CA2362970A1 - Hormone-hormone receptor complexes and nucleic acid constructs and their use in gene therapy - Google Patents

Abstract

The invention relates to the use of a nucleic acid contruct comprising at least one hormone responsive element and a transgene for preparing an agent for gene transfer. It further relates to particular nucleic acid contructs comprising at least one hormone responsive element and a transgene, wherein one of said at least one hormone responsive elements is not functionally linked to the transgene, vectors comprising such nucleic acid contructs and compositions of matter comprising such nucleic acid constructs wherein the hormone responsive elements of the constructs are coupled to a hormone-hormone receptor complex. The nucleic acid constructs, plasmids, and compositions of matter of the invention have applications in gene therapy, particularly in the treatment of human blood clotting disorders, such as hemophilia. They may also be used to up- or down-regulate target genes and for the delivery of vaccines.

Description

Hormone -Hormone Receptor Complexes and Nucleic Acid Constructs and Their Use in Gene Therapy Background of the Invention 1. Object of the Invention The invention relates to the use of a nucleic acid construct to comprising at least one hormone responsive element and a transgene for preparing an agent for gene transfer. It further relates to particular nucleic acid constructs comprising at least one hormone responsive element and a transgene, wherein one of said at least one hormone responsive elements is not functionally linked to the is transgene, vectors comprising such nucleic acid constructs and compositions of matter comprising such nucleic acid constructs wherein the hormone responsive elements of the constructs are coupled to a hormone-hormone receptor complex. The nucleic acid constructs, plasmids, and compositions of matter of the invention Zo have applications in gene therapy, particularly in the treatment of human blood clotting disorders, such as hemophilia. They may also be used to up- or down-regulate target genes and for the delivery of vaccines.
2s 2. Summary of the Related Art Gene therapy is a method that holds great promise for many diseases and disorders. In general, it involves the transfer of recombinant genes or transgenes into somatic cells to replace proteins with a genetic defect or to interfere with the pathological process of CONFIRMATION COPY

an illness. In principle, gene therapy is a simple method. In practice, many disadvantages must still be overcome.
Research in gene therapy has concentrated on ways to most effectively incorporate DNA into cells of a patient. Viral vectors are s currently the widely used vehicles in clinical gene therapy approaches.
In terms of efficacy in gene expression, the viral delivery systems have major advantages over techniques using DNA-lipid formulations as delivery vehicles or over mechanical methods, such as the gene gun. Although there are a variety of viral systems tested for gene ~o therapeutical strategies, retroviral vectors and adenoviral vectors are presently the most widely used vehicles (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. ). Still, these systems have major disadvantages, such as Is potential viral contamination. Other safety concerns continue- to hamper the development of clinical application of gene therapy using these viral systems. For example, recombinant retroviruses have the disadvantage of random chromosomal integration, which may lead to activation of oncogenes or inactivation of tumor-suppressor genes.
Zo Also, repetitive use of recombinant adenoviruses has caused severe immunological problems (Elkon, K. B. et al., Proc. Natl. Acad. Sci.
USA, Vol. 94, 9814, 1997). The humoral response resulted, in the production of antibodies to adenovirus proteins preventing subsequent infection. Immunosuppressive drugs may ameliorate these effects, but as they place an additional burden on the patient (Dai, Y., et al., Proc.
Nat/. Acad. Sci. USA, Vol. 92, 1401, 1995).
Yet another viral delivery system involves adenoassociated virus (AAV). The AAV requires coinfection with an unrelated helper virus.
Although such recombinant AAV virions have proven useful for 3o introducing several small gene sequences into host cells, gene J
delivery systems based on those particles are limited by the relative small size of AAV particles. This feature greatly reduces the range of appropriate gene protocols. Moreover, the need to also use a helper virus adds a complicating factor to this delivery system (Muzyczka, N., s Curr. Top. Microbiol. Immunol., Vol. 158, 97, 1992).
Though safer, non-viral gene therapy approaches are also unsatisfactory. Problems with inefficient gene delivery or poor sustained expression are major drawbacks. Yet the methods available such as the direct injection of DNA into cellular compartments, or the ~o application of mixtures of DNA with cationic lipids or polylysine allowing the transgene to cross the cell membrane more easily, have not overcome these hurdles (Felgner, P., et al., Proc. Nat/. Acad. Sci.
USA, Vol. 84, 7413, 1987; Behr, J.-P., Bioconjugate Chemistry, Vol. 5, 382, 1994).
is Introduction of naked DNA (polynucleotide) sequences (including antisense DNA) into vertebrates, is reported to be achieved by injection into tissues such as muscle, brain or skin or by introduction into the blood circulation (Wolff, J. A., et al., Science, Vol. 247, 1990;
Lin, H., et al., Circulation, Vol. 82, 2217, 1990; Schwartz, B., et al., Zo Gene Ther., Vol. 3, 405, 1996). Also, a direct gene transfer into mammals has been reported for formulations of DNA encapsulated in liposomes and DNA entrapped in proteoliposomes containing receptor proteins. Although injected naked DNA leads to transgene expression, the efficiency is by far not comparable to viral-based DNA delivery 2s systems. A limitation of the method of naked DNA injection is the fact that transgene expression is dose-dependent. The gene expression is saturable, and an increase in the amount of DNA injected leads to decreased protein production per plasmid. Thus, protein expression can dramatically decrease, if the amount of DNA injected is above a 3o certain threshold.

