DE19907099A1 - Novel nucleic acid construct useful in gene therapy comprising an hormone responsive element and transgene in which the hormone responsive element is not functionally linked to the transgene - Google Patents

Abstract

A nucleic acid construct (I) comprising an hormone responsive element (HRE) and a transgene (T), is new. Independent claims are also included for the following: (1) a nucleic acid construct (II), comprising at least one HRE and a (T), where one of the HREs is not functionally linked to the transgene; (2) a vector (III) comprising (I), or (II); (3) a transformed cell or transgenic organism comprising (I), (II) or (III); (4) a composition of matter (IV) comprising (II), and/or (III), and in which HRE is coupled to a hormone-hormone receptor complex; (5) preparing (IV) involves admixing (II) with the hormone receptor and the hormone; (6) a pharmaceutical composition comprising (II), (III) and/or (IV); (7) gene transfer comprising administering a gene transfer agent (GTA) comprising (I), or (IV) to an organism or to a cellular system; (8) delivering (D) a nucleic acid construct encoding (T) to be expressed in the cells of the organism or the cells of the cellular system, into an organism or into a cellular system involves administering GTA comprising (I), or (IV) to the organism or cellular system so that the hormone in the composition interacts with the cell membrane and enhances diffusion and transport of the nucleic acid that is coupled to the hormone-hormone receptor complex; (9) use of a steroid hormone for preparing GTA; and (10) gene transfer comprising administering (I) to a organism or a cellular system in which (I) contains a transgene and is encapsulated in a steroid hormone.

Description

Background of the Invention 1. Subject of the invention

The invention provides a composition of matter which contains a nucleotide sequence which comprises at least one hormone-responsive element (HRE), this hormone-responsive element is coupled to a hormone-hormone receptor complex. she further relates to nucleic acid constructs that have at least one hormone-responsive element include, as well as vectors containing such constructs, wherein the hormone responsive Element regulates a gene that codes for a human blood coagulation factor. The nucleic acid constructs, plasmids and substance compositions according to the invention are used in gene therapy, especially in treatment human coagulation disorders, for example hemophilia. You can also do this used to up or down target genes, as well as to vaccines administer.

2. Summary of the state of the art

Gene therapy represents an extreme for numerous diseases and defects promising method. Generally speaking, it involves the transfer recombinant genes or transgenes in somatic cells to match with a genetic Defective proteins to replace or the pathological process of a disease influence. In principle, gene therapy is a simple method. In practice there are still many disadvantages to be overcome.

Research in gene therapy has focused on pathways, DNA to be incorporated into a patient's cells as effectively as possible. There are currently virals Vectors the carriers that are commonly used in clinical gene therapy approaches  become. In terms of gene expression efficiency, the viral delivery systems have great advantages compared to techniques that use DNA lipid formulations as carriers Use administration, or compared to mechanical methods, for example the "gene gun". Although there are a variety of viral systems available for gene therapy Strategies that have been tested are retroviral vectors and adenoviral vectors currently the most widely used carriers (Salmons, B. and Gunzburg, W.H., Hum. Gene Ther., Vol. 4, 129, 1993; Kasahara, N.A., et al., Science, Vol. 266, 1373, 1994; Ali, M., et al. Gene Ther., Vol. 1, 367, 1994). However, these systems have major drawbacks such as the possibility of viral contamination. Other security concerns remain before developing a clinical application of using gene therapy of these viral systems in the way. For example, recombinant retroviruses have the Disadvantage of the random chromosomal integration, which leads to the activation of Oncogenes or can inactivate tumor suppressor genes. Furthermore repeated use of recombinant adenoviruses is serious causes immunological problems (Elkon, K.B. et al., Proc. Natl. Acad Sci. USA, Vol. 94, 9814, 1997). The humoral answer led to the production of antibodies against Adenovirus proteins that prevented subsequent infection. Immunosuppressive Medications can mitigate these effects, but they pose to the patient an additional burden (Dai, Y., et al., Proc. Natl. Acad. Sci. USA, Vol. 92, 1401, 1995).

Another viral delivery system relies on the adeno-associated virus (AAV). The AAV requires co-infection with an unrelated helper virus. Although such recombinant AAV virions are smaller for the introduction of numerous Gene sequences in host cells have been shown to be useful Gene delivery systems based on such particles through which relatively small size of the AAV particles limited. This property diminishes the range of suitable genetic protocols to a large extent. In addition, the ' Need to use a helper virus, another complicating factor of this Administration system. (Muzyczka, N., Curr. Top. Microbiol. Immunol., Vol. 158, 97, 1992).  

Non-viral gene therapy approaches are also unsatisfactory, although they are safer are. Problems related to inefficient gene delivery or weaker Continuous expression are major disadvantages. Methods such as for example the direct injection of DNA into cellular compartments up to Mixtures of DNA and cationic lipids or polylysine, which are lighter Allowing the transgene to pass through the cell membrane does not yet have these hurdles overcome. (Felgner, P., et al., Proc. Natl. Acad. Sci. USA, Vol. 84, 7413, 1987; Behr, J.-P., Bioconjugate Chemistry, Vol. 5, 382, 1994).

With the introduction of naked DNA (polynucleotide) sequences (including antisense DNA) in vertebrates a controlled expression of a protein is possible. It will reports that administration of the polynucleotide sequences in tissues such as for example muscles, brain or skin by injection or by insertion can be reached in the bloodstream. (Wolff, J.A., et al., Science, Vol. 247, 1990; Lin H., et al., Cirulation, Vol. 82, 2217, 1990; Schwartz, B., et al., Gene Ther., Vol. 3, 405, 1996). Furthermore, a direct gene transfer into mammals has been linked with DNA encapsulated in liposomes and with DNA enclosed in proteosomes which receptor proteins contained was reported. Although the injection of naked DNA is used to Expression of the transgene, the efficiency is far from that of DNA Administration systems based on viruses comparable. Still has bare DNA the advantage that there are no pathogenic effects. A limitation of Method of injecting naked DNA means the fact that transgene expression is dose dependent. Gene expression can reach a saturation point and one Increasing the amount of DNA injected leads to decreased protein production per Plasmid. As a result, protein expression can decrease dramatically when the amount of injected DNA is above a certain threshold.

Among the genetic diseases, which the person skilled in the art with the help of these Prior art methods have sought to overcome those in the Associated with blood clotting defects, and especially hemophilia (Lozier, J.N. and Brinkhous, K.M., JAMA, Vol. 271, 1994; Hoeben, R.C., Biologicals, Vol. 23, 27, 1995). Hemophilia A and B, for example, are X-coupled, recessive  Bleeding disorders caused by defects in blood coagulation factors VIII or IX (Sadler, J.E. et al., in: The Molecular Basis of Blood Diseases, 575, 1987). Hemophilia occurs with a frequency of approximately one in 5000 in males Newborn on. Hemophilia patients suffer from excessive bleeding due to lack of Blood clotting at wound sites. The inability to properly clot also causes and causes damage to joints and internal tissues risks related to the correct treatment of cuts.

