptide activator (Gly- Glu) from the active polypeptide of Granzyme B. Like caspases, Granzyme B specifically recognizes substrates in aspartic acid for cleavage. TNFa is a cytokine, mainly synthesized by monocytes, macrophages and lymphocytes in response to activation. The classical elements that govern its expression are located in the region of the proximal or distal promoter (reviewed in Pauli, U. Critical Rev. in Eukaryotic Gene Expression, 1994, 4, 323-344). Next, we summarize regions that have been described by playing an important role in the activity of the TNFa promoter: a) it was shown that the elements that respond to TNFa were located between the base pairs -100 to -125. The region of -108 to -101 bp contains a palindrome, TGAGCTCA, which is similar to an AP-1 sequence that contains elements that respond to PMA. Multiple copies of -125 to -85 bp confer an induction of expression, from 7 to 11 times of the reporter gene (Leitman, D. et al., J. Biol. Chem. 266, 9343, 1991). b) It was demonstrated that the elements that respond to PMA were present between the base pairs -101 to -286 (Hensel, G. et al, Lymphokine Res. 8, 347, 1989). c) It was shown that the response elements induced by anti-CD3 antibodies (as well as induced by Ca ionophore) were between the base pairs -118 to -80. The kappaB3 sequence (GGGTTTCTCC) in this region is of great importance for the CsA-sensitive activation of the TNFa promoter by the Ca ionophore. It is suggested that these elements are optically functional in the context of their own promoter.

(Goldfield, et al., J. Exp. Med. 178, 1356, 1993), d) in U937 cells the element that responds to PMA is located between the bp -95 to -36 and the element that responds to cAMP (CRE) is represented on the map in the position -107 to -99 bp . This region does not respond to PMA (Economou, J. S. et al., J. Exp. Med. 170, 321, 1989). e) All three kappaB sites [namely kappa Bl (-587 to 577), kappaB2 (-210 to -202) and kappaB3 (-98 to -87)] linked virus-inducible protein, although the deletion of these sites did not affect inducibility by the virus (Goldfield, A. et al, PNAS, 87, 9769, 1990). In addition, deletion mutants of kappaB sites demonstrated that they are not the main targets for PMA stimulation of the human TNFa gene (Goldfield, A. et al J. Exp. Med. 174, 73, (1991). the murine system, the promoter constructs of TNFa -1059, -695 and -655 bp are strongly inducible opr LPS. This inducibility by LPS was greatly reduced in a construction of -451 bp and, in addition, between bp -301 and -241. The -1059 bp fragment of the TNFa promoter was quiescent in macrophages and was strongly expressed after stimulation with LPS. The greatest activation drop occurred in bp -695 to -655 which contains a kappaB element in the murine TNFa promoter (Shakhov, A.N. et al., J. Exp. Med., 1990, 171, 35; Drouet, C. et al., J. Immunol. 1991 147, 1694). It is known that the elements in the 3 'untranslated region (3'UTR) of the TNFa gene are important for post-trans-transcriptional regulation. The analysis of the influence of 3'UTR has been carried out in the murine system, in which, in conjunction with the homologous promoter, the lducibility by LPS was very intense. By using the murine TNFa promoter system, it was shown that 3'UTR effectively inhibits CAT activity in three non-macrophage cell lines, namely HeLa, NIH3T3 and L929. The TTATTTAT sequence was repeated several times in the 3'UTR, and it was proposed that it was involved in the regulation (Han, J., et al., J. Immunology, 1991, 146, 1843-1848; Cra ford, F.K., et al., J. Biol.

Chem., 1996, 271, 22383-22390). A variety of cells are present in inflamed joints such as activated macrophages, activated T cells, macrophage-like smoviocytes as well as fibroblast-like smoviocytes, and synoviocytes similar to transformed macrophages (also referred to as pancreatic cells). A mvasora structure, called the pannus, is derived of the hyperplastic nature of smoviocitos and panocitos. Pannus can result from excessive proliferation of cells and / or decreased apoptosis in these cells. It was shown that the proliferation index in these cells was relatively low. Therefore, hyperplasia in synoviocytes could be due to abnormalities in the apoptosis of the smovial lining. In the synovium, the frequency of cells with end-stage apoptosis is low. Abnormalities of p53 mutations, which could result in resistance to apoptosis, are reported in the smovial fibroblasts of RA. Additionally, excessive amounts of pro-mflamatory cytokines such as TNFa and IL-1β are produced in the smovial tissue by a variety of cartilage-pannus joint cell types, including macrophage lineage cells, macrophage-like smoviocytes, cells Activated T and, possibly, fibroblast-like synoviocytes (Chu CQ et al., Arthptis &Rheumatism, 1991, 34, 1125-1132, Delehan BW, et al., Arthritis S Rheumatism, 1992, 35, 1170-1178). This perpetuates the infiltration of inflammatory cells and the production of more cytokines and pro-inflammatory factors that are responsible for the proliferation of cells synovial. In addition to the inflammatory effects described above, TNFa plays a ubiquitous and major role in a variety of pro-mflamatory events. TNFa induces the activity of IL-lß in 5 monocytes. In fact, anti-TNFα neutralizing antibodies have been shown to reduce the overall production of IL-1β (Portillo, et al., Immunol., 1989, 66, 170-175; Brennan FM, et al., Bptlsh Medical Bulletm 1995, 51 / , 368-38). So, an added benefit to blocking of the effect of the inflammatory cytokine TNFα was the reduction of the production of the inflammatory mediator inflammation, IL-1β. In addition to this, it is well known that TNFa is a transcriptional activator of other genes related to inflammation. For example, the presence of TNFa stimulates the production of other cytokines (such as GM-CSF) and cell surface receptors, including HLA class II antigens and adhesion molecules (Alvaro-García JM, et al., J Exp. Med., 1989, 146, 865-875) that perpetuate the recruitment of activated T cells and neutrophils, resulting in smoovial inflammation and hyperplasia and, ultimately, increased destruction of cartilage and bone (Alien JB, J. Exp. Med., 1990, 171, 231).

Conventional therapy against inflammatory disorders is typically directed against symptomatic inflammation. A therapy of this type provides only temporary relief without significantly slowing the progression of the disease. In contrast, it is likely that therapies directed against TNFa and other factors induced in the inflammatory process are more promising. For example, in models with animals with collagen-induced arthritis, an anti-TNFα antibody and soluble TNFα-IgG receptor chimera effectively reduced leg swelling, joint complications, and cartilage and bone destruction (Williams RO et al. al., Proc. Nati. Acad.? ci., 1992, 89, 9784-9788). Assays with humans using both humanized anti-TNFα antibodies and chimeric TNFα-IgG receptor molecules produced dramatic results (Elliot MJ, et al., Arthirtis and Rheumatism, 1993, 36, 1681-1690, Elliot MJ, et al., Lancet, 343, 1105-1110). Although treatment with these TNFa antagonists appears to be well tolerated, it also results in the production of antibodies against the recombinant proteins. Thus, these therapies may not be suitable for long-term treatment and do not achieve a real decrease in the disease.

WO 97/07828 describes methods for treating, by gene therapy, a patient with a cellular accumulation or a chronic inflammatory disease that was the result of a gene deficient in the regulation of apoptosis, more specifically p53. The treatment re-establishes the defect with a wild-type gene attached to a promoter that drives the expression of the regulatory gene of apoptosis. In order to truly modify the progress of the disease, TNFa must be continuously targeted using specific therapies for TNFa. A continuous therapeutic protocol of this type was not practical with these biological agents and would be difficult to administer in the long term. In an alternative therapeutic option, inflamed smovio that can be removed using a surgical smovectomy (Herold N. Y? Chroder HA, Acta Orthop.Scand., 1995, 66, 252-S54; Ogilvie-Harps DJ and Weisleder L., Arthroscopy , 1995, 11, 91-95), chemistry (Cruz-Esteban C. and Wilke WS, Bailliere's Clinical Rheumatol., 1995, 9, 787-801) or radiation-induced (Cruz-Esteban C. and Wilke WS, Bailliere's Clmical Rheumatol., 1995, 9, 787-801). After arthroscopic surgery marginal to good results were obtained. The non-surgical synovectomy is it effects using various chemical agents such as osmic acid, alkylating agents such as mustard nitrogen and thiotepa, methotrexate. Unfortunately, non-surgical smovectomies (including chemistry and radiation-induced smovectomies) are procedurally complicated, provide only a short-term relief and show only a non-uniform reduction of synovial hyperplasia. In addition, most non-surgical alternatives are potential teratogens. In addition, an innate inflammatory response is concomitant with tissue injury arising from chemical or surgical intervention. Finally, it should be noted that these methods suffer from the risks and side effects commonly associated with conventional pharmaceutical therapy and hostile surgical procedures, including the expense and discomfort of hospitalization and rehabilitation. Accordingly, there remains a need for an effective therapeutic method for treating inflammatory disorders in general, and RA in particular.

Summary of the Invention The invention provides methods and compositions related to a new destruction of cells producing TNFa, induced by apoptosis. Therefore, it was an object of the invention to provide unique chimeric nucleic acid molecules having at least one region of the TNFa promoter enhancer (comprising SEQ ID N0: 4, SEQ ID N0: 5; SEQ ID NO: 11 , SEQ ID N0: 12 of nucleic acids or a conservative substitution or allelic variants thereof) linked to a TNFa promoter, the TNFa promoter also being linked to a nucleic acid sequence encoding the Granzyme B protein or a conservative substitution or allelic variants thereof, which, in turn, was additionally linked to a 3 'UTR nucleic acid sequence. Another objective of this invention was to provide TNFp-AIG and similar chimeric nucleic acid constructs, procedures for producing them, methods of using them and preparations containing them.

It was a further object to provide a method for treating an inflammatory disorder by administering to a patient in need of such a treatment a pharmaceutically effective amount of the composition having the chimeric nucleic acid molecule described herein.

It was still a further objective of this invention to provide a method for the induction of apoptosis in cells transfected with the chimeric nucleic acid TNFp-AIG, a method for the selection of somatic variants of non-TNFa somatic cells in a population, a method to identify dominant / negative genes responsible for the genesis of a non-TNFa producing population and a method to identify the products responsible for the regulation of TNFa production. These and other objects will be readily appreciated by one of ordinary skill in the art based on the following detailed description of the invention.