Among the genetic disorders that the skilled artisan has sought to overcome using these prior art methods are those relating to blood clotting disorders, and in particular, hemophilia (Lozier, J. N. and Brinkhous, K. M., JAMA, Vo1.271, 1994; Hoeben, R. C., Biologicals, s Vol. 23, 27, 1995). For example, hemophilia A and B are X-linked, recessive bleeding disorders caused by deficiencies of clotting factors VIII and IX, respectively (Sadler, J. E. et al., in: The Molecular Basis of Blood Diseases, 575, 1987). The incidence of hemophilia is about 1 in 5,000 male births. Hemophiliacs suffer from excessive bleeding due io to the lack of clotting at the site of wounds. The inability to clot properly causes damage to joints and internal tissues as well as posing risks to the proper treatment of cuts.
Treatment of hemophilia A is possible by the administration of the blood clotting factor VIII. Until recently, factor VIII preparations is had to be prepared by concentrating blood from donors, posing -the risk of contamination by infectious agents, such as HIV and hepatitis.
The gene for factor VIII has been cloned (e.g., Vehar et al., Nature Vol. 312, 337 1984) allowing for the production of a recombinant product. Although recombinant methods provide factor VIII of higher ao purity than blood concentrates, the exogenous supply of factor VIII to a patient still requires repeated doses throughout the lifetime of the patient, an inconvenient and expensive solution.
Other forms of hemophilia include hemophilia B, caused by a defect in the gene coding for Factor IX. The gene therapy systems Zs described above have been attempted for the treatment of hemophilia A and B with factors VIII and IX, respectively. (See e.g., WO
94/29471). However, these systems have the disadvantages already discussed above.
On the other hand, the classical model of the action of hormones 3o is based on the concept of binding interaction of the hormone to an intracellular receptor, located in the cytoplasm or the nucleus (Evans, R., Science, Vol. 240, 889, 1988). These intracellular receptors remain latent until exposed to their target hormone. When so exposed, the hormone receptor changes its conformation after the s hormone is bound and translocates in the activated form into the cell nucleus where it binds as a dimer to hormone responsive elements in the promoter region of hormone-regulated genes (Beato, M., Cell, Vol.
56, 335, 1989; O ~ Mallet', B., et al., Biol. Reprod., Vol. 46, 163, 1992). The hormone responsive elements are enhancer elements io usually located in the 5 ~ flanking region of the specific hormone-induced gene, i.e., are functionally linked to the specific hormone induced gene. DNA constructs comprising a hormone responsive element and a nucleic acid sequence encoding a protein of interest are disclosed in U.S. Pat. Nos. 5,688,677 and 5,580,722 and are taught to Is be suitable for expression of the protein of interest.
An example of such intracellular receptors is the steroid receptor. Steroid receptors belong to a superfamily of ligand-dependent transcription factors characterized by a unique molecular structure. The centrally located highly conserved DNA-binding domain Zo defines this superfamily. The second important and relatively invariant region is the COOH-terminal ligand-binding domain. An example of such a receptor is the progesterone receptor mediated by the steroid progesterone. At the progesterone receptor, progesterone acts as a natural agonist whereas it displays potent antimineralocorticoid as properties both at the molecular and the systemic level. Besides classical effects on the uterus, antiepileptic, anxiolytic, hypnotic and anesthetic properties have been attributed to progesterone according to numerous studies.
Methods have been proposed for the use of mutant hormone 3o receptors, including mutant steroid receptors for gene therapy. For G
example, such methods are disclosed in WO 93/23431, WO 98/18925, WO 96/40911. Moreover, WO 98/33903 discloses a genetic construct comprising a steroid responsive element from a tissue specific gene, a coding sequence, and an SV40 enhancer.
s Brief Description of the Invention The object of the present invention is to overcome the disadvantages of the previous gene therapy delivery systems. It was found that a hormone-hormone receptor complex possesses the io ability to drag a nucleic acid construct having one or more hormone responsive elements) through the cell membrane into a cell. It was also found that if the construct comprises further functional sequences besides the hormone responsive elements (hereinafter "transgenes"), the functional sequences exert their function. The hormone responsive ~s element may also enhance the expression of the transgene. Moreover, it was found that steroid hormones are very effective mediators for the transfer of nucleic acid constructs through the cell membranes into a cell. The present invention thus provides (1) the use of a nucleic acid construct comprising at least one 2o hormone responsive element (hereinafter referred to as "HRE") and a transgene for preparing an agent for gene transfer (said at least one HRE being functionally linked to the transgene or not);