Hemophilia A can be treated by administering blood coagulation factor VIII. Until recently, factor VIII preparations had to be done by concentrating donor blood recovered, increasing the risk of contamination with infectious agents, such as HIV or hepatitis. The factor VIII gene has been cloned (e.g. Vehar et al., Nature, Vol. 312, 337, 1984), which enables the production of a recombinant Product was made possible. Although factor VIII in rec a higher purity than can be provided from the blood concentrates the exogenous care of a patient with factor VIII still the administration repeated doses throughout the patient's life, an impractical and expensive solution. Other forms of hemophilia include hemophilia B, which are characterized by a Defect of the gene coding for factor IX is caused. The ones described above Gene therapy systems have been associated with the treatment of hemophilia A and B with factor VIII or IX (see e.g. WO 94/29471). These systems point however, the disadvantages already discussed above.

The classic model of hormonal action is based on the concept of Binding interaction of the hormone with an intracellular receptor, which in the Cytoplasm or located in the nucleus (Evans, R. Science, Vol. 240, 889, 1988). This intracellular receptors remain in the latent state until they become their respective Target hormone are exposed. At such an exposure, the Hormone receptor after binding the hormone its conformation and translocates into the activated form in the cell nucleus, where it acts as a dimer on hormone-responsive elements (HRE) binds in the promoter region of hormone-regulated genes (Beato, M., Cell, Vol. 56, 335, 1989; O'Malley, B., et al., Biol. Reprod., Vol. 46, 163, 1992). The HRE are  "enhancer" elements that are usually in the 5'-flanking region of each hormone-induced gene.

The steroid receptor is an example of such intracellular receptors. Steroid receptors belong to a superfamily of ligand-dependent Transcription factors that are characterized by a unique molecular structure. The centrally located, highly conserved, DNA-binding domain defines this Super family. The second important and comparatively invariant region is the COOH terminal ligand binding domain. An example of such a receptor is Progesterone receptor, which is controlled by the steroid progesterone. In which Progesterone receptor acts as a natural agonist, and as such it shows strong anti-mineralocorticoid properties on both the molecular and the systemic level. In addition to the classic uterine effects, the progesterone in numerous studies including anti-epileptic, anxiolytic, hypnotic and anesthetic Assigned properties.

Methods for using mutant receptors, including Steroid receptor mutants for gene therapy have been proposed. Such Methods are for example in WO 93/23431, WO 98/18925 and WO 96/40911 disclosed. WO 98/33903 discloses a genetic construct, which a steroid-responsive element of a tissue-specific gene, a coding sequence and includes an SV40 enhancer.

Brief description of the invention

The aim of the present invention is to overcome the disadvantages of the previous ones Overcome gene therapy delivery systems. The delivery system according to the present invention is a composition of matter containing a nucleic acid which comprises at least one hormone-responsive element (HRE), this hormone-responsive element is coupled to a hormone-hormone receptor complex. A preferred embodiment of the composition of matter according to the present  Invention is one in which the hormone-responsive element is a steroid-responsive element (SRE) and the receptor is a steroid receptor. In the most preferred In one embodiment, the hormone-responsive element is a progesterone-responsive element (PRE) and the receptor is a progesterone receptor.

The present invention provides a gene therapy delivery system which should overcome the disadvantages of the prior art. The presence of the hormone-responsive element in conjunction with the nucleic acid responsible for a gene of interest codes, promotes and enhances gene expression and what else more importantly, promotes the binding of a hormone-hormone receptor complex. On Hormone-responsive element is preferably in the form of a dine or multimer Nucleic acid sequence available. Even an inverse orientation of the hormone-responsive Elements will function properly. The hormone-hormone receptor complex contains a hormone receptor that activates after its specific hormone is bound becomes. When activated, the hormone receptor is able to be specific hormone-responsive element, which according to the present invention with a Combined nucleic acid that the desired gene, for example a human Blood clotting factor contains, to recognize and bind to it.

Another aspect of the present invention is a nucleic acid construct that contains at least one hormone-responsive element (HRE) and in particular at least a hormone-responsive element for the regulation of a gene that is responsible for a human Blood coagulation factor coded. A preferred embodiment is one in which the hormone-responsive element is a steroid-responsive element (SRE). In one particularly preferred form, the hormone-responsive element is a progesterone-responsive element (PRE).

Another aspect of the present invention are vectors that Contain nucleic acid constructs according to the present invention. Embodiments the present invention further include transfected and transformed cells which contain these vectors and / or nucleic acids.  

Another embodiment of the present invention are pharmaceutical Compositions containing a therapeutically effective dose of the invention Contain nucleic acid constructs and a hormone. The hormone is preferably a Steroid, particularly preferred progesterone. Another aspect of the invention is Use of the substance compositions and nucleic acid constructs as medicines against genetic defects and diseases, for example hemophilia, and the Use of the substance compositions and nucleic acid constructs for the Manufacture of a medicament against genetic defects and diseases, for example hemophilia.

The invention also includes a method for introducing a Nucleic acid construct that is for a gene of interest, for example a human coagulation factor, encoded into a cell to insert the gene of interest into to express the cell. This procedure introduces a nucleic acid into the cell (for example by means of a vector) so that the cell contains the gene for which the foreign Nucleic acid encoded, expressed. In this method, the nucleic acid which is used for encodes the gene, for example a human blood coagulation factor, with a Combined nucleic acid construct, which has at least one hormone-responsive element (HRE), preferably a progesterone-responsive element. The presence of the hormone-responsive element in connection with the nucleic acid, which is responsible for the gene, for example a human blood coagulation factor, encodes, promotes and reinforces the Gene expression and promotes the binding of a hormone-hormone receptor complex in the Target cell.

Another aspect of the invention is a method for treating a Blood clotting defect by administering a therapeutically effective amount of the substance composition according to the invention to an organism.

Another embodiment according to the inventive method is the Vaccination. The introduction of a nucleic acid construct or Composition of the invention comprising a gene for an antigen or viral sequences  contains, into a cell (DNA or mRNA vaccine) using the above mentioned method can also be a way to the cellular immune response stimulate.

Brief description of the pictures

Figure 1 is a diagram of the vector pTGFG1.

Figure 2 is a diagram of the vector pTGFG4.

Fig. 3 is a diagram of the vector pTGFG5.