Brief Description of the Figures Figure 1 is a schematic representation of chimeric TNFp-AIG nucleic acids of this invention. The apoptosis-inducing gene (AIG) is Granzyme B. Figure 2 is a schematic illustration showing the results of gene therapy using chimeric TNFp-AIG nucleic acids of this invention.

Figure 3 is a summary of the deletion constructs used for the identification of the inducible cis elements of the TNFa promoter using the expression of the luciferase gene (Luc) as a reporter system. Figure (a) shows the results of the representative experiment carried out to establish the expression of the luciferase gene driven by the deletion constructs of the TNFa promoter in transiently transfected Jurkat cells. Histograms represent the stimulation index as a measure of inducibility by the PMA activating agent. Jurkat cells produce TNFa after stimulation with PMA. Figure 4 (b) shows the results of the representative experiment carried out to establish the expression of the luciferase gene driven by the deletion constructs of the TNFa promoter in transiently transfected THP-1 cells. The histograms represent the stimulation index as a measure of inducibility by the activating agent, LP? . THP-1 cells produce TNFa after stimulation with LPS.

Figure 5 is a flow diagram for the preparation of TNFpGB using native elements selected from the TNFα and Gfanzyme B promoter. Figure 6 (a and b) provide a summary of results from representative experiments performed to observe the expression of chimeric Granzyme B TNFp (TNFpGB). The expression of various clones of TNFpGB constructs are expressed in transiently transfected Jurkat cells (Figure 6 a). The expression of Granzyme B was established by Western blot analysis using anti-Granzyme B antibodies. Bands representing Granzyme B in transfected cells are identified by arrows. The induction of apoptosis by the expression of chimeric nucleic acids TNFpGB was established by transient transfection in Jurkat cells (Figure 6b). Apoptosis was established by cell death ELISA. In both experiments, the histograms with dashes represent the unstimulated control, in which the cells were transfected with a chimeric nucleic acid shown, and the transfected cells were not stimulated with PMA. The black histograms represent the induction of apoptosis after stimulation with PMA, in the control transfections (transfected with -1005Luc3'UTR) or chimeric nucleic acids expressing Granzyme B. Figure 7 (a and b) is a schematic representation of a chimeric TNFp-AIG nucleic acid of this invention comprising multiple copies of the transducible cis elements of the promoter of TNFa that, in turn, drive the expression of the AIG (figure 7a). A schematic representation of a TNFpAIG chimeric nucleic acid comprising multiple copies of the cisducible elements of the TNFa promoter, which drives the expression of the AIG, in which, located below it, is the 3'-untranslated region of the gene TNFa (TNF3'UTR) (figure 7b). 3'UTR of the TNFa gene was involved in the regulation of the transducing expression of TNFa (Han, J., et al., J. Immunology, 1991, 146, 1843-1843, Cra ford, EK, et al., J. Biol. Chem., 1997, 272, 21120-21137, and Figure 9). Figure 8 (a and b) are flow diagrams of reaction schemes for preparing chimeric superpromotor constructs of TNFa-Granzyme B. Figure 9 shows a summary of the results of the experiments to demonstrate the regulatory effect of TNF3'UTR on the Infusible expression of the luciferase reporter gene. The Transient transfection was performed on a fibroblast cell line. The dot histograms represent the inducibility of TNFpLuc in the absence of TNF3'UTR, and the black histograms represent the inducibility of TNFpLuc in the presence of TNF3'UTR. Similar results are obtained in the Jurkat cells.

Figure 10 is a schematic representation for the selection of non-TNFa somatic cell variants within a population of TNFa-producing cells and the identification of dominant negative suppressor genes responsible for the inhibition of TNFa production. Fig. 11 (a) is a schematic representation of the reinforcer regions (ER) identified ER1 (SEQ ID NO: 4), ER2 (? EQ ID NO: 5), ER3 (SEQ ID NO: 11) and ER4 (? EQ ID NO: 12) of the TNFα promoter and the insertion of two copies of said ER 's above the native TNFα -120 promoter. Figure 11 (b) shows the results of experiments carried out to establish the expression of the luciferase gene driven by the native TNFa-120 promoter to which at the 5 'end two copies of reinforcer regions ER1, ER2, ER3 and ER4 are bound. . The construction that drives the expression of the luciferase gene was in Jurkat cells transiently transiected. The histograms represent the stimulation index as a measure of inducibility by the PMA activating agent. Jurkat cells produce TNFa after stimulation with PMA. Figure 11 (c) shows the results of experiments carried out to establish the expression of the luciferase gene driven by the native TNFα-120 promoter to which at the 5 'end two copies of reinforcer regions ER1, ER2, ER3 and ER4 are bound. . The construct that drives the expression of the luciferase gene was in transiently transfected THP-1 cells. The histograms represent the stimulation index as a measure of inducibility by the activating agent LPS. THP-1 cells produce TNFa after stimulation with LPS. Figure 12 is a graphical representation of the sequence -706TNFpGB3 'UTR of the chimeric nucleic acid, wherein the sequence of the promoter fragment is indicated by the lowercase alphabet, the sequence of the linker fragment is indicated by the italic alphabet in uppercase, the sequence of the Granzyme B fragment is indicated by the alphabet in capital letters and the sequence of the fragment TNFa3'UTR is indicated by the lowercase alphabet underlined.

Figure 13 is a graphic representation of the sequence -1005TNFpGB3 'UTR of the chimeric nucleic acid, wherein the sequence of the promoter fragment is indicated by the lowercase alphabet, the sequence of the linker fragment is indicated by the italic alphabet in uppercase, the sequence of the fragment of Granzyme B is indicated by the alphabet in capital letters and the sequence of the fragment TNFa3'UTR is indicated by the lowercase alphabet underlined.

Detailed Description of the Invention The invention described herein overcomes the drawbacks in the art by providing novel chimeric nucleic acid molecules for use in therapeutic compositions and methods for using such compositions. The compositions are directed to selectively induce apoptosis in TNFa producing cells by causing the destruction of these cells. As used herein, the abbreviation 3'UTR means 3 'untranslated region. The abbreviation "AIG" refers to gene that induces apoptosis. An AIG includes Granzyme B.

The abbreviation "CsA" refers to cyclosporin A, a biologically active fungal metabolite with immunosuppressive properties. The abbreviation "DN" refers to dominant / negative gene products that have a negative effect on the expression or function of other genes or gene products. The abbreviation "ER" refers to reinforcer region, where ER1 has the? EQ ID N0: 4, ER2 has the SEQ ID NO: 5, ER3 has the SEQ ID NO: 11 and ER4 has the SEQ ID NO: 12 . The abbreviation "GB" refers to Granzyme B.

The abbreviation "PMA" refers to phorbol myristate acetate. The abbreviation "AR" refers to rheumatoid arthritis. The abbreviation "TNFa" refers to the alpha factor of tumor necrosis. The terms "TNF promoter", "TNFa promoter" and "TNFp" are used interchangeably herein. Unless stated otherwise, these terms refer to the complete nucleotide sequence corresponding to a minimal promoter sequence of native TNFα linked to one or more enhancer elements located above, whether natural, native or are made by genetic engineering in the laboratory. "Substitutions" of amino acids are defined as having amino acid replacements one by one. They are conservative in nature when the substituted amino acid has a similar structure and / or chemical properties. Examples of conservative replacements are the substitution of a leucma with an ísoleucma or valine, an aspartate with a glutamate or a tremone with a serma. "Conservative variants" refer to substitutions of amino acids in a polypeptide. "Allelic variants" refer to variation in the level of nucleic acid and protein due to conservative or non-conservative substitutions that result in an alternative form of the same gene. "Reporter" molecules are chemical residues used to mark a nucleic acid or an amino acid sequence. These include, but are not limited to radionucleos, enzymes, fluorescent agents, chemiluminescers or chromogens. Reporter molecules are associated with, establish the presence of and may allow the quantification of a particular nucleic acid or amino acid sequence.

"Reporter genes" are nucleic acids and fragments thereof that encode a functional protein such as luciferase, which can be used to establish the activity of heterologous promoters. A "functional fragment" of a polynucleotide or nucleic acid comprises all or any part of the nucleotide sequence having fewer nucleotides, which can be used as sufficient genetic material to initiate the transcription of a gene or to encode a functional subunit of a polypeptide . This invention was based on the evidence that apoptosis of inflammatory cells in certain inflammatory diseases was therapeutically beneficial. The invention relates, specifically, to self-regulated apoptosis by gene therapy. In general terms, in the practice of the invention, a chimeric nucleic acid comprising at least one promoter of the promoter linked to at least one functional copy of a minimal promoter, the promoter being a gene or a combination of genes activated in inflammatory cells or in cells at a site of inflammation, it was attached to at least one copy of an apoptosis-inducing gene (AIG), so that the expression of AIG was driven by the promoter, thus directing the inflammatory cells. The promoters of the putative genes activated in the inflammation include the nucleic acid sequence of the TNFa promoter and the conservative substitution or allelic variants thereof. Chimeric nucleic acids according to the invention comprise reinforcer, promoter and AIG elements in direct, distal or proximal binding, and combinations thereof. As mentioned above and discussed in more detail below, in some embodiments multiple copies of the enhancer, promoter and / or AIG were used for maximum efficiency.