(2) a preferred embodiment of (1) above, wherein the agent further comprises a hormone-hormone receptor complex;
as (3) a nucleic acid construct comprising at least one HRE and a transgene, wherein one of said at least one HREs is not functionally linked to the transgene;
(4) a vector comprising the nucleic acid construct of (3) above;

(5) a transformed cell or transgenic organism comprising the nucleic acid construct as defined in (3) above or the vector as defined in (4) above;
(6) a composition of matter comprising a nucleic acid construct s comprising at least one HRE and a transgene as defined in (3) above and/or a vector as defined in (4) above, said at least one HRE being coupled to a hormone-hormone receptor complex;
(7) a preferred embodiment of (6) above, wherein the transgene is a gene encoding a blood clotting factor;
to (8) a preferred embodiment of (7) above, wherein the blood clotting factor is factor IX;
(9) a preferred embodiment of (7) above, wherein the blood clotting factor is factor VIII;
(10) a pharmaceutical composition comprising the nucleic acid ~s construct as defined in (3) above and/or the composition of matter as defined in (6) to (9) above;
(11) a method for preparing the composition of matter as defined in (6) above, which method comprises admixing the nucleic acid construct with the hormone receptor and the hormone;
ao (12) a method for gene transfer which comprises administering the agent as defined in (1) and (2) or the composition of matter as defined in (6) to (9) above to an organism or to a cellular system;
(13) a method for delivering into an organism or into a cellular system a nucleic acid encoding a transgene to be expressed in the 2s cells of the organism or the cells of the cellular system, which method comprises administering an agent as defined in (1) above or composition of matter as defined in (6) to (9) above to the organism or to the cellular system so that the hormone in the composition interacts with the cell membrane and therewith enhances diffusion and transport of the nucleic acid that is coupled to the hormone-hormone receptor complex across the membrane and into the cell;
(14) a method of treating blood clotting disorders comprising administering a therapeutically effective amount of the composition of s matter as defined in (7) above to an organism or to a cellular system;
(15) a method of treating hemophilia B, comprising administering a therapeutically effective amount of the composition of matter as defined in (8) above to an organism or to a cellular system;
(16) method of treating hemophilia A, comprising administering ~o a therapeutically effective amount of the composition of matter as defined in (9) above to an organism or to a cellular system;
(17) use of a steroid hormone for preparing an agent for gene transfer; and (18) a method for gene transfer which comprises administering is a nucleic acid construct to an organism or to a cellular system;
wherein the nucleic acid construct contains a transgene and is encapsulated in a steroid hormone.
In a preferred embodiment of (1) to (16) above the hormone ao responsive element is a steroid responsive element (SRE), most preferably a progesterone responsive element (PRE). In embodiments (2) and (6) to (16) the receptor preferably is a steroid receptor, most preferably, a progesterone receptor. Similarly, the hormone is preferably a steroid, most preferably, progesterone.
2s The present invention thus provides a delivery system for gene therapy that should overcome the prior art disadvantages. The presence of the hormone responsive element on the nucleic acid carrying a transgene encourages the binding of a hormone-hormone receptor complex. Thus, the present invention uses the activated 3o hormone receptor as a link (or binding compound) between the nucleic acid carrying the transgene and the hormone known to interact with the cell membrane. The general known biological activity mediated by the HREs is not the primary effect utilized in the present invention, but might k-e an additional effect when regulation of the s transgene is desired. The general principle is depicted in Figure 1. The hormone responsive element is preferably present as a nucleic acid dimer sequence or nucleic acid multimer sequence. Even in an inverse orientation, the hormone responsive element will exert its proper function. The hormone-hormone receptor complex contains a ~o hormone receptor that becomes activated after binding of its specific hormone. The hormone receptor in the activated state is able to recognize and bind to its specific hormone responsive element, which in the present invention is present within the nucleic acid comprising the desired transgene, e.g., a human blood-clotting factor.
is Vaccination is another aspect of the embodiment (12) defined above. Introducing a nucleic acid construct or composition of matter of the invention comprising a gene for an antigen or containing a viral sequence into a cell (DNA vaccines) using the method mentioned above may also provide a way to stimulate the cellular immune Zo response.
Brief Description of the Drawings Fi_ u~ shows the concept of gene transfer of the present invention Zs (with HRE = hormone responsive element, HR = hormone receptor, H
= hormone, blank circles = lipophilic matrix).
Fig~~ure 2 is a diagram of the vector pTGFGl.
Figure 3 is a diagram of the vector pTGFGS.
Figure 4 is a diagram of the vector pTGFG20.
3o Figure 5 is a diagram of the vector pTGFG33.