Figure 4 is a diagram of the vector pTGFG6.

Fig. 5 is a diagram of the vector pTGFG7.

Figure 6 is a diagram of the vector pTGFG8.

Figure 7 is a diagram of the vector pTGFG9.

Figure 8 is a diagram of the vector pTGFG11.

Fig. 9 is a diagram of the vector pTGFG11.

Fig. 10 is a diagram of the vector pTGFG13.

Figure 11 is a diagram of the vector pTGFG14.

Figure 12 is a diagram of the vector pTGFG15.

Figure 13 is a diagram of the vector pTGFG16.

Figure 14 is a diagram of the vector pTGFG18.

Figure 15 is a diagram of the vector pTGFG19.

Figure 16 is a diagram of the vector pTGFG20.

Figure 17 is a diagram of the vector pTGFG21.

Figure 18 is a diagram of the vector pTGFG22.

Figure 19 is a diagram of the vector pTGFG23.

Fig. 20 is a diagram of the vector pTGFG24.

Fig. 21 is a diagram of the vector pTGFG25.

Fig. 22 is a diagram of the vector pTGFG26.

Fig. 23 is a diagram of the vector pTGFG27.

Fig. 24 is a diagram of the vector pTGFG28.

Fig. 25 is a diagram of the vector pTGFG29.

Fig. 26 is a diagram of the vector pTGFG30.

Fig. 27 is a diagram of the vector pTGFG31.

Fig. 28 is a diagram of the vector pTGFG32.

Fig. 29 is a diagram of the vector pTGFG33.

Fig. 30 is a diagram of the vector pTGFG2.

Fig. 31 is a diagram of the vector pTGFG34.

Fig. 32 is a diagram of the vector pTGFG35.

Fig. 33 is a diagram of the vector pTGFG36.

Fig. 34 is a diagram of the vector pTGFG37.

Fig. 35 is a diagram of the vector pTGFG38.

Fig. 36 is a diagram of the vector pTGFG53.

Fig. 37 is a diagram of the vector pTGFG64.

Fig. 38 is a diagram of the vector pTGFG0.

Fig. 39 is a diagram of the vector pTGFG66.

Fig. 40 is a diagram of the vector pTGFG67.

Fig. 41 is a diagram of the vector pTGFG68.

Fig. 42 is a diagram of the vector pTGFG69.

Fig. 43 is a diagram of the vector pTGFG82.

Fig. 44 is a diagram of the vector pTGFG95.

Fig. 45 is the DNA sequence of the vector pTGFG36 (SEQ ID No. 1).

Fig. 46 is the DNA sequence of the vector pTGFG67 (SEQ ID No. 2).

Fig. 47 shows a GFP concentration curve for cell homogenates after transfection with pTGFG5 or pTGFG20.

Fig. 48 shows corresponding light (a and c) and fluorescence microscopic (b and d) images of HeLa cells after transfection with pTGFG5 (a and b) and pTGFG20 (c and d).

Detailed description of the invention 1. Definitions

"Nucleic acid" means DNA, cDNA, mRNA, tRNA. The nucleic acid can be linear or be circular, double or single-stranded.  

"Nucleic acid construct" refers to a composite of related Nucleic acid elements. The nucleic acid elements of the construct can be in one orientation incorporated in a vector such that the gene of interest can be transcribed and, if desired, expresses a desired protein can be.

"Hormone Responsive Element" (HRE) refers to nucleic acid and in particular Areas of DNA that, as a result of hormone activation, cause gene transcription regulate. Typically, HREs are about 10-40 nucleotides in length, and especially from about 13-20 nucleotides. As explained above, HREs are activated when a hormone binds to its corresponding intracellular receptor and one Conformational change causes the receptor to have an increased affinity for the HRE exhibits and binds to it. The HRE in turn stimulates transcription. On "Steroid Responsive Element" (SRE) is an HRE that is in response to the Steroid activation regulates the transcription of genes. A "progesterone-responsive Element "(PRE) is an HRE / SRE that controls the transcription of genes as a result of Progesterone activation regulated.

"Hormone receptor" refers to a receptor that binds to or through a hormone a hormone is activated. "Steroid Receptor" means a receptor that is linked to a Steroid hormone binds or is activated by this. A "progesterone receptor" is a Receptor that binds to or is activated by the steroid hormone progesterone.

"Gene" means DNA that is involved in the expression of a polypeptide that optionally "leader" and "trailer" sequences, as well. May contain introns and exons.

"Vector" refers to any genetic construct, such as a plasmid, a phage Kosmid etc. that can replicate if associated with the right controls and that can transfer gene sequences between cells. This term includes Cloning and Expression Vehicle.  

"Promoter" denotes a region of regulatory DNA sequences for the control of the Gene transcription to which the RNA polymerase binds. The promoter forms with RNA Polymerase an initiation complex to initiate and drive transcription. Enhancers can activate the complex or silencers can inhibit it. On "Tissue-specific promoter" is a promoter which is in the DNA of a tissue for found the transcription of genes expressed in specific tissues becomes.

"Therapeutically effective dose" of the products according to the invention denotes a dose, which is effective for the purpose of treatment or prophylaxis, for example one Dose, which is an effective treatment or reduction of the symptoms of the Induces hemophilia. It is also a dose which expresses a target gene measurably activated, which can be determined by measurements of the levels of the target protein can, or a dose of which can be obtained by extrapolating in vitro or in vivo data can predict that they will be effective for treatment or prophylaxis purposes becomes. The determination of a therapeutically effective dose is within the Experience area of the specialist.

"Coded" or "coding" refers to a nucleic acid sequence which (in the case of of DNA) or (in the case of mRNA) in vitro or in vivo into a polypeptide is translated when under the control of appropriate regulatory sequences is set.

For the purposes of this application, "express", "express" or "expression" refers to on the transcription and translation of a gene encoding a protein.

2. Detailed description and examples

As stated above, an object of the present invention is to develop a new and to provide an improved delivery system for gene therapy. So puts the In one embodiment, the invention provides a composition of matter comprising a  Contains nucleic acid which comprises at least one hormone-responsive element (HRE), this hormone-responsive element to a hormone-hormone receptor complex is coupled. A preferred embodiment of the composition of the Invention is one in which the HRE is a steroid-responsive element (SRE) and the Receptor is a steroid receptor. A progesterone-responsive is particularly preferred Element (PRE) as a hormone-responsive element and a progesterone receptor as a receptor.