In order that the invention described herein may be more fully understood, the following detailed description is gathered which emphasizes chimeric nucleic acids comprising at least one enhancer of the TNFa promoter linked to at least one functional copy of a promoter. minimum of TNFa, and additionally linked to at least one copy of an AIG only for illustrative purposes. Although the following examples also employ these types of constructions, it will be appreciated by experts that the basic constructions described herein can be altered to provide other embodiments using products, methods. methods and compositions of the invention with other promoters comprising motif genes activated in inflammation such as the types listed above that exhibit similar functions that can be used to direct cells at the site of infection. The apoptosis-inducing gene (sometimes referred to herein as AIG) was driven by a TNFa promoter (TNFp) or other inducible gene activated in inflammation. In one embodiment, apoptosis was selectively induced in cells capable of producing TNFa. TNFp-AIG or other chimeric nucleic acid can be conveniently introduced m vi vo using conventional gene therapy techniques. Advantageously, in the embodiment in which the chimeric nucleic acid was TNFp-AIG, this was expressed only in the cells that produced the inflammatory cytokine, TNFa. In addition, since the chimeric TNFp-AIG nucleic acid contains the elements of the TNFa promoter, it also sequesters inducible, selective TNFp transcription factors. A sequestration of this type results in a reduction in the endogenous production of TNFa. The present invention relates specifically to TNFp-AIG and constructs of similar genes, to cells that contains chimeric nucleic acids, methods for the induction of apoptosis in cells transfected with chimeric nucleic acids, pharmaceutical compositions containing chimeric nucleic acids, methods for the selection of somatic cell variants not producing TNFa within a population of TNFa-producing cells and the like, to a method for identifying dominant negative / dominant suppressor genes, responsible for the inhibition of TNFα production and to therapeutic methods using chimeric nucleic acid. In order to clarify the comment below of chimeric TNFp-AIG nucleic acids by way of example of this invention, illustrate the following sequences:? EQ ID NO: 1 is the nucleotide sequence corresponding to the sequence of the full length human TNFa promoter, as published (Takashiba S., et al., Gene, 1993, 131 , 307-308). The nucleotide numbers used in this specification refer to the numbering of this sequence. SEQ ID NO: 2 is the sequence of the native TNFa promoter of the gene that was used in this invention (-1077 nucleotides from the start site of the transcript, TSS). There are a few differences in the TNFp sequence in SEQ ID NO: 1 and SEQ ID NO: 2. Such differences in the nucleotide sequences of the TNFa promoter have been reviewed (Takashiba S., et al., Gene, 1993, 131, 307-308). ? EQ ID NO: 3 is the minimal promoter sequence of native TNFα (nucleotides -120 to T ??), which includes at least one enhancer element (site k3); see Pauli, U., Crit. Rev. in Eucaryotic Gene Expression, 1994, 4, 323-344; Rhoades K.L., et al., J. Biol. Chem., 1992, 267, 22102-22107; and Takashiba S., et al., Gene, 131, 307-308). SEQ ID NO: 4 is the reinforcer region 1 (ER1) of the TNFa promoter spanning nucleotides -1005 to -905. SEQ ID NO: 5 is the enhancer region 2 (ER2) of the TNFa promoter spanning nucleotides -706 to -517. ? EQ ID NO: 6 are the additional multiple cloning sites (MC?), Engineered upstream of the minimal TNFa promoter -120 in the -20pGL3 construct. SEQ ID NO: 7 is the 3 'untranslated region (3'UTR) of the TNFa gene (Nedm, G.E., et al., Nucleic Acid Research, 1985, 13, 6361-6373).

SEQ ID NO: 8 is the full-length Granzyme B. SEQ ID NO: 9 is truncated Granzyme B, devoid of the nucleotides encoding the conductive peptide and activating the dipeptide. SEQ ID NO: 10 is the full-length Granzyme B, which contains the nucleotides encoding the conductive peptide, but devoid of the nucleotides encoding the activating dipeptide. SEQ ID NO: ll is region 3 of reinforcer (ER3) of the TNFa promoter spanning nucleotides -234 to -120. SEQ ID NO: 12 is the enhancer region 4 (ER4) of the TNFa promoter spanning nucleotides -234 to -65. SEQ ID NO: 13 is the chimeric nucleic acid -706TNFpGB3'UTR. SEQ ID NO: 14 is the chimeric nucleic acid -1005TNFpGB3'UTR. The TNFa promoter elements for the preparation of chimeric nucleic acid constructs according to this invention were selected from elements that were capable of inducing the expression of a therapeutic gene driven by the TNFa promoter. These elements of the promoter are they will be referred to herein as "cis-inducible elements", "cis-inducible elements" or "enhancer elements" of the TNFa promoter. The enhancer elements may be physically bound to the minimal promoter sequence or separated from the minimal promoter by a linker sequence that may or may not have unique restriction sites. Thus, as outlined above, the enhancer elements can be joined directly, distally or proximally, or any combination thereof, to chimeric nucleic acids of the invention. These were typically built higher than the promoter. Examples of TNFa enhancer elements are set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 11 and SEQ ID NO: 12; Fragments or functional variants and combinations thereof can be used. Some preferred gene constructs according to this invention include those that have multiple copies of the enhancer elements, ie two or more copies. Some embodiments have about 2 to 25, more closely 2 to 10 and, even more closely, 2 to 5 copies. The terms "TNF promoter", "TNFa promoter" and "TNFp" are used interchangeably in this memory. Unless stated otherwise, these terms refer to the complete nucleotide sequence corresponding to a minimal promoter sequence of native TNFα linked to one or more enhancer elements located above (already occurring naturally, ie native , or are made by genetic engineering in the laboratory). Examples include, but are not limited to, SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and fragments, vanants and functional mixtures of any of these. Many functional fragments and variants of these TNFa and other sequences described herein share a sequence homology of at least about 80% and, in some cases, greater than 90% with respect to their native and engineered antagonists, but these are known to the experts and are defined in the references cited in this specification. This invention provides a novel therapeutic method comprising the step of introducing into the cells of a mammal a chimeric nucleic acid comprising an apoptosis-inducing gene (AIG) driven by the TNFa promoter (TNFp). Examples of chimeric nucleic acids of the invention are listed in SEQ ID NOs: 13 and 14; they can also be used fragments or vanishing functional of these. Without wishing to be bound by theory, as a result of being controlled by TNFp, AIG was expressed only in the cells that produced the inflammatory cytokine, TNFa. Therefore, any cells that express TNFa will be self-destructive, while cells that do not express TNFa will not be affected. Advantageously, this methodology can direct any TNFa producing cells such as activated macrophages, activated T cells, synoviocytes similar to macrophages and similar to fibroblasts and primary cells that reside in the joints with RA. In fact, the targeted TNF [alpha] producing cell may be one that does not usually carry or express, or normally does not, an apoptosis gene in its native and unaltered form. Therefore, by using the chimeric nucleic acids and methods of this invention, cellular sources of TNFα can be destroyed in a highly selective manner. Another advantage of using the TNFp-AIG chimeric nucleic acid of this invention was that TNFp sequesters transcription factors required by endogenous TNFp, thereby leading to a reduction in the production of endogenous TNFα. In one example, TNFp was present in a large excess in the cell therapeutically directed. This can be achieved by introducing multiple copies of the transfected gene into the cell. Alternatively, the TNFp-AIG chimeric nucleic acid according to this invention can contain multiple copies of the transducing cis elements of the TNFa promoter. As mentioned above, multiple copies of the "inducible enhancer elements" of TNFp are present in some embodiments of the TNFp-AIG chimeric nucleic acids of this invention. By including multiple copies of the cisducible elements of the TNFp construct, the transcpptional factors required by the transfected cell to produce TNFa were sequestered by the exogenously introduced sequence. This preferred TNFp-AIG chimeric construct was characterized by increased efficacy by competing for the TNFp-specific transcription factors compared to chimeric nucleic acids of this invention that contained only a single enhancer element linked to TNFp. The "super-promoter muctible" constructed in this way was able to (1) compete more effectively for the specific transcription factors of TNFa, (2) boost the expression of the apoptosis-inducing gene in an augmented manner by virtue of multiple reinforcing elements. In rheumatoid arthritis patients, synovectomy, ie the separation of the smovial tissue, has proven to be clinically beneficial. Unlike conventional and surgical synovectomy processes, the therapeutic method directed to the cells described herein is only directed to TNFa producing cells. Thus, advantageously, the introduction and expression of the TNFp-AIG chimeric gene and the subsequent induction of apoptosis do not induce an inflammatory response. Accordingly, the methods of this invention were comparatively selective and resulted in minimal tissue injury and reduced inflammation. The products and methods described herein are also useful for the treatment of other inflammatory disorders. Inflammatory disorders of this type include, but are not limited to multiple sclerosis, Guillam-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft-versus-host disease, lupus eptematosus, insulin-dependent diabetes mellitus, psoriatic arthritis, sarcoidosis , hypersensitivity pneumonitis, ankylosing spondylitis and related spoldiloarthropathies, Reiter syndrome and systemic sclerosis. Thus, this invention encompasses methods for treating an inflammatory disorder in a patient, when administering to a patient in need of such a treatment a pharmaceutically effective amount of a pharmaceutical composition having the chimeric nucleic acid according to the invention. Apoptosis was induced in inflammatory cells or cells at a site of the patient's inflammation by introducing into the cells at least one chimeric nucleic acid of the invention. This was achieved, typically, by preparing a pharmaceutical composition containing at least one chimeric nucleic acid of the invention and, typically, a pharmaceutically acceptable vehicle, and administering the composition to a patient using standard means known to those skilled in the art. . The pharmaceutical composition can be delivered directly to the site of inflammation using the topical, intravenous, mpentaneal and similar methods. In the following, an additional methodology is discussed. In addition to the therapeutic indications, the chimeric nucleic acids according to this invention can be used in a variety of useful screening and selection methods. In one such Methods, non-TNFa somatic cell variants within a population of TNFa producing cells can be selected in vi tro by introducing a chimeric TNFp-AIG nucleic acid into the population of TNFa producing cells. The cells that produce TNFa will suffer apoptois. Cells that do not produce TNFa will survive. The selection of those variants of cells that possess the survival phenotype was an easy way to identify non-TNFa-producing cells. A selection process of this type can be used to determine the expression of genes that act in trans to regulate the activity of the TNFa promoter, thereby reducing the production of TNFa. Such genes were characterized as dominant negative (DN) / dominant suppressor genes in other systems (Behrends S., et al., J. Biol. Chem. 1995, 270, 21109-21113; Zhang S., et al., J. Biol. Chem., 1995, 270, 23934-23936; Wato ich S.?., Et al., Mol. Cell Biol., 1994, 14/6, 3535-3549). In a further in vi tro method, a chimeric TNFp-AIG nucleic acid according to this invention can be used to identify negative dominant genes responsible for the genesis of a population of cells not producing TNFa. According to this method, a chimeric TNFp-AIG nucleic acid according to this invention was introduced into cells that produced TNFa. Except for the presence of a dominant negative gene, these cells should undergo apoptosis upon activation. Therefore, it can be reduced that the surviving variants possess a dominant negative gene capable of upregulating the induction of TNFa. The negative dominant gene can be easily identified by producing a cDNA library and by transfecting cell lines (eg, Jurkat and THP-1). These cells were stable transfectants of an inducible TNFp-AIG chimeric nucleic acid or TNFp-AIG cells transfected with a TNFp-luciferase gene will be selected for the survival phenotype after m vi tro activation; the survival phenotype was indicative of the effect of the DN genes. In cells transfected with the TNFp-luciferase gene, the reduction in luciferase activity will be indicative of the effect of the DN gene. Negative dominant genes identified using this protocol can be used as the future therapeutic agents themselves. Such genes will be candidates for gene therapy in order to reduce the production of TNFa. The methods used for gene transfer were grouped into two broad categories: 1. Direct approach: transduction m si t u of the therapeutic gene into target cells such as synoviocytes using a suitable vector as a vehicle for the therapeutic gene. The vector containing the therapeutic gene was injected directly into the affected area (for example, an arthritic joint). 2. Indirect approach: Ex vivo transfection of the therapeutic gene in target cells such as smoviocytes. In this approach, the smovio was removed from the joints, smoviocytes were isolated and cultured m vi tro. The cultured cells were transfected with the therapeutic gene, and genetically modified synoviocytes were transplanted back into the smovio. For the transfer, several vectors have been evaluated for their efficiency in the supply of genes (Nita, et al., Arthritis &Rheumatism, 1996, 39/5 820-828). Among the vectors used for gene therapy, vectors derived from retroviruses were by far the best developed. These were able to insert the genetic material into the host's genome and produce stable transfectants. However, these vectors were capable of infesting non-dividing cells and, since these were inserted into the host genome, the possibility of a insertional mutagenesis. In comparison, adenovirus-derived vectors infest dividing as well as non-dividing cells and episomally deliver DNA. The drawback of adenovirus-based vectors was that these vectors continue to produce vinca proteins in infested cells making them potentially antigenic. A third type of virus-based vector was derived from the Herpes simplex virus (HSV), which were also capable of infesting dividing cells as well as not dividers. Among the non-binding vector systems, cationic liposomes and simple plasmid DNA have been evaluated. The liposomes were in the most advanced phase of development, although certain types of cells, such as muscle and skin, absorbed, retained and expressed DNA from the plasm gone simple. A particle-mediated gene delivery system is also possible (Rakhmilevich, et al., PNAS, 1996, 93, 6291) and it is a promising approach. The following "m v vo" gene delivery protocols can be used to deliver the chimeric nucleic acids of this invention: (1) Nita et al., Arthritis and Rheumatism, 1996, 39, 820-823 Experiments in vi in rabbits: Each vector was injected intra-articularly into a knee joint. For viral vectors, between 10 β and 109 particles suspended in 0.5 ml of equilibrium saline were injected by knee. By knee, liposome-DNA complexes (200 nmol of DC-Chol complexed with 20 μg of DNA / ml) were injected into 1 ml of equilibrium saline. (2) Methods in Molecular Medicine: Gene Therapy Protocols, Paul Robbins, ed., 1997, Barr et al., Pages 205-212 Provision of adenovirus-based vectors to hepatocytes: rat hepatocytes 1X1011 PFU in 100 g of animal. In dogs (12-17 kg), the portal vein was perfused with approximately 1.5X10 11 PFU / kg giving a copy of the adenovirus genome per diploid copy of host DNA. In rabbits (2-4 kg), 1.5X1013 viral particles (approximately 1.5X1011 PFU) gives a 100% transduction in hepatocytes; 4X1012 viral particles give a 50-75% transduction. Yang N-S, et al., 281-296 Gene delivery mediated by gold particles: transfection of mammalian skin tissue 0.1, 0.5, 1.0 and 2.5 μg DNA / g particle in a linear relationship with expression levels in transgenes. Nabel, et al., 297-305 Gene delivery mediated by liposomes in humans: Protocol 1: 15 nmol of DC-Chol / Dope liposomes combined with 1 μg of DNA in 0.7 ml, 0.2 ml of the above mixture was injected in the nodule of the patient's melanoma. For delivery by catheter, 0.6 ml of the solution was delivered to the artery. Protocol 2: 15 nmol of DMRIE / Dope liposomes combined with 5 μg of DNA in 1.0 ml. For direct intratumoral injections, range of DNA concentrations from 3 μg complexed with 4.5 nM of DMRIE / Dope up to 300 μg complexed with 450 nM of DMRIE / Dope. (3) Roessler, et al. 369-374 Gene transfer to smovio: A dose range, 109-1012 adenovirus particles containing therapeutic gene / joint was used. However, the optimal dose for any experimental series Particular needs to be determined empirically and depended both on the properties of the genomic backbone of the recombinant adenovirus that was being used and also on the transgene that was being expressed. For the indirect approach, a variety of methods were well established, including the use of transfection based on cationic lipid or cationic polymer and electroporation. Any of the above refined techniques can be altered to suit the particular needs of ordinary persons skilled in the art. Such modifications are within the level of experience possessed by ordinary practitioners and do not require undue experimentation. These obvious variations are within the scope of this invention.