~'O 00/49147 PCT/EP00/01368 Fi uq re 6 is a diagram of the vector pTGFG36.
Figure 7 is a diagram of the vector pTGFG53.
Figure 8 is a diagram of the vector pTGFG64.
Fi uq re 9 is the DNA sequence of vector pTGFG36 (SEQ ID NO: 1).
s Figure 10 shows the protein sequence of factor IX encoded by vector pTGFG36 (SEQ ID N0: 2).
Figure 11 shows a GFP concentration curve for cell homogenates after transfection with pTGFG5 and pTGFG20, respectively.
Figure 12 shows corresponding light (a and c) and fluorescent (b and to d) micrographs of Hel_a cells transfected with pTGFG5 (a and b) and pTGFG20 (c and d), respectively.
Figure 13 shows the amount of GFP expressed by utilizing the favoured vectors of the invention in a transfection experiment.
Relative fluorescence units from mock and background can be clearly ~s separated.
Figure 14 shows the additive effect of human clotting factor IX on clotting activity of mouse blood.
Figure 15: hPR (A-form) was expressed in insect cells and purified by cobaltz+ affinity chromatography as described in Example 5. The final Zo preparation (85pg protein) was separated on a denaturing 7,5% SDS
polyacrylamid gel, followed by staining with coomassie° 8250 (lane A) or western blotting with hPR-specific staining (lane C).
Lane B: Molecular mass standard. Arrows indicate the two highly enriched protein species (94 and 74 kDa) accessible to Zs immunodetection.
Figure 16: Domain structure of hPR-B (numbers on the top of the bar represent amino acid positions within the polypeptide sequence).
Figure 17 shows the mean values of the difference in the clotting time of Example 9.
3o Figure 18 shows the clotting time detected in Example 9.

Figiure 19 shows the activity of human progesterone receptor as determined in Example 8.
Figure z0: shows the amino acid sequence of the hPR B-Form. The start methionine 165 of the hPR A-Form is underlined (SEQ ID N0:
s 18).
Figure z1 shows the nucleic acid sequence of the mRNA coding for hPR. The reading frame for the hPR B-form starts at position 176, the reading frame for the hPR A-Form at position 668. The respective start codons ATG are underlined (SEQ ID NO: 19). The sequences of to Figures 20 and 21 are taken from Genbank, accession number AF016381.
Detailed Description of the Invention is 1. Definitions "Nucleic acid" means DNA, cDNA, mRNA, tRNA, rRNA. The nucleic acid may be linear or circular, double-stranded or single-stranded.
"Nucleic acid construct" refers to a composite of nucleic acid 2o elements in relation to one another. The nucleic acid elements of the construct may be incorporated into a vector in such an orientation that a desired gene may be transcribed, and if desired, a desired protein may be expressed.
"Transgene" refers to a functional nucleic acid sequence which is Zs transcriptionally active (with or without regulatory sequences).
"Gene transfer" includes "gene therapy".
"Hormone responsive element" (HRE) refers to regions of nucleic acids, and in particular, DNA, which regulate transcription of genes in response to hormone activation. HREs are typically about 10-30 40 nucleotides in length, and more usually, about 13-20 nucleotides in length. As explained above, HREs become activated when a hormone binds to its corresponding intracellular receptor causing a conformational change, so that the receptor has increased affinity for the HRE and binds to it. The HRE, in turn, stimulates transcription. A
s "steroid responsive element" (SRE) is an HRE that regulates transcription of genes in response to steroid activation. A
"progesterone responsive element" (PRE) is an HRE/SRE that regulates transcription of genes in response to progesterone activation.
to A "hormone receptor" refers to a receptor which binds to and is activated by a hormone. A "steroid receptor" refers to a receptor which binds to and is activated by a steroid hormone. A "progesterone receptor" is a receptor which binds to or is activated by the steroid hormone progesterone.
is "Functionally linked" refers to configurations of the nucleic acid construct, where the HRE (or SRE/or PRE) is located within the construct so that it can stimulate transcription of the transgene. "Not functionally linked" refers to configurations where the HRE is so remotely located from the transgene that it cannot stimulate its 2o transcription.
"Gene" refers to DNA sequence encoding a polypeptide, optionally including leader and trailer sequences and introns and exons.
"Vector" refers to any genetic construct, such as a plasmid, 2s phage, cosmid, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. The term includes cloning and expression vehicles.
"Promoter" refers to a region of regulatory DNA sequences for 3o the control of transcription of a gene to which RNA polymerise binds.