HREs that can potentially be used in the present invention are already have been described, for example GREs (Scheidereit, C., et al., Nature, Vol. 304, 749, 1983; von der Ahe, D., et al., Proc. Natl. Acad. Sci. USA, Vol. 83, 2817, 1986), EREs or PREs (Chambon, P., et al., Rec. Prog. Horm. Res., Vol., 40, 1, 1984; Klock, G., et al., Nature, Vol. 329, 734, 1987). It has already been found above that according to the invention most preferred HRE is a PRE. In particular, the PRE is preferred, which in Example 1 is described. The nucleic acid used in the invention contains at least one hormone-responsive element. A nucleic acid is preferred, which contains an HRE, but can also contain more than one HRE. So it can Nucleic acid for example contain 3 or 5 HREs. The most preferred The embodiment is a nucleic acid which contains a PRE.

Hormone receptors that can potentially be used in the present invention are, for example, estrogen receptors, mineral corticoid receptors, glucocorticoid receptors, retinoic acid receptors, androgen, calcitriol, thyroid hormone or progesterone receptors, as well as "orphan" receptors. Such receptors have been described in the past (Green, S., et al., Nature, Vol. 320, 134, 1986; Green, GL, et al., Science, Vol. 231, 1150, 1986; Arriza, JL, et al., Science, Vol. 237, 268, 1987; Hollenberg, SM, et al., Nature, Vol. 318, 635, 1985; Petkovitch, M., et al., Nature, Vol. 330, 444, 1987 ; Giguere, V., et al., Nature, Vol. 330, 624, 1987; Tilley, W., et al., Proc. Natl. Acad. Sci. USA, Vol. 86, 327, 1989; Baker, AR , et al., Proc. Natl. Acad. Sci. USA, Vol. 85, 3294, 1988; Weinberger, C., et al., Nature, Vol. 324, 641, 1986; Sap, J., et al. , Nature, Vol. 324, 635, 1086; Misrahi, M., et al., Biochem. Biophys. Res. Commun., Vol. 143, 740, 1987; Kastner, P., et al., Cell, Vol. 83, 859, 1995). These receptors can be from humans or other mammals, although human are preferred. Nucleotide and / or amino acid sequences of human steroid receptors are available from GenBank: Mineral Corticoid Receptor: M16801; Glucocorticoid receptor α: M10901; Glucocorticoid receptor α ₂ : U01351; Glucocorticoid receptor β: M11050; Retinoic acid receptor α: AF088888 (exon 1), AF088889 (exon 2), AF088890 (exon 3), AF088891 (exon 4), AF088892 (exon 5 and 6), AF088893 (exon 7), AF088894 (exon 8), AF088895 (exon 9 and the complete cDNA); Retinoic acid receptor γ: M24857; Androgen Receptor: M27423 (Exon 1), M27424 (Exon 2), M27425 (Exon 3), M27436 (Exon 4), M27427 (Exon 5), M27428 (Exon 6), M27429 (Exon 7), M27430 (Exon 8); Thyroid hormone receptor α ₁ : M24748, thyroid hormone receptor α ₂ : J03239; Progesterone receptor: AF016381; Somatotropin receptor: J00148; Vitamin D receptor (calcitriol receptor): J03258.

Those skilled in the art will understand that the receptor proteins use standard methods can be expressed, for example by PCR cloning of the known cDNAs from cDNA libraries and overexpression of the corresponding proteins in suitable ones Expression vectors, such as in the vectors of the present invention, in suitable host cells, for example COS cells. In a corresponding manner, a subsequent purification of the cytosolic fraction using routine methods such as can be achieved, for example, by affinity chromatography purification. To this Purpose are various suitable antibodies against the desired receptor commercially available. For example, polyclonal antibodies against the Mouse progesterone receptor, which has a sufficiently high cross-reactivity for the have human protein, obtained from Dianova (Hamburg, Germany). Further purification can also be achieved using standard methods, for example by means of chromatographic methods such as ion exchange chromatography and / or FPLC.

The most preferred receptor is the progesterone receptor. The is preferably Receptor a human progesterone receptor. Such a human progesterone receptor (from human T47D breast cancer cells) is disclosed in U.S. Patent No. 4,742,000, and Cells that express this receptor are deposited (ATCC accession number HTB, 133). As already described above, this would be a purification Receptor from the cytosol with the help of receptor-specific antibodies  Routine matter. Furthermore, US Patent No. 4,742,000 discloses a method to purify the human progesterone receptor using a specific Steroid affinity resin (see Grandics et al., Endocrinology, Vol. 110, 1088, 1982). Short said the cytosolic fraction of the T47D cells is given via sterogel commercial preparation of deoxycorticosterone coupled to Sepharose 2B and selectively binds the progesterone receptor. After washing steps with order buffer, the bound receptor eluted with a buffer containing progesterone. The eluted Steroid receptor complex is then chromatographed on DEAE-Biogel and gradually eluted with a 0.2 molar NaCl buffer. Then the bound progesterone can be released easy to replace. As described above, further purification can be carried out using Achieve routine methods well known to those skilled in the art.

Mutated versions of these receptors and their derivatives that me the function of Have receptors to bind a ligand and thereby be activated, and which bind DNA and regulate transcription can also be used in the invention be used. Such a derivative can be a chemical derivative, a variant as well as a chimera, hybrid, analog or a fusion product.

The hormone in the composition of matter can be synthetic or natural hormones include, for example, estrogen, testosterone, glucocorticoid, carrion drugs, thyroid hormone, Progesterone or derivatives thereof. These are generally available. Progesterone is on most preferred. For example, natural micronized progesterone has been used since Distributed in France in 1980 under the brand name UTROGESTAN®. His Properties are similar to those of endogenous progesterone, in particular it shows anti-estrogenic, gestagenic, weakly anti-androgenic and anti-mineral corticoid properties on.

Micronized progesterone has advantages that make it a suitable carrier to bring genes or nucleic acid constructs into their target cells. In particular, the synergistic effect of the double process leads to micronization and suspension in long chain fatty acids for increased progesterone absorption. It is  been shown that after oral administration of 100 mg UTROGESTAN® maximum Plasma progesterone levels in most cases were reached after 1 to 4 hours (Padwick, M.L., et al., Fertil. Steril., Vol. 46, 402, 1986). Then the mirrors sank greatly decreased, although they were still elevated after 12 hours. They were even after 84 hours Mirror a little above the baseline. A kinetic study in the United States has earlier Results confirmed which micronized the bioavailability of orally ingested Progesterone has shown. It was shown there that a maximum after two hours is reached and that thereafter the plasma progesterone level drops rapidly (Simon, J.A., et al., Fertil., Steril., Vol., 60, 26, 1993).