EXAMPLES In order to more fully understand this invention, the following examples are collected. These examples are intended to illustrate some preferred embodiments of this invention, and are not to be considered in any way limiting the scope of this invention.

EXAMPLE 1 Production of TNFp-Granzyme B constructs In order to construct chimeric Granzyme B driven by cis enhancer elements of the TNF promoter, either in a single copy or in multiple copies of the same region or various regions, it was effected the identification of the regions of indices responsible for the inducible optimal induction of the reporter gene.

Selection of the elements of the TNFa promoter to construct a chimeric nucleic acid The regions of the TNFa promoter were amplified by polymerase chain reaction (PCR) using primers spanning various TNFa promoter deletion constructs (Figure 3). As reference, regions identified by other investigators in various other cellular systems were used (Rhoades, et al., J. Biol. Chem., 1992, 267, 22102-22107; Leitman, et al., Mol. Cell Biol., 1992 , 12, 1352-1356; Pauli U., Cpt. Reviews Eukaryotic Gene Expression, 1994, 4, 323-344). The PCR amplified genes were then cloned in a position upstream of a reporter gene, such as luciferase, in a commercially-free promoter vector. available. These constructs were tested for their constitutive and inducible expression in various cell lines such as Jurkat (lymphoblastoid T), U973 (myelomonocytic), THP-1 (monocytic), fibroblasts and in human synoviocytes cultured in vi tro . The identification of the regions responsible for the inducible expression of the reporter gene was based mainly on the results obtained using two lines of TNFa-producing cells, namely Jurkat (after stimulation with PMA) and THP-1 (after stimulation). with LPS) (figures a and b). These cells were transiently transfected using well established and commercially available reagent methods, for example DEAE-dextran and Superfect. The cis elements of the TNFa promoter that were responsible for the inducible expression of the reporter gene were then used to construct chimeric nucleic acids TNFp-Granzyme B.

Construction of chimeric nucleic acids TNFp-Granzyme B The coding region of Granzyme B was amplified using cDNA primed with oligo-dT as template, which was obtained from lymphocytes of the Human peripheral blood activated with PHA / ant? -CD3 that were maintained in a medium containing IL-. Sense primers corresponding to codons 1-7 and 21-27 were used for the amplification of the full-length forms of Granzyme B (? EQ ID NO: 8) or truncated (? EQ ID N0: 9). The anti-sense primer used was the same in the two amplifications to give corresponding products up to the stop codon, ie residue 248. Constructs of Granzyme B were prepared with the dipeptide deleted (? EQ ID NO: 10) using Granzyme Full length B (? EQ ID N0: 8) in mold quality. Mutagenic sense and antisense complementary primers were used to create the deletion, flanking 15 nucleotides on either side of, but not including the six nucleotides corresponding to codons 19 and 20 (dipeptide mactant). The constructions were produced using a QuikChange mutagenesis kit (Stratagene). Fragments of nucleic acids encoding Granzyme B devoid of dipeptides were subcloned below the TNFa promoter by replacing the luciferase gene in the deletion constructs -706 and -1005 of the TNFa promoters (Figure 5). The complete 3 'untranslated region of the TNFa gene (SEQ ID NO: 7) was amplified by PCR and inserted downstream of the nucleic acid fragment encoding the Granzyme B gene devoid of dipeptide driven by the deletion fragments -706 and -1005 of the TNFa promoter. The complete sequence of the chimeric nucleic acids is found in SEQ ID NO '? : 13 and 14.

Construction of superpromotor chimeric nucleic acids of TNFa-Granzyme B Two broad preferred regions, namely ER1 (-1005 to -905) (SEQ ID NO: 4), ER2 (-706 to -517) (? EQ ID NO: 5) of the TNFa promoter, which contains the elements responsible for the inducible expression of the reporter gene described above (Figures 4 a and b) were amplified by PCR and ligated above the minimal native promoter (-120 to TSS,? EQ ID NO: 3) in the form of a single copy or multiple copies. Two regions plus ER3 (-234 to -120) (? EQ ID NO: ll) and ER4 (-234 to -65) (? EQ ID NO: 12) of the TNFa promoter were also identified as reinforcer regions that were used in chimeric constructions using the strategies described below. The superpromotor contains multiple cassettes (2-10) of the aforementioned regions containing inducible promoter elements (Figure 7). This was achieved by PCR amplification of the regions of interest using primers synthesized with restriction sites inserted at the 5 'end of each of the primers. These unique restriction sites flank the amplified gene product of interest. Preferably, AIG PCR amplified was cloned downstream of the TNFa superpromotor, replacing the luciferase reporter gene in the original construct as described (Figure 5) for the native TNFα promoter. The reaction schemes for the construction of a superpromotor of TNFa and the linker sequences representing unique restriction sites (these restriction sites were absent in the selected elements of the TNFa promoter and Granzyme B) for an effective direct insertion are outlined in what follows and are described in figure 8: Reaction Scheme 1: STAGE 1: Insertion of the minimal promoter of TNFa (-120 to T ??) in the vector luciferase basic (without promoter) pGL3 (Promega): In the following the elements of the basic vectors pGL3 are shown. were used for the construction of the chimeric nucleic acid TNFp-AIG. _KpnI.Sacl .Mlul.Nhel.? Mal.Xhol.BglII.HindIII. [luciferase] .Xbal_ The minimal promoter was amplified by PCR using primers containing Xhol and BglII sites. HindIII, so that Xhol was at the 5 'end and the BglII sites. HindIII were at the 3 'end of the amplified product. This fragment was inserted into the polylinker of the basic vector pGL3 using these same restriction sites. This construction was called "Construction Al" and was as follows: Ifnl. Sacl .Mul. NheISroalXhol. (-120 to TS? BglII) .HindIII. [luciferase] Xbal STAGE 2: The enhancer fragment (ER1, ER2, ER3 or ER4) was amplified by PCR using the primer containing several restriction sites. The resulting fragment will have the restriction sites Kpnl .AatlI .BssHII at the 5 'end and Nsil. ? epl .Mul at end 3 'as follows: 5' Kpnl. AatlI. BssHII. (ER1, ER2, ER3 or ER) .Nsil. Spel. Mlul 3"The fragment was inserted in the" Al construction "generated in STAGE 1 using the restriction sites Kpnl and Mlul, this construction was called" Construction Bl "and was as follows: pnl.AatlI.BssHII. (ER1, ER2, ER3 or ER4) .Nsil. ? pel .Mul.