The promoter forms an initiation complex with RNA polymerise to initiate and drive transcription activity. "Enhancers" may activate the complex or "silencers" may inhibit the complex. A "tissue-specific promoter" is a promoter found in the DNA of tissue for transcription of s genes expressed in this specific tissue.
"Organism" refers to a multicellular living entity including vertebrates such as mammals (especially humans, cattle, rodents, dogs) and invertebrates.
"Cellular system" includes cell cultures, e.g., primary cell to cultures (especially those suitable for reimplantation), stem cells, blood cells, tissue samples and whole organs and immortalized cell cultures.
"Therapeutically effective dose" of the products of the invention refers to a dose effective for treatment or prophylaxis, for example, a Is dose that yields effective treatment or reduction of the symptoms of hemophilia. It is also a dose that measurably activates expression of a target gene as determined by measurements of target protein levels, or a dose that is predictable to be effective for treatment or prophylaxis by extrapolating from in vitro or in vivo data. The 2o determination of a therapeutically effective dose is within the purview of one skilled in the art.
"Encodes" or "encoding" refers to a property of the nucleic acid sequence of being transcribed (in case of DNA) or translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under Zs the control of appropriate regulatory sequences.
For the purposes of this application, "express", "expressing" or "expression" shall refer to 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 provide a new and improved delivery system for gene therapy. The invention thus provides nucleic acid constructs comprising at least one HRE and s a transgene wherein one of said at least one HREs is not functionally linked to the transgene, and compositions of matter comprising such nucleic acid construct wherein said at least one HRE is coupled to a hormone-hormone receptor complex (embodiments (3) and (6) defined above). A preferred embodiment of the nucleic acid construct io and of the composition of matter of the invention is one where the hormone responsive element is a steroid responsive element (SRE), and the receptor is a steroid receptor. Most preferably, the hormone responsive element is a progesterone responsive element (PRE), and the receptor is a progesterone receptor.
is Potential HREs for use in the present invention have been previously 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, ao Vol. 329, 734, 1987). As already stated above, the most preferred HRE for the invention is a PRE. Specifically, the preferred PRE is described in Example 1, i.e., is the double stranded DNA sequence comprised of SEQ ID NOs: 3 and 4. The nucleic acid for use in the invention comprises at least one hormone responsive element.
Zs Preferred is a nucleic acid comprising more than one HRE. For example, the nucleic acid may comprise three to ten, preferably three to five HREs. The most preferred embodiment is a nucleic acid comprising three to five PREs.
Potential hormone receptors for use in the present invention 3o are, for example, estrogen receptors, mineralocorticoid receptors, glucocorticoid receptors, retinoic acid receptors, androgen, calcitriol, thyroid hormone or progesterone receptors and orphan receptors.
Such receptors have been previously described. (Green, S., et al., Nature, Vol. 320, 134, 1986; Green, G. L.,et al., Science, Vol. 231, s 1150, 1986; Arriza, J. L., et al., Science, Vol. 237, 268, 1987;
Hollenberg, S. M., 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, A. R., et al., Proc. Natl. Acad. Sci. USA, Vol. 85, ~0 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 may be from human or other mammalian sources, although human is preferred. Nucleotide and/or is amino acid sequences of human steroid receptors are available in the GenBank: mineralocorticoid receptor: M16801; glucocorticoid receptor a: M10901; glucocorticoid receptor a2: 001351; glucocorticoid receptor (3: M11050; retinoic acid receptor a: AF088888 (exon 1), AF088889 (exon 2), AF088890 (exon 3), AF088891 (exon 4), 2o AF088892 (exon 5 and 6), AF088893 (exon 7), AF088894 (exon 8), AF088895 (exon 9 and complete cDNA); retinoic acid receptor y:
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 al:
Zs M24748, thyroid hormone receptor a2: J03239; progesterone receptor: AF016381; somatotropin receptor: J00148; vitamin D