Another advantage is the use of progesterone as a carrier in that it has little unwanted side effects. Orally administered Progesterone did not respond to plasma lipids (Jensen, J. et al., Am. J. Obstet. Gynecol., Vol. 156, 66, 1987) on carbohydrate metabolism (Mosnier-Pudar, H. et al., Arch. Times. Coeur, Vol 84, 1111, 1991) an unfavorable influence. Furthermore, have an effect Daily progesterone doses of 200 mg and 300 mg not on liver enzymes (ASAT, ALAT, AFOS), the synthesis of sex hormone binding globulin (SHBG) or on the HDL cholesterol level. Although the deoxycorticosterone plasma level can increase significantly during treatment with UTROGESTAN® clear evidence that the mineral corticoid effects of this Progesterone metabolites due to the antimineral corticoid effects of the progesterone itself be fully compensated. This can be seen from a comparative study (Corvol, P., et al., In: Progesterone and progestins. Raven Press, New York, 179, 1983), in the oral administered UTROGESTAN® the mineral corticoid effects of 9-alpha Could balance fluorohydrocortisone.

It will be clear to the person skilled in the art that the composition contains further components. may be the introduction of the nucleic acid into a cell for the purpose of gene therapy can support. In particular, the composition can also contain β-cyclodextrin, Contain glycerin, lecithin or corn oil. For example, the composition of the hormone-hormone-receptor-nucleic acid complex according to the invention humans or Animals are administered orally in the form of a gelatin capsule. Progesterone could be in there  a 35% or 40% β-cyclodextrin solution or in corn oil or glycerin Peanut oil combined with lecithin is present in a concentration of 200-300 mg his.

Another aspect of the present invention is a nucleic acid construct which contains at least one hormone-responsive element (HRE), and in particular at least an HRE for regulating the expression of a gene such as, for example, a gene, which codes for a human blood coagulation factor. A preferred one Embodiment then exists that the hormone-responsive element is a steroid-responsive Element (SRE). The hormone-responsive element is particularly preferably a progesterone-responsive element (PRE). This nucleic acid construct can be used as Nucleic acid used in the composition of matter of the first aspect of the invention become.

In addition to the HREs, SREs or PREs already described above, the nucleic acid of the present invention can also contain promoter, "enhancer" or "silencer" sequences. The promoter can be ubiquitous or tissue specific. The CMV promoter is the most preferred of the ubiquitous promoters. However, a tissue specific promoter is preferred over a ubiquitous promoter. Tissue-specific promoters that can be imagined in the context of the present invention include, for example, alpha ₁ -antitrypsin. The nucleic acid construct can also additional sequences such. B. contain the ampicillin resistance gene. Other reporter sequences known to those skilled in the art may also be included, such as e.g. B., the green fluorescent protein (GFP), luciferase, β-galactosidase or chloramphenicol transferase (CAT). The SV40 intron and the SV40 poly-A are particularly preferred as the enhancer sequence. A preferred nucleic acid construct contains, successively from the 5 'to the 3' end: a PRE, a CMV promoter, a gene of interest, the SV40 intron and an SV40 poly A enhancer sequence and an ampicillin resistance gene.

The gene of interest can be selected from those for proteins encode that are lacking in various genetic defects or involved in conditions are related to disproportionate responses to hormone signals there are, for example, hormone-dependent forms of cancer such as breast cancer, Ovarian cancer, endometrial cancer and prostate cancer. The interesting thing Gen can also be used to identify a defective gene that is too genetic Diseases such as hemophilia, von Willebrand's disease or cystic fibrosis leads to replace. The gene of interest also includes mutations in such genes or Gene that codes for a fusion product. The nucleic acid construct of the present Invention can include more than one gene of interest.

In particular, the gene of interest can have genes for a blood coagulation factor, preferably a human coagulation factor. The genes for Code factor VIII and factor IX, which are associated with hemophilia A and B, respectively are good candidates for the invention. Other candidates include the gene which codes for von Willebrand factor, factor N, factor X or protein C.

Other useful genes include, but are not limited to, hormone genes, such as the genes encoding insulin, parathyroid hormone, luteinizing hormone releasing factor (LHRH), alpha and beta seminal inhibin, and human growth hormone; Hormone receptor genes such as the glucocorticoid receptor, the estrogen receptor, the progesterone receptor, the retinoic acid receptor; Growth factors such as vascular endothelial growth factor (VEGF), nerve growth factor, epidermal growth factor; Genes for enzymes; Genes for cytokines or lymphokines such as. B. encode interferon, granulocytic macrophage colony stimulating factor (GM-CSF), colony stimulating factor-1 (CSF-1), tumor necrosis factor (TNF), and erythropoietin (EPO); Genes coding for inhibitor substances such as alpha ₁ -antitrypsin and genes; which code for substances which act as medicaments, for example genes which code for the diphtheria and cholera toxins, ricin or cobra venom (cobra venom factor). In addition, antisense sequences can also be administered as genetic material.

Another aspect of the present invention relates to vectors that Contain nucleic acid constructs of the present invention. These vectors can be used in the composition of matter of the present invention. Preferably is the nucleic acid sequence used in the present invention, however linear rather than circular. The vectors can be transient, permanent or episomal Expression of the nucleic acid in the nucleic acid construct will be able to. As above mentioned, the nucleic acid construct may further contain additional elements.

Embodiments of the invention further include transfected and transformed cells containing these vectors and / or nucleic acid constructs. In the context of this invention, a transfected cell is one in which foreign DNA has been incorporated. Transfection methods can include microinjection, CaPO ₄ precipitation, electroporation, liposome fusion, or "gene gun" methods. Most preferred is transfection by electroporation.

Transformation refers to the introduction of genetic material into a cell, such as that vectors or nucleic acid constructs according to the invention, whereby the cell is transient, stable, or permanently changed so that it expresses a specific gene product or is otherwise altered in expression. transformation can be achieved by in vivo or in vitro techniques, although an in vivo Transformation is preferred.

Another embodiment of the present invention are pharmaceutical Compositions containing a therapeutically effective dose of the invention Contain nucleic acid constructs and a hormone. The hormone is preferably a Steroid, and progesterone is particularly preferred as described above. The dose is depending on the condition to be treated, the parameters of the patient and the desired result. The determination of the dose is within the range of experience of the specialist.  

The pharmaceutical composition (or the substance composition that Nucleic acid construct or the vector) of the present invention can be administered orally, intravenously, administered intramuscularly, subcutaneously, topically or by the "gene gun". Oral administration using a micronized hormone is preferred. Administration can be systemic or targeted to a particular tissue.

The invention further comprises a method for introducing a nucleic acid construct, for a gene of interest, for example a human blood coagulation factor, encoded into a cell to express the clotting factor in that cell. At This procedure uses the nucleic acid necessary for a human coagulation factor encoded, combined with a nucleic acid construct that contains at least one hormone-responsive element (HRE), preferably a progesterone-responsive element, includes.