Nhel ? mal.Xhol (-120 to TS? BglII) .HindIII. [luciferase] .Xbal_ STEP 3: The TNFa enhancer fragment (ER1, ER2, ER3 or ER4) was amplified using the restriction sites containing AatlI and BssHII primers to generate the PCR product as follows: 5 'AatII. (ER1, ER2, ER3 or ER4) BssHII 3 '. This fragment was cloned into the "Bl Construction" using these same restriction sites. This construction was called "Construction Cl" and was as follows: Kpnl .AatlI. (ER1 or ER2). BssHI I. (ER1, ER2, ER3 or ER4) .Nsil .Spel .Mul .NHel.? Mal .Xhol (-120 to T? BglII) .HindIII. [luciferase] -Xbal STAGE 4: The TNFa enhancer fragment (ER1, ER2, ER3 or ER4) was amplified using the restriction sites containing primers Nsil and? Pel to generate the PCR product as follows: 5'NsiI. (ERl, ER2, ER3 or ER4).? 3 '. This fragment was cloned into the "Cl Construction" using these same restriction sites. This construction was called "Construction DI" and was as follows: Kpnl .AatlI. (ER1, ER2, ER3 or ER4). BssHII. (ER1, ER2, ER3 or ER4). Nsil (ER1, ER2, ER3 or ER4). pei .Mul. Nhel Smal Xhol (1-20 to TSS BglII). HindIII. [luciferase] -Xbal STEP 5: The Granzyme B coding regions were PCR amplified using the primers containing the BglII and Xbal restriction sites that generate the fragment as follows: 5 'EcoRI .BglII. [Granzyme B] XbaI 3 '. This fragment was inserted into the "DI Construction" using BglII and Xbal. The construction was called "Construction El" and was as follows: Kpnl .AatlI. (ER1, ER2, ER3 or ER4). BssHI I. (ER1, ER2, ER3 or ER4) .Nsil (ER1, ER2, ER3 or ER4). Spel. Mlul. Nhel Smal Xhol (-120 to TSS BglII). HindIII. [Granzyme B] .EcorI Xbal Alternatively, Reaction Scheme 2 was as follows: Reaction Scheme 2: STAGE 1: The same as in Reaction Scheme 1 that gives rise to the "Al Construction", which was as follows: barrel ? acl .MLuI. Nhel anal. hol. (-120 to T ?? BglII) .HindIII. [luciferase] .Xbal STAGE 2: Insertion of additional multiple cloning sites (SEQ ID NO: 6).

Using commercial sources, two complementary oligonucleotides (5 'fos-selos) providing Nhel were synthesized. ? acII. EcorV.AfIII .AatlI .Avrll.

Spel. PvuII. Xhol. These oligonucleotides were annealed and then cloned into the Nhel and Xhol sites of the "Al Construction". The resulting construction was named "Construction B2" and was as follows: Kpnl. Sacl. Mlul. Nhel Sacll. EcorV.AfIII. Aatil .Avrll.

Spel .PvuII .Xhol. (-120 to T ?? BglII) .HindII. [luciferase] Xbal STAGE 3: The TNFa enhancer fragment (ER1, ER2, ER3 or ER4) was amplified using the primers containing the Spel.PvuII restriction sites at the 5 'end, and Xhol at the 3' end to generate the PCR product as follows: 5 'Spel. PvuII. (ER1, ER2, ER3 or ER4) .Xh 3 '. This fragment was cloned in the "Construction B2" using the restriction Spel and Xhol. This construction was called "Construction C2" and was as follows: _KpnI.SacI.MluI. Nh el. Sacll. EcorV. Afl II. Aat II. Avrll.

Spel.PvuII. (ER1, ER2, ER3 or ER4). Xhol. (-120 to TSS BglII) .HindIII. [luciferase] Xbal STAGE 4: The TNFa enhancer fragment (ER1, ER2, ER3 or ER4) was amplified using the primers containing the Avrll restriction sites. Spel at the 5 'end, and PvuII at the 3' end to generate the PCR product as follows: 5 'Avr II. Spel. (ER1, ER2, ER3 or ER4) .PvuII 3 '. This fragment was cloned into the "C2 Construction" using the Avrll and PvuII restriction sites. This construction was called "Construction D2" and was as follows: Kpnl. Sacl. Mlul. Nhel Sacll. EcorV.Af III. Aat 11. Avr II. Spel.

(ER1, ER2, ER3 or ER4). PvuII. (ER1, ER2, ER3 or ER4) .Xhol. (-120 to TSS BglII) .HindIII. [luciferase] .Xbal Thus, using this strategy, at least seven copies of the reinforcer regions (ER1, ER2, ER3"or ER4, individually or in combination), at one time, can be added using one or more restriction sites located higher than the one precedes in a PCR amplification of the booster regions of choice. desired number of copies of the reinforcer regions, AIG was inserted below the superpromotor as described in STEP 5 of Reaction Scheme 1. The. Inducible expression of the chimeric TNFp-Granzyme B gene was assayed by transient transfection of the cell lines mentioned above. The expression of the TNFp-Granzyme B nucleic acid was measured by detecting the apoptosis of transfected cells, making proteins expressing AIG in Western blots using commercially available antibodies and establishing the protease activity using a well-documented and commercially available specific synthetic tetrapeptide substrate.

Regulation of TNFp-driven expression of a reporter gene The 3 'untranslated region of the TNFa gene plays an important role in the regulation of TNFa biosynthesis. This was involved in the translational expression of the TNFa gene in normal non-activated states. Importantly, these elements allow for de-repression to occur when TNFa producing cells were activated by external stimuli (Han, J., et al., J. Immunology, 1991, 146, 1843-1848; Cra ford, F.K., et al., J. Biol. Chem., 1996, 271, 22383-22390). Genetic constructs were produced in which the complete 3 'untranslated region (SEQ ID NO: 13) was inserted downstream of the luciferase gene driven by deletion fragments, namely -120, -706 and -1005 of the TNFa promoter. The results of the transient expression of these constructions are summarized in figure 9. Using the strategy, 3'UTR was also inserted further under the chimeric construction Granzyme B-TNFpGB (figures 6 (a), 6 (b), 12 and 13).

EXAMPLE 2 Assay protocols Methods in vi tro: Luciferase assay: Luciferase activity was determined using commercially available reagents (Promega).

Expression of the Granzyme B gene: a) Western blots of the transfected cell lysates were developed using anti-Granzyme B antibody. B) Apoptosis of transfected cells: the apoptosis of transfected cells due to Granzyme B was determined by nucleating by propidium iodide (Krishan, A., J. Cell Biol., 66, 1994, 188-193) and by an ELI? A kit of commercially available cell death (Boehringer Manngeim).

Models with animals Model of rabbits of arthritis induced by IL-lß (Pettipher E.R., et al., Proc. Nati. Acad.? Ci., 1986, 83, 8749-8753): IL-1β was injected into the knee joints of New Zealand white rabbits. Intra-articular injection of IL-1β causes a dose-dependent infiltration of leukocytes into the joint space and loss of proteoglycan of the articular cartilage. Antigen-induced arthritis: The m-articular injection of antigens (ovalbumin) into the knee joints induces the accumulation of leukocytes and the degradation of the cartilage that is closely resembles rheumatoid arthritis in humans. Swelling of the joint after the injection was maintained for 14 days. Model with smoviocytes from mice? Cid-human beings (Houp J.M., et al., Current Opmions Rheumatol., 1995, 7, 201-205; Sack U., et al., J.

Autoimmunity, 1995, 9, 51-58; Geiler T., et al., Arthptis S Rheumatism, 1994, 37, 1664-1671): These were recently developed models of arthritis in which fresh smovial tissue from RA patients was implanted with normal human cartilage in mice? subcutaneous, under the renal capsule (Geiler T., et al., Arthptis S Rheumatism, 1994, 37, 1664-1671) or in knee joints (? ack U., et al., J. Autoimmuni ty, 1995, 9 , 51-58). The implants developed with characteristics similar to arthritis, including pannus tissue formation of high cell density, bone and cartilage erosion, development of giant tmuclear giant cells and invasion of cartilage by smovial fibroblasts. Indirect method: the smoviocytes were transfected with the therapeutic gene and re-transplanted into rabbits. Arthritis was induced in these rabbits by injecting IL-1β and the expression of the therapeutic gene was confirmed after activation. Expression induced by the activation of chimeric nucleic acid induces apoptosis in transplanted cells. Direct method: intra-articular injection of the chimeric nucleic acids. Can you use any of the gene delivery methods described above, including the cationic liposome-mediated delivery of single plasmid DNA. For the use of the viral vector-based delivery, the chimeric nucleic acids were cloned into suitable vectors. Then, the vectors were modified by suppressing the eucapotic promoter present in these vectors. Medicular-articular injection of the therapeutic genes inserted into appropriate vectors can then be performed to confirm the therapeutic efficacy as well as the prophylactic.

EXAMPLE 3 Selection of somatic cell variants not producing TNFa Cells (THP-1, Jurkat) were stably transfected with the chimeric nucleic acid TNFp-AIG. After several stimulation cycles, which induce apoptosis in the cells expressing the TNFp-AIG gene, the surviving cells were then harvested. A cDNA library was constructed from these cells, which was used for functional cloning (Legerski R and Peterson C., Nature, 1992, 359, 70-73; Jaattela M., et al., Oncogene, 1995, 10, 2297-2305).