receptor (calcitriol receptor): J03258.
The skilled person will understand that expression of the receptor proteins can be achieved by standard methods, e.g. via PCR-3o cloning of the known cDNAs from cDNA libraries and overexpression of the corresponding proteins in suitable expression vectors, such as, for example, the vectors of the present invention, in suitable host cells, e.g., COS cells. Accordingly, subsequent purification of the cytosolic fraction can be achieved by routine methods such as affinity s chromatography purification. For this purpose, various suitable antibodies against the desired receptor are commercially available.
For example, polyclonal antibodies against the mouse progesterone receptor that have a sufficiently high cross-reactivity for the human protein are available from Dianova (Hamburg, Germany). Likewise, to further purification can be achieved by standard methods, e.g., chromatographical methods such as ion-exchange chromatography and/or FPLC.
The most preferred receptor is the progesterone receptor.
Preferably, the receptor is a human progesterone receptor. Such a Is human progesterone receptor (from T47D human breast cancer cells) is disclosed in US Patent No. 4,742,000, and cells expressing this receptor have been deposited (ATCC deposit number HTB, 133). As already described above, it would be routine to purify such a receptor from the cytosol using receptor specific antibodies. In addition, US
2o Patent No. 4,742,000 discloses a method for purification of the human progesterone receptor using a specific steroid affinity resin (cf.
Grandics et al., Endocrinology, Vol. 110, 1088, 1982).
Briefly, the cytosolic fraction of the T47D cells is passed over Sterogel, a commercial preparation of deoxycorticosterone coupled to 2s Sepharose° 2B that selectively binds the progesterone receptor.
After washing with loading buffer, the bound receptor is eluted with a buffer containing progesterone. The eluted steroid-receptor complex is then chromatographed on DEAE-Biogel and eluted stepwise with a buffer containing 0.2M NaCI. Subsequently, the bound progesterone can be readily exchanged. As described above, further purification can be achieved by routine methods well-known to the skilled person.
An alternative method is disclosed in Example 5.
The structure of the hPR polypeptide is depicted in Fig. 16. The hPR
s polypeptide is composed of distinct structural domains. Naturally the human progesterone receptor (hPR) is expressed as two different sized proteins termed hPR-B (120 kDa) and hPR-A (94 kDa). HPR-A is a truncated but otherwise identical form of hPR-B, that is missing 165 the N-terminal amino acids (see Fig. 20, SEQ ID NO: 18). Both forms to seems to be indistinguishable regarding their progesterone or DNA
binding properties. In human cells the A and B forms of hPR are produced from the same gene by alternate initiation of translation at two different AUG start sites within the same RNA transcript. As it was reported earlier hPR-A and B can be expressed in Spodoptera Is frugiperda (Sf9) cells as biological fully active polypeptides (Christensen et ai., Mol. Endocrinol. 5, 1755ff (1991); Elliston et al., JBC 267, 5193-5198 (1992)).
The carboxyl terminus of the hPR polypeptide as shown in Fig.
16 comprises a progesterone binding domain (PBD) but also contains Zo sequences responsible for the association with heat shock proteins and receptor dimerization. The hinge region provides a flexible link between the DNA-binding domain (DBD) and the PBD but is also thought to contain elements for receptor dimerization as well as nuclear localization. Binding of the hPR to its corresponding target 2s sites at the chromosomal DNA (PREs, Progesterone Responsive Elements) is known to be mediated by the DBD. The remaining N-terminal trans-activation domain (TAD) consists of regions specific for the in vivo function of the hPR as a transcriptional gene activator.
Even though the N-terminus also seems to contribute directly to 3o the homodimerization of hPR after progesterone binding, it has been I ii demonstrated that a fragment comprising only the hinge region and the PBD was the minimal C-terminal fragment to mediate progesterone dependent hPR-hPR-interaction (Tetel et al., Mol.
Endocrinol. 11, 1114ff. (1997). It is believed that genetically s engineered hPR polypeptides lacking either in part or completely the