The mixture of nucleic acids attached to the hormone-hormone receptor complex is bound with an excess of the hormone, preferably progesterone used to isolate the nucleic acid by various methods known to those skilled in the art known and outlined above to be brought into a cell. The admission to the cell will stimulated by the interaction of the hormone at the level of the cell membrane. The Hormone or steroid not only interacts with the lipid bilayer of the cell membrane due to perturbation of the membrane; but also by activating certain hormone or steroid sensitive membrane receptors. This is for progesterone and others Steroids have been proven. Furthermore, it is also known to use hormones Diffusion can pass through the cell membrane. In the present invention, the Nucleic acid bound to the hormone-hormone receptor complex in the course of the Diffusion process are transported through the membrane.

Another aspect of the invention is a method for treating a Blood coagulation defect, by administering a therapeutically effective dose of the substance composition according to the invention to an organism. This method  includes the previously discussed considerations regarding administration and Dosage.

Experiments were used to illustrate the technical aspects of the present invention carried out. These experiments are described in Examples II to IV.

In the following, the present invention is explained using examples. The Those skilled in the art will readily recognize that the invention is not limited to these examples is.

Example I Construction of the vectors Preparation of the vector pTGFG1

The vector pUC19 (MBI Fermentas) was digested with XbaI, with Klenow enzyme treated and religated. This XbaI deleted vector was then digested with EcoRI, with Klenow enzyme treated and religated to delete the EcoRI site. To one To insert the XbaI interface into the SacI interface of this vector, this was included SacI digested, treated with T4 polymerase, dephosphorylated with alkaline phosphatase and ligated with the XbaI linker CTCTAGAG (Biolabs # 1032). Another Xbal Interface was introduced in that the newly created vector with Hindill was digested, treated with Klenow, dephosphorylated with alkaline phosphatase and was ligated with the XbaI-left CTCTAGAG (Biolabs # 1032). This vector was called pUC19 / X.

In order to delete the XbaI interface in the vector phGFP-S65T (Clontech), this vector was digested with XbaI, treated with Klenow enzyme and religated to the vector pGFP / 0 thus obtained. A 2.3 kb fragment containing the GFP gene was isolated after pGFP / 0 was digested with MluI, treated with Klenow enzyme and digested with BamHI. This fragment was inserted into the multiple cloning region of the vector pUC19 / X after it had been digested with SalI, treated with Klenow enzyme and digested with BamHI. The resulting vector was named pTGFG1 ( Fig. 1).

Production of the insert PRE (ds)

The oligonucleotides (Metablon) PRE-S (5'-GGG GTA CCA GCT TCG TAG CTA GAA CAT CAT GTT CTG GGA TAT CAG CTT CGT AGC TAG AAC ATC ATG TTC TGG TAC CCC-3 ') (SEQ ID No. 3) and PRE-AS (5'-GGG GTA CCA GAA CAT GAT GTT CTA GCT ACG AAG CTG ATA TCC CAG AAC ATG ATG TTC TAG CTA CGA AGC TGG TAC CCC-3 ') (SEQ. ID No. 4) were hybridized and by means of kinase reaction phosphorylated, resulting in the inserts PRE (ds).

Preparation of the vector pTGFG4

The vector pTGFG1 was digested with PstI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. It was then ligated to the PRE (ds) fragment, the resulting vector was called pTGFG4 ( Fig. 2).

Preparation of the vector pTGFG5

The vector pTGFG1 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE (ds) insert, the resulting vector was called pTGFG5 ( Fig. 3).

Preparation of the vector pTGFG6

The vector pTGFG1 was digested using EheI and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG6 ( Fig. 4).

Preparation of the vector pTGFG7

The vector pTGFG1 was digested with KpnI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG7 ( Fig. 5).

Preparation of the vector pTGFG8

The vector pTGFG7 was digested using EheI and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG8 ( Fig. 6).

Preparation of the vector pTGFG9

The vector pTGFG7 was digested with SapI, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG9 ( Fig. 7).

Preparation of the vector pTGFG10

The vector pTGFG7 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was then ligated to the PRE (ds) fragment and the resulting vector was called pTGFG10 ( Fig. 8).

Preparation of the vector pTGFG11

The vector pTGFG7 was digested with PstI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE (ds) insert and the resulting vector was called pTGFG1 l ( Fig. 9).

Preparation of the vector pTGFG13

The vector pTGFG6 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG13 ( Fig. 10).

Preparation of the vector pTGFG14

The vector pTGFG6 was digested with PstI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG14 ( Fig. 11).

Preparation of the vector pTGFG15

The vector pTGFG5 was digested with SapI, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG15 ( Fig. 12).

Preparation of the vector pTGFG16

The vector pTGFG5 was digested with PstI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG16 ( Fig. 13).

Preparation of the vector pTGFG18

The vector pTGFG9 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG18 ( Fig. 14).

Preparation of the vector pTGFG19

The vector pTGFG10 was digested using EheI and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG19 ( Fig. 15).

Preparation of the vector pTGFG20

The vector pTGFG11 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG20 ( Fig. 16).

Preparation of the vector pTGFG21

The vector pTGFG15 was digested using EheI and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRB (ds) insert, the resulting vector was called pTGFG21 ( Fig. 17).

Preparation of the vector pTGFG22

The vector pTGFG14 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG22 ( Fig. 18).

Preparation of the vector pTGFG23

The vector pTGFG11 was digested using EheI and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG23 ( Fig. 19).

Preparation of the vector pTGFG24

The vector pTGFG9 was digested with PstI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG24 ( Fig. 20).

Preparation of the vector pTGFG25

The vector pTGFG14 was digested with SapI, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG25 ( Fig. 21).

Preparation of the vector pTGFG26

The vector pTGFG16 was digested with SapI, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG26 ( Fig. 22).

Preparation of the vector pTGFG27

The vector pTGFG9 was digested using EheI and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG27 ( Fig. 23).

Preparation of the vector pTGFG28

The vector pTGFG18 was digested using EheI and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG28 ( Fig. 24).

Preparation of the vector pTGFG29

The vector pTGFG25 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG29 ( Fig. 25).

Preparation of the vector pTGFG30

The vector pTGFG27 was digested with PstI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. It was then ligated to the PRE (ds) fragment and the resulting vector was called pTGFG30 ( Fig. 26).

Preparation of the vector pTGFG31

The vector pTGFG24 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG31 ( Fig. 27).

Preparation of the vector pTGFG32

The vector pTGFG19 was digested with PstI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. Then it was ligated with the PRE (ds) insert, the resulting vector was called pTGFG32 ( Fig. 28).