EXAMPLE i Identification and characterization of negative dominant genes (DN) THP-1 and Jurkat cells stably transfected with TNFp-AIG underwent repeated stimulation cycles to activate the expression of TNFp-AIG. The cells, which do not express negative regulatory genes, undergo apoptosis, while those that express negative dominant genes survive. In these surviving cells, the DN gene products act in trans with the TNFa promoter, thereby inhibiting their activities to transcribe AIG, ultimately resulting in a surviving phenotype. A cDNA library was constructed using polyadenylated mRNA from these cells. The DN genes that rescue THP-1 or Jurkat cells transfected with TNFp-AIG from apoptosis were identified by functional cloning as described for other genes (Legerski R. and Peterson C, Nature, 1992, 359, 70-73; Jaattela; M., et al., Oncogene, 1995, 10, 2297-2305). The above description was for the purpose of teaching a person of ordinary experience in the The method used to carry out the present invention, and did not intend to detail all the modifications and obvious variations thereof that will be evident to the expert after reading the description. However, it was intended that all obvious modifications and variations of this type will be included within the scope of the present invention, which was defined by the following claims. The claims are intended to cover the claimed components and the steps in any sequence that is effective to meet the intended objectives, unless the context specifically indicates otherwise. The documents cited herein are expressly incorporated by reference in their entirety.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Boehringer Ingelheim Pharmaceuticals, Inc. (B) STREET: 900, Ridgebury Road (C) CITY: Ridgefield (E) COUNTRY: USA. (F) POSTAL CODE (CP): CT-06877 (G) TELEPHONE: ++ 1/203 / 798-9988 (H) TELFAX: ++ 1/203 / 791-6183 (ii) TITLE OF THE INVENTION: APOPTOSI? AUTO-REGULATED CELL? INFLAMMATORY? THROUGH GENE THERAPY (iii) NUMBER OF? ECUENCE? : 14 (iv) LEGIBLE FORM BY COMPUTER: (A) TYPE OF MEANS: Floating disk (B) COMPUTER: IBM PC compatible (C)? I? OPERATIONAL THEME: PC-DOS / MS-DO? (D) LOGICAL SYSTEM: Patentln Reléase n ° 1.0, version n ° 1.30 (EPO) (v) DATA OF THE PREVIOUS APPLICATION: (A) NUMBER OF APPLICATION: 60 / 076.316 (B) DATE OF SUBMISSION: FEB 27, 1998 (2) INFORMATION FOR SEQ. ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1178 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: native minimal TNF-alpha promoter (B) LOCATION: 1..1178 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: ggggaagcaa aggagaagct gagaagatga aggaaaagtc agggtctgga ggggcggggg 60 tcagggagcc cctgggagat atggccacat gtagcggctc tgaggaatgg gttacaggag 120 acctctgggg agatgtgacc acagcaatgg gtaggagaat gtccagggct atggaagtcg 180 agtatcgggg accccccctt aacgaagaca gagggcccca gggccatgta gggagtgaaa 240 gagcctccag gacctccagg tatggaatac aggggacgtt tggccacaca taagaagata 300 ctggggccct gagaagtgag agcttcatga aaaaaatcag gttccttgga ggaccccaga 360 gaaaccagca agccaagact ttatgagtct ccgggtcaga atgaaagaag aaggcctgcc 420 ccagtggtct gtgaattccc gggggtgatt tcactccccg ggctgtccca ggcttgtccc 480 tgctaccccc acccagcctt tcctgaggcc tcaagctgcc accaagcccc cagctccttc 540 cccaaacaca tccccgcaga ggcctcagga ctcaacacag cttttccctc caaccccgtt 600 caaggactca ttctctccct gctttctgaa gcccctccca gttctagttc tatctttttc 660 ctgcatcctg tctggaagtt agaaggaaac agaccacaga cctggtcccc aaaagaaatg 720 gaggcaatag gttttgaggg gcatggggac ggggttcagc ctccagggtc ctacacacaa 780 atcagtcagt ggcccagaag acccccctcg gaatcggagc agggaggatg gggagtgtga 640 ggggtatcct tgatgcttgt gtgtccccaa ctttccaaat ncccgccccc gcgatggaga 900 agaaaccgag acagaaggtg cagggcccac taccgcttcc tccagatgag cttatgggtt 960 tctccaccaa ggaagttttc cgctggttga atgattcttt ccccgccctc ctctcgcccc 1020 agggacatat aaaggcagtt gttggcacac ccagccagca gacgctccct cagcaaggac 1080 agcagaggac cagctaagag ggagagaagc aactgcagac cccccctgaa aacaaccctc 1140 tcccctgaca agacgccaca agctgccagg caggttct 1178 (2) INFORMATION FOR SEQ. ID NO: 2: (i) CHARACTERISTICS? OF THE SEQUENCE: (A) LENGTH: 1096 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: promoter of human TNF-alpha (B) LOCATION: 1..1096 (xi) DESCRIPTION OF THE SEQUENCE:? EQ ID NO: 2; gaggccgcca ggggaagcaa gactgctgca aggagaagct gagaagatga aggaaaagtc 60 agggtctgga ggggcggggg tcagggaget cctgggagat atggccacat gtagcggctc 120 tgaggaatgg gttacaggag acctctgggg agatgtgacc acagcaatgg gtaggagaat 180 gtccagggct atggaagtcg agtatgggga ccccccctta acgaagacag ggccatgtag 240 agggccccag ggagtgaaag agcctccagg acctccaggt atggaataca ggggacgttt 300 aagaagatat ggccacacac tggggccctg agaagtgaga gcttcatgaa aaaaatcagg 360 gaccccagag ttccttggaa gccaagactg aaaccagcat tatgagtctc cgggtcagaa 420 tgaaagaaga aggcctgccc cagtggggtc tgtgaattcc cgggggtgat ttcactcccc 480 ggggctgtcc caggcttgtc cctgctaccc ccacccagcc tttectgagg cctcaagcct 540? ccaccaagc ccccagctcc ttctccccgc agggacccaa acacaggcct caggactcaa 600 cacagctttt ccctccaacc ccgttttctc tccctcaagg actcagcttt ctgaagcecc 660 tcccagttct agttctatct ttttcctgca tcctgtctgg aagttagaag gaaacagacc 720 acagacetgg tccccaaaag aaatggaggc aataggtttt gaggggcatg gggacggggt 780 gggtcctaca tcagcctcca cacaaatcag tcagtggccc to? aagacccc cctcggaatc 840 ggagcaggga ggatggggag tgtgaggggt atecttgatg ettgtgtgtc cccaactttc 900 caaatccccg cccccgcgat ggagaagaaa ccgagacaga aggtgcaggg cccactaccg 960 cttcctccag atgagctcat gggtttctcc accaaggaag ttttccgctg gttgaatgat 1020 tctttccccg ccctcctctc gccccaggga catataaagg cagttgttgg cacacccagc 1080 cagcagacgc tccctc 1096 (2) INFORMATION FOR SEQ. ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 139 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (i) ORIGINAL SOURCE: (A) ORGANI: MO: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: promoter of native minimal TNF-alpha (B) LOCATION: 1..139 (xi) DE? CRIPTION OF THE SEQUENCE: SEQ ID NO: 3; ccgcttcctc cagatgagct catgggcttc tccaccaagg aagttttccg ctggttgaat 60 gattctttcc ccgccctcct ctcgccccag ggacatataa aggcagttgt atggcacacc 120 cgccagcaga cgctccctc j39 (2) INFORMATION FOR SEQ. ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 123 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: region 1 of enhancer (ER1) of the TNF-alpha promoter (B) LOCATION: 1..123 (xi) DE? CRIPTION OF THE? ECUENCE:? EQ ID NO: 4; ggggcggggg tcagggagct cctgggagat atggccacat gtagcggctc tgaggaatgg 60 gttacaggag acctctgggg agatgtgacc acagcaatgg gtaggagaat gtccagggct 120 atg "3 (2) INFORMATION FOR THE? EQ. ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 190 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANI: MO: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: enhancer region 2 (ER2) of the TNF-alpha promoter (B) LOCATION: 1..190 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: tccttggaag ccaagactga aaccagcatt atgagtctcc gggtcagaat gaaagaagaa 60 ggcctgcccc agtggggtct gtgaattccc gggggtgatt tcactccccg gggctgtccc 120 aggcttgtcc ctgctacccc cacccagcct ttcctgaggc ctcaagcctg ccaccaagcc 180 eccagctcct 190 (2) INFORMATION FOR THE? EQ. ID NO: 6: (i) CHARACTERISTIC? OF THE SEQUENCE: (A) LENGTH: 223 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: multiple cloning sites elaborated by genetic engineering above the minimum TNF-alpha promoter in the construction -120pGL3 (B) LOCATION: 1..223 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: ggtaccgagc tcttacgcgt gctagccgcg gatatcttaa gacgtcctag gactagtcag 60 ctgctcgage cgcttcctcc agatgagctc atgggtttct ccaccaagga agttttccgc 120 tggttgaatg attctttccc cgccctcctc tcgccccagg gacatataaa ggcagttgtt 1T0 ggcacaccca gccagcagac gctccctcag cagatctaag ctt 223 (2) INFORMATION FOR SEQ. ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 787 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: untranslated region of TNF-alpha (B) LOCATION: 1.787 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: tctagaggag gacgaacatc caaccttccc aaacgcctcc cctgccccaa tccctttatt 60 accccctcct tcagacaccc tcaacctctt ctggctcaaa aagagaattg ggggcttagg 120 gtcggaaccc aagcttagaa ctttaagcaa caagaccacc acttcgaaac ctgggattca 180 ggaatgtgtg gcctgcacag tgaagtgct-g gcaaccacta agaattcaaa ctggggcctc 240 cagaactcac tggggcctac agctttgatc cctgacatct ggaatctgga gaccagggag 300 cctttggttc tggccagaat gctgcaggac ttgagaagac aattgacaca ctcacctaga 360 agtggacctt aggccttcct ctctccagat gtttccagac ttccttgaga cacggagccc 420 agccctcccc atggagccag ctccctctat ttatgtttgc acttgtgatt atttattatt 480 tatttattat ttatttattt acagatgaat gtatttattt gggagaccgg ggtatcctgg 540 gggacccaat gtaggagctg ccttggctca gacatgtttt ccgtgaaaac ggagctgaac 600 aataggctgt tcccatgtag ccccctggcc tctgtgcctt cttttgatta tgttttttaa 660 aatatttatc tgattaagtt gtctaaacaa tgctgatttg gtgaccaact gtcactcatt 720 gctgagcctc tgctccccag gggagttgtg tctgtaatcs ccctactatt cagtggcgag atctaga 780 787 (2) INFORMATION FOR SEQ. ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 839 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) FEATURE: (A) NAME / KEY: Granzyme B full length (B) LOCATION: 1.,839 (xi) DE? CRIPCION DE LA? ECUENCIA: SEQ ID NO: 8: atgcaaccaa tcctgcttct gctggccttc ctcctgctgc ccagggcaga tgcaggggag 60 atcatcgggg gacatgaggc caagccccac tcccgcccct acatggctta tcttatgatc 120 agtctctgaa tgggatcaga ggcttcctga gaggtgcggt tacaagacga cttcgtgctg 180 acagctgctc actgttgggg aagctccata aatgtcacct tgggggccca caatatcaag 240 cgacccagca gaacaggagc gtttatccct gtgaaaagag ccatccccca tccagcctat 300 acttctccaa aatcctaaga tgacatcatg ctactgcagc tggagagaaa ggccaagcgg 360 accagagctg tgcagcccct caggctacct agcaacaagg cccaggtgaa gccagggcag 420 acatgcagtg tggccggctg ggggcagacg gcccccctgg gaaaacactc acacacacta 480 caagaggtga agatgacagt gcaggaagat cgaaagtgcg aatctgactt acgccattat 540 tacgacagta ccattgagtt gtgcgtgggg gacccagaga ttaaaaagac ttcctttaag 600 ggggactctg gaggccctct tgtgtgtaac aaggtggccc agggcattgt ctcctatgga 660 cgaaacaatg gcatgcctcc acgagcctgc accaaagtct caagctttgt acactggata 720 tgaaacgcta aagaaaacca ctaactacag gaagcaaact aagccccgc tgtaatgaaa 780 caccttctct ggagccaagt ccagatttac actgggagag gtgccagcaa ctgaataaa 839 (2) INFORMATION FOR THE? EQ. ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 782 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (li) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTIC: (A) NAME / KEY: Granzyme B truncated (devoid of the conductive peptide and inactivating dipeptide) (B) LOCATION: 1.782 (xi) DE? CRIPCION DE LA? ECUENCIA: SEQ ID NO: 9: atgatcatcg ggggacatga ggccaagccc cactcccgcc cctacatggc ttatcttatg 60 atctgggatc agaagtctct gaagaggtgc ggtggcttcc tgatacaaga cgacttcgtg 120 ctgacagctg ctcactgttg gggaagctcc ataaatgtca ccttgggggc ccacaatatc 180 aaggaacagg agccgaccca gcagtttatc cctgtgaaaa gagccatccc ccatccagcc 240 tataatccta agaacttctc caatgacatc atgctactgc agctggagag aaaggccaag 300 cggaccagag ctgtgcagcc cctcaggcta cctagcaaca aggcccaggt gaagccaggg 360 cagacatgca gtgtggccgg ctgggggcag acggcccccc tgggaaaaca ctcacacaca 420 ctacaaga ? g tgaagatgac agtgcaggaa gatcgaaagt gcgaatctga cttacgccat 480 gtaccattga tattacgaca gttgtgcgtg ggggacccag agattaaaaa gacttccttt 540 aagggggact ctggaggccc tcttgtgtgt aacaaggtgg cccagggcat tgtctcctat 600 ggacgaaaca atggcatgcc tccacgagcc tgcaccaaag tctcaagctt tgtacactgg 660 ataaagaaaa ccatgaaacg .ctactaacta caggaagcaa actaagcccc cgctgtaatg 720 tctggagcca aaacaccttc agtccagatt tacactggga gaggtgccag caactgaata 780 aa 782 (2) INFORMATION FOR SEQ. ID NO: 10: (i) CHARACTERISTIC? OF THE SEQUENCE: (A) LENGTH: 833 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTIC: (A) NAME / KEY: Granzyme B truncated without the dipeptide inactively (B) LOCATION: 1 .. 833 (xi) DE? CRIPCION DE LA? ECUENCIA:? EQ ID NO: 10: atgcaaccaa tcctgcttct gctggccttc ctcctgctgc ccagggcaga tgcaatcatc 60 gggggacatg aggccaagcc ccactcccgc ccctacatgg cttatcttat gatctgggat 120 cagaagtctc tgaagaggtg cggtggcttc ctgatacaag acgacttcgt gctgacagct 180 gctcactgtt ggggaagctc cataaatgtc accttggggg cccacaatat caaggaacag 240 gagccgaccc agcagtttat ccctgtgaaa agagccatcc cccatccagc ctataatcct 300 aagaacttct ccaatgacat catgctactg gaaaggccaa cagctggaga gcggaccaga 360 gctgtgcagc ccctcaggct acctagcaac aaggcccagg tgaagccagg gcagacatgc 420 agtgtggccg gctgggggca gacggccccc ctgggaaaac actcacacac actacaagag 480 cagtgcagga gtgaagatga agatcgaaag tgcgaatctg acttacgcca ttattacgac 540 agtaccattg agttgtgcgt gggggaccca gagattaaaa agacttcctt taagggggac 600 tctggaggcc ctcttgtgtg taacaaggtg gcccagggca ttgtctccta tggacgaaac 660 aatggcatgc ctccacgagc ctgcaccaaa gtctcaagct ttgtacactg gataaagaaa 720 accatgaaac gctactaact acaggaagca aactaagccc ccgctgtaat gaaacacctt 780 ctctggagcc aagtccagat ttacactggg agaggtgcca gcaactgaat aaa 833 (2) INFORMATION FOR SEQ. ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 114 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: reinforcer region 3: promoter of TNF-alpha -234 a -120 (B) LOCATION: 1..114 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: ll: gcagggagga tggggagtgt gaggggtate cttgatgctt gtgtgtcccc aactttccaa 60 atccccgccc ccgcgatgga gaagaaaccg agacagaagg tgcagggcce act 114 (2) INFORMATION FOR SEQ. ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 169 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ll) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) CHARACTERISTICS: (A) NAME / KEY: reinforcer region 4: promoter of TNF-alpha -234 to -65 (B) LOCATION: 1.169 (xi) DE? CRIPTION OF THE? ECUENCE:? EQ ID NO: 12: gcagggagga tggggagt? t gaggggtatc cttgatgctt gtgtgtcccc aactttccaa 60 atccccgccc ccgcgatg? a gaagaaaccg agacagaagg tgcagggccc actaccgctt 120 cctccagatg agctcatggg tttctccacc aaggaagttt tccgctggt 169 (2) INFORMATION FOR SEQ. ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2270 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) FEATURE: (A) NAME / KEY: - 706TNFpGB3 'UTR (B) LOCATION: 1..2270 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: ctcgagtcct tggaagccaa gactgaaacc agcattatga gtctccgggt cagaatgaaa 60 gaagaaggcc tgccccagtg gggtctgtga attcccgggg gtgatttcac tccccggggc 120 tgtcccaggc ttgtccctgc tacccccacc cagcctttcc tgaggctcaa gcctgccacc 180 aagcccccag ctccttctcc ccgcagggac ccaaacacag gcctcaggac tcaacacagc 240 ttttccctcc aaccccgttt tctctccctc aaggactcag ctttctgaag cccctcccag 300 ttctagttct atctttttcc tgcatcctgt ctggaagtta gaaggaaaca gaccacagac 360 ctggtcccca aaagaaatgg aggcaatagg ttttgagggg catggggacg gggttcagcc 420 tccagggtcc tacacacaaa tcagtcagtg gcccagaaga cccccctcgg aatcggagca 480 gggaggatgg ggagtgtgag gggtatcctt gatgcttgtg tgtecccaac tttccaaatc 540 cgatggagaa cccgcccccg gaaaccgaga cagaaggtgc agggcccact acegcttcct 600 ccagatgagc tcatgggttt ctccaccaag gaagttttcc gctggttgaa tgattctttc 660 tctcgcccca cccgccctcc gggacatata aaggcagttg ttggcacacc cagccagcag 720 agcagatcta acgctccctc tgcaaccaat cctgcttctg ctggccttcc tcctgctgcc 780 cagggcagat gcaatcatcg ggggacatga ggccaagccc cactcccgcc cctacatggc 840 ttatcttatg atctgggatc agaagtctct gaagaggtgc ggtggcttcc tgatacaaga 900 cgacttcgtg ctgacagctg ctcactgttg gggaagctec ataaatgtca ccttgggggc 960 ccacaatatc aaagaacagg agccgaccea gcagtttatc cctgtgaaaa gacccatccc 1020 ccatccagcc tataatccta agaacttctc caacgacatc atgctactgc agctggagag 1080 áaaggccaag cggaccagag ctgtgcagcc ccteaggcta cctagcaaca aggcccaggt 1140 cagacatgca gaagccaggg gtgtggccgg ctgggggcag acggcccccc tgggaaaaca 1200 ctcacacaca ctacaagagg tgaagatgac agtgeaggaa gatcgaaagt gcgaatctga 1260 tattacgaca cttacgccat gtaccattgá gttgtgcgtg ggggacccag agattaaaaa 1320 gacttccttt aagggggact ctggaggccc tcttgtgtgt aacaaggtgg cccagggcat 1380 tgtctcctat ggacgaaaca atggcatgcc tccacgagcc tgcaccaaag tctcaagctt 1440 tgtacactgg ataaagaaaa ccatgaaacg ctactaagaa ttctctagag gaggacgaac 1500 atccaacctt cccaaacgcc tcccctgccc caatcccttt attaccccct ccttcagaca 1560 ccctcaacct cttctggctc aaaaagagaa ttgggggctt agggtcggaa cccaagctta 1620 gaactttaag caacaagacc accacttcga aacctgggat tcaggaatgt gtggcctgca 1680 ctggcaacca cagtgaagtg ctaag AATTC aaactggggc ctccagaact cactggggcc 1740 tacagctttg atccctgaca tctggaatct ggagaccagg gagcctttgg ttctggccag 1800 aatgctgcag gacttgagaa gacctcacct agaaattgac acaagtggac cttaggcctt 1860 gatgtttcca cctctctcca gacttccttg agacacggag cccagccctc cccatggagc 1920 cagctccctc tatttatgtt tgcacttgtg attatttatt atttatttat tatttattta 19B0 tttacagatg aatgtattta tttgggagac cggggtatcc tgggggaccc aatgtaggag 2040 ctgccttggc tcagacatgt tttccgtgaa aacggagctg aacaataggc tgttcccatg 2100 tagccccctg gcctctgtgc cttcttttga ttatgttttt taaaatattt atctgattaa 2160 gttgtctaaa caatgctgat ttggtgacca actgtcactc attgctgagc ctctgctccc 2220 caggggagtt gtgtctgtaa tcgccctact attcagtggc gagatctaga 2270 (2) INFORMATION FOR SEQ. ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2570 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear . { ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) FEATURE: (A) NAME / KEY: - 1005TNFpGB3 'UTR (B) LOCATION: 1. .2570 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: ctcgagggcg ggggtcaggg agctcctggg agatatggcc acatgtagcg gctctgagga 60 atgggttaca ggagacctct? gggagatgt atgggtagga gaccacagca gaatgtccag 120 ggctatggaa gtcgagtatg gggaeccecc cttaacgaag acagggccat gtagagggcc 180 ccagggagtg aaagagcctc caggacctcc aggtatggaa tacaggggac gtttaagaag 240 atatggccac acactggggc cctgagaagt gaga c tea tgaaaaaaat cagggacccc 300 agagttcctt ggaagccaag actgaaacca gcattatgag tctccgggtc agaatgaaag 360 aagaaggcct gccccagtgg ggtctgtgaa ttcccggggg tgatttcact ccccggggct 420 gtcccaggct tgtccctgct acccccaccc agcctttcct gaggcctcaa gcctgccacc 480 aagcccccag ctccttctcc ccgcagggac ccaaacacag gcctcaggac tcaacacagc 540 ttttccctcc aaccccgttt tctctccctc aaggactcag ctttctgaag cccctcccag 600 ttctagttct atctttttcc tgcatcctgt gaaggaaaca ctggaagtta gaccacagac 660 ctggtcccca aaagaaatgg aggcaatagg ttttgagggg catggggacg gggttcagcc 720 cccagggtcc tacacacaaa tcagtcagtg geccagaaga cccccctcgg aat ggagca 780 gggaggatgg ggagtgtgag gggtatcctt gatgcttgtg tgtccccaac tttccaaatc 840 cccgcecccg cgatggagaa gaaaccgaga cagaaggtgc agggcccact accgcttcct 900 ecagatgage tcatgggttt ctccaccaag gaagttttcc gctggttgaa tgattctttc 960 tctcgcccca eccgccctcc gggacatata aaggcagttg ttggcacacc cagccagcag 1020 agcagatcta acgctccctc tgcaaccaat cctgcttctg ctggccttcc tcctgctgcc 1080 cagggcagat gcaatcatcg ggggacatga ggccaagccc cactcccgcc cctacatggc 1140 ttatcttatg atctgggatc agaagtctct gaagaggtgc ggtggcttcc tgatacaaga 1200 cgacttcgtg ctgacagctg ctcactgttg gggaagctcc ataaatgtca ccttgggggc 1260 ccacaatatc aaagaacagg agcc gaccca gcagtttatc cctgtgaaaa gacccatccc 1320 ccatccagcc tataatccta agaacttctc caacgacatc atgctactgc agctggagag 1380 aaaggccaag cggaccagag ctgtgcagcc cctcaggcta cctagcaaca aggcccaggt 1440 gaagccaggg cagacatgea gtgtggccgg ctgggggcag acggcccccc tgggaaaaca 1500 ctcacacaca ctacaagagg tgaagatgac agtgcaggaa gatcgaaagt gcgaatctga 1560 tattaegaca cttacgccat gtaccattga gttgtg gtg ggggacccag agattaaaaa 1620 gacttccttt aagggggact ctggaggccc tcttgtgtgt aacaaggtgg cccagggcat 1680 ggacgaaaca tgtctcctat atggcatgcc tccacgagcc tgcaccaaag tetcaagett 1740 tgtacactgg ataaagaaaa ccatgaaacg ctactaagaa ttctctagag gaggacgaac 1800 atccaacctt cccaaacgcc tcccctgccc caatcccttt attaccccct ccttcagaca 1860 ccctcaacct cttctggctc aaaaagagaa ttgggggett agggtcggaa cccaagctta 1920 gaactttaag caacaagacc accacttcga aacctgggat tcaggaatgt gtggcctgca 1960 cagtgaagtg ctggcaacca ctaagaattc aaactggggc ctccagaact cactggggcc 2040 tacagctttg atccctgaca tctggaatct ggagaccagg gagcctttgg ttctggccag 2100 aatgctgcag gacttgagaa gacctcacct agaaattgac acaagtggac cttaggcctt 2160 gatgtttcca cctctctcca gacttccttg agacacggag cccagccctc cccatggagc 2220 cagctccctc tatttatgtt tgcacttgtg attatttatt atttatttat tatttattta 2280 tttacagatg aatgtattta tttgggagac cggggtatcc tgggggaccc aatgtaggag 2340 ctgccttggc tcagacatgt tttccgtgaa aacggagctg aacaataggc tgttcccatg 2400 tagccccctg gcctctgtgc cttcttttga ttatgttttt taaaatattt atctgattaa 2460 gttgtctaaa caatgctgat ttggtgacca actgtcactc attgctgagc ctctgctccc 2520 caggggagtt gtgtctgtaa tcgccctact attcagtggc gagatctaga 2570 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (18)