Preparation of the vector pTGFG33

The vector pTGFG28 was digested with PstI, treated with T4 polymerase and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE (ds) insert and the resulting vector was called pTGFG33 ( Fig. 29).

Preparation of the vector pTGFG2

The vector pUC19 (MBI Fermentas) was digested with SalI, with Klenow enzyme treated and dephosphorylated with alkaline phosphatase. He was awarded the NotI  Left GCGGCCGC (Biolabs # 1045) ligated, the resulting vector became pUCI9 / N called.

A 1.4 kb fragment containing the open reading frame of human coagulation factor IX and isolated from a human cDNA library was inserted into the PstI site of the vector pUC19 / N digested with PstI treated with T4 polymerase and had been dephosphorylated with alkaline phosphatase. A 1.4 kb fragment, which contained the open reading frame of human blood coagulation factor IX, was cut out of the resulting vector pUC19 / N-FIX with the aid of a double digest with HindIII and NotI. This fragment was ligated to the 4.3 kb fragment of the HindIII / NotI double-digested vector pTGFG1. The resulting vector was called pTGFG2 ( Fig. 30).

Preparation of the vector pTGFG34

A 1.4 kb fragment which contained the open reading frame of the human blood coagulation factor IX was cut out of the vector pUC19lN-FIX by means of a HindIII / NotI double digest. This fragment was ligated to the 4.3 kb fragment of the HindIII / NotI double-digested vector pTGFG3. The resulting vector was called pTGFG34 ( Fig. 31).

Preparation of the vector pTGFG35

A 1.4 kb fragment was cut from the vector pUC19 / N-FIX by HindIII / NotI double digestion, which contained the open reading frame of the human blood coagulation factor IX. This fragment was ligated to the 4.3 kb fragment of the HindIII / NotI double-digested vector pTGFG4. The resulting vector was called pTGFG35 ( Fig. 32).

Preparation of the vector pTGFG36

A 1.4 kb fragment was cut out of the vector pUC19 / N-FIX by HindIII / NotI double digestion and contained the open reading frame of the human blood coagulation factor IX. This fragment was ligated to the 4.3 kb fragment of the HindIII / NotI double-digested vector pTGFG5. The resulting vector was called pTGFG36 ( Fig. 33). This vector is preferred for the introduction of factor IX into the cell. Its DNA sequence is shown in Fig. 46.

Preparation of the vector pTGFG37

A 1.4 kb fragment was cut from the vector pUC19 / N-FIX by HindIII / NotI double digestion, which contained the open reading frame of the human blood coagulation factor IX. This fragment was ligated to the 4.3 kb fragment of the HindIII / NotI double digested vector pTGFG6. The resulting vector was named pTGFG37 ( Fig. 35).

Preparation of the vector pTGFG38

A 1.4 kb fragment was cut from the vector pUC19 / N-FIX by HindIII / NotI double digestion, which contained the open reading frame of the human blood coagulation factor IX. This fragment was ligated to the 4.3 kb fragment of the HindIII / NotI double-digested vector pTGFG7. The resulting vector was named pTGFG38 ( Fig. 35).

Preparation of the vector pTGFG53

A 1.4 kb fragment was cut from the vector pUC19 / N-FIX by HindIII / NotI double digestion, which contained the open reading frame of the human blood coagulation factor IX. This fragment was ligated to the 4.3 kb fragment of the HindIII / NotI double-digested vector pTGFG20. The resulting vector was called pTGFG53 ( Fig. 36).

Preparation of the vector pTGFG64

A 1.4 kb fragment was cut out of the vector pUC19 / N-FIX by HindIII / NotI double digestion and contained the open reading frame of the human blood coagulation factor IX. This fragment was ligated to the 4.3 kb fragment of the HindIII / NotI double-digested vector pTGFG33. The resulting vector was called pTGFG64 ( Fig. 37).

Production of the insert ALLG (ds)

The oligonucleotides (Metablon) ALLG1 / 1 (5'-AGC TTG ACC TCG AGC AAG C-3 ') (SEQ. ID NO: 6) and ALLG2 (5'-GGC CGC TTG CTC GAG GTC A-3 ') (SEQ. ID NO: 7) were hybridized and phosphorylated by means of a kinase reaction, the resulting insert was called ALLG (ds). This insert ALLG (ds) was constructed for the purpose in any vector, a sequence with a multiple cloning area for the to introduce possible insertion of other transgenes.

Preparation of the vector pTGFG0

The vector pTGFG1 was double digested with HindIII and NotI. The 4.3 kb fragment was ligated with the insert ALLG (ds), the resulting vector was called pTGFG0 ( Fig. 38).

Preparation of the vector pTGFG66

The vector pTGFG4 was double digested with HindIII and NotI. The 4.3 kb fragment was ligated to the ALLG (ds) fragment, the resulting vector was called pTGFG66 ( Fig. 39).

Preparation of the vector pTGFG67

The vector pTGFG5 was double digested with HindIII and NotI. The 4.3 kb fragment was ligated with the insert ALLG (ds), the resulting vector was called pTGFG67 ( Fig. 40). This vector is preferred for the administration of any gene inserted into the cloning site; its DNA sequence is shown in Fig. 47.

Preparation of the vector pTGFG68

The vector pTGFG6 was double digested with HindIII and NotI. The 4.3 kb fragment was ligated with the insert ALLG (ds), the resulting vector was called pTGFG68 ( Fig. 41).

Preparation of the vector pTGFG69

The vector pTGFG7 was double digested with HindIII and NotI. The 4.3 kb fragment was ligated with the insert ALLG (ds), the resulting vector was called pTGFG69 ( Fig. 42).

Preparation of the vector pTGFG82

The vector pTGFG20 was double digested with HindIII and NotI. The 4.3 kb fragment was ligated with the insert ALLG (ds), the resulting vector was called pTGFG82 ( Fig. 43).

Preparation of the vector pTGFG95

The vector pTGFG33 was double digested with HindIII and NotI. The 4.3 kb fragment was ligated to the ALLG (ds) fragment, the resulting vector was called pTGFG95 ( Fig. 44).

Example II Isolation of human factor IX cDNA

Factor IX cDNA was determined using two primers that started and stopped Factor IX reading frame codon overlapped from human liver cDNA (Clontech) amplified. The resulting 1387 base pair fragment contained the full open reading frame. Interfaces for EcoRI (upstream) and BamHI (downstream) were placed at the end of the respective primer in order to clone facilitate. The amplification was carried out with Pwo polymerase (Boehringer Mannheim) in a 50 µl reaction volume [10 mM Tris HCl pH 8.85, 25 mM KCl, 5 mM  Ammonium sulfate, 2 mM magnesium sulfate] with 30 incubation cycles (1 min. 96 ° C., 1 min. 60 ° C, 2 min. 72 ° C, followed by a final extension step of 10 min at 72 ° C).

The reaction products were in the EcoRI and BamHI sites of pUC19 ligated and used to transform E. coli DH5-a. Clones were positive selected. The correctness of the sequences was checked by cyclic sequencing (Amersham) from both ends with labeled primers (IR-700) and automated Analysis confirmed using the LiCor sequencing system (MWG, Biotech).

The following primers were used:

Example III Expression and quantification of the marker protein GFP ("Green Fluorescent Protein ")

HeLa cells were electroplated with the plasmids pTGFG5 and pTGFG20 transfected. The transfected cells were harvested, the cell pellets homogenized and in lysed a buffer, the philosophy buffered saline (pH 7.5) and 10 mM PMSF contained. The concentration of the green fluorescent protein (GFP) in the Cell homogenate was determined by competitive ELISA.

For this purpose, GFP was used in a defined concentration on microtiter plates upset. GFP samples were then taken in the presence of anti-GFP antibodies added. After various washing steps, a labeled second antibody became added to enable detection of the first antibody. The color reaction was measured photometrically (extinction). In general there was less antibody left to bind to the applied GFP the more GFP had been added.  

As a result, a reduced absorbance correlates with a higher GFP concentration in the rehearsal.

A GFP concentration curve was determined by linear regression ( Fig. 47), with bovine serum albumin (BSA) being used as the standard. An average of 2.4 µg GFP / ml was found for pTGFG5 (1 PRE) and 5.2 µg GFP / ml for pTGFG20 (3 PREs).

Fig. 48a-d show microscopic images of HeLa cell cultures after transfection with pTGFG5 ( Fig. 48a and b) or TGFG20 ( Fig. 48c and d).

Fig. 46a and c show light microscopic images as a control, and Figs. 48b and d show the corresponding sections in fluorescence images. By default, more than 50% of the cells expressed GFP, indicating very efficient expression, with more efficient expression in the presence of a single PRE.

Example IV Quantification of human factor IX using ELISA

HeLa cells were either by electroporation or by using the Liposome reagent DOTAP (Boehringer Mannheim) with the plasmids pTGFG36, pTGFG53 and pTGFG64 transfected. These plasmids contain the cDNA of the human coagulation factor IX. Recombinant human Blood coagulation factor IX was secreted into the cell culture supernatant and with the help of a Sandwich ELISA method quantified.

0.11 M sodium citrate and 10 mM PMSF were added to adjust the degradation of the to prevent human factor IX. The concentration determination of the expressed human factor IX was determined using the enzyme immunological in vitro Procedure "Asserachrom IX: AG" carried out by Boehringer-Mannheim. As standard was the factor IX standard from Octapharma AG in aqueous solutions of 28 IU / ml used.  

In six different transfection experiments, in which HeLa cells with plasmids transfected and the human factor IX cDNA (pTGFG36, 53 and 64) under Use of electroporation or the lipid transfection reagent (DOTAP, Boehringer Mannheim) were transfected, a concentration range of 3-25 ng / ml human coagulation factor IX reached.

Claims (33)

1. A composition of matter that contains a nucleic acid that is a hormone responsive element (HRE), said hormone-responsive element a hormone-hormone receptor complex is coupled. 2. A composition of matter according to claim 1, wherein the nucleic acid additionally contains a gene that codes for a blood coagulation factor. 3. A composition of matter according to claim 2, wherein the human blood coagulation factor from the group of factors VIII, IX and von Willebrand factor (vWF) is selected. 4. A composition of matter according to claim 2, wherein the hormone responsive Element is a steroid-responsive element (SRE). 5. A composition of matter according to claim 4, wherein the steroid responsive Element (SRE) is a progesterone-responsive element (PRE). 6. A composition of matter according to claim 1, wherein the complex is a steroid Is steroid receptor complex. 7. A composition of matter according to claim 6, wherein the receptor is a Progesterone receptor and the steroid is progesterone. 8. A composition of matter according to claim 3, wherein the human blood coagulation factor is factor IX. 9. A composition of matter according to claim 7, wherein the human blood coagulation factor is factor IX. 10. A nucleic acid construct that contains at least one hormone-responsive element (HRE) for the regulation of the expression of a gene for human blood coagulation factor coded, contains. 11. A nucleic acid construct according to claim 10, wherein the hormone responsive Element is a steroid-responsive element (SRE). 12. A nucleic acid construct according to claim 11, wherein the steroid responsive Element is a progesterone-responsive element (PRE).   13. A nucleic acid construct according to claim 10, wherein the human blood coagulation factor from the group of factors VIII, IX and von Willebrand factor (vWF) is selected. 14. A nucleic acid construct according to claim 10, further comprising a tissue contains specific promoter. 15. A nucleic acid construct according to claim 10, wherein the hormone responsive Element (HRE) a progesterone-responsive element (PRE) and the Blood coagulation factor is factor IX. 16. A nucleic acid construct according to claim 15, further comprising a tissue contains specific promoter. 17. A vector containing a nucleic acid construct according to claim 10. 18. A vector containing a nucleic acid construct according to claim 11. 19. A vector containing a nucleic acid construct according to claim 12. 20. A vector containing a nucleic acid construct according to claim 13. 21. A vector containing a nucleic acid construct according to claim 14. 22. A vector containing a nucleic acid construct according to claim 15. 23. A vector containing a nucleic acid construct according to claim 16. 24. A cell that transfects with a nucleic acid construct according to claim 10 is. 25. A cell that is transformed by a nucleic acid construct according to claim 10 has been. 26. A pharmaceutical composition containing a therapeutically effective dose of a nucleic acid construct according to claim 10 and contains a hormone. 27. A pharmaceutical composition containing a therapeutically effective dose of a nucleic acid construct according to claim 15 and progesterone. 28. A pharmaceutical composition containing a therapeutically effective dose contains a composition of matter according to claim 1.   29. A pharmaceutical composition containing a therapeutically effective dose contains a composition of matter according to claim 9. 30. A method of introducing a nucleic acid necessary for an expression Gene encoded, in a cell, comprising the provision of the substance mixture according to Claim 1 to an organism so that the hormone of the mixture Cell membrane through diffusion and attached to the hormone Hormone receptor complex coupled nucleic acid through the membrane and into the Cell transported. 31. A method according to claim 30, wherein a nucleic acid is for human Coded factor IX, is brought into the cell. 32. A method of treating a blood coagulation defect which involves the administration of Delivery of a therapeutically effective amount of a composition of matter according to claim 1 to an organism. 33. A method of treating hemophilia B which involves administering a therapeutically effective amount of a composition of matter according to claim 9 to an organism.