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

1. A chimeric nucleic acid molecule, characterized in that it comprises: at least one enhancer region of the TNFa promoter linked to a TNFa promoter, the enhancer region comprising the? EQ ID NO: 4,? EQ ID NO: 5, SEQ ID NO: ll, SEQ ID NO: 12 of nucleic acid or a conservative substitution or allelic vandals thereof; the TNFa promoter being further linked to a nucleic acid sequence encoding the protein Granzyme B or a conservative substitution or allelic variants thereof which, in turn, are additionally linked to a 3'UTR nucleic acid sequence.

2. The chimeric nucleic acid molecule according to claim 1, characterized in that two to five enhancer regions of the TNFa promoter are present, and wherein any of the reinforcer regions comprises SEQ ID NO: 5, SEQ ID NO: 12 or combinations thereof.

3. The chimeric nucleic acid molecule according to claim 2, characterized in that the TNFa promoter comprises the? EQ ID NO: l, SEQ ID NO: 2, SEQ ID NO: 3 nucleic acid or a conservative substitution or allelic variants thereof.

4. The chimeric nucleic acid molecule according to claim 1, characterized in that the sequence of the nucleic acid molecule is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and a conservative substitution or allelic variants of the same

5. The chimeric nucleic acid molecule according to claim 1, characterized in that the 3'UTR is linked downstream of the Granzyme B nucleic acid sequence.

6. A pharmaceutical composition, characterized in that it comprises a chimeric nucleic acid molecule according to claim 1.

7. Use of the composition according to claim 6 for the preparation of a medicament for treating an inflammatory disorder.

8. The use in accordance with the claim 7, characterized in that the inflammatory disorder is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft-versus-host disease, lupus erythematosus, insulin-dependent diabetes mellitus. , psoriatic arthritis, sarcoidosis, hypersensitivity pneumonitis, ankylosing spondylitis, Reiter's syndrome and systemic sclerosis.

9. The use in accordance with the claim 8, characterized in that the inflammatory disorder is rheumatoid arthritis.

10. A method for constructing a chimeric nucleic acid molecule comprising at least one enhancer of the TNFct promoter linked to a functional copy of a minimal TNFa promoter linked to at least one copy of a functional apoptosis-inducing nucleic acid molecule, which is additionally linked to a nucleic acid sequence TNFa-3'UTR, wherein the expression of the nucleic acid induces apoptosis is driven by the TNFa promoter, characterized in that it comprises the steps of: (a) amplifying a nucleic acid molecule that comprises a TNFa promoter by a polymerase chain reaction using primers encompassing deletion constructs of the TNFa promoter; (b) cloning the amplified nucleic acid by PCR obtained in step (a) above a reporter nucleic acid sequence to produce a construct; (c) testing the constructs obtained in step (b) for constitutive and inducible expression in at least one TNFa-producing cell line; (d) selecting the TNFa promoter responsible for the inducible expression of the reporter in the cell line; (e) PCR amplifying regions of the TNFa promoter that reinforce the expression of the reporter to obtain a booster and ligate at least one copy of the enhancer upstream of the promoter; (f) inserting at least one copy of an apoptosis-inducing nucleic acid molecule downstream of the TNFa promoter by replacing the reporter with the apoptosis-inducing nucleic acid deletion constructs; and (g) PCR amplifying a TNFa-3'UTR and ligating it downstream of the apoptosis-inducing nucleic acid molecule to obtain the chimeric nucleic acid molecule.

11. The method according to claim 10, characterized in that two or more copies of the reinforcer are inserted above the promoter.

12. The method according to claim 10, characterized in that the reporter nucleic acid sequence is luciferase.

13. The method according to claim 10, characterized in that the enhancer comprises a sequence selected from the group consisting of SEQ ID NO: 4,? EQ ID NO: 5, SEQ ID NO: ll and SEQ ID NO: 12.

14. The method according to claim 13, characterized in that the nucleic acid induces apoptosis is Granzyme B or a conservative substitution or allelic variants thereof.

15. The method according to claim 10, characterized in that it produces a chimeric nucleic acid molecule selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 1 and a conservative substitution or allelic variants thereof.

16. The method according to claim 10, characterized in that the cell lines for testing constructs are selected from the group consisting of T lymphoblastoid, myelomonocytic, monocytic, fibroblast, cultured human synoviocytes and primary cells residing in the RA.

17. A method for screening negative dominant genes, characterized in that it comprises: transferring a non-TNFa producing population of cells with a sequence comprising the chimeric nucleic acid according to claim 1; stimulate the expression of the inserted sequence; and select the surviving cells that possess negative dominant genes.

18. The method according to claim 17, characterized in that the sequence comprises the chimeric nucleic acid molecule according to claim 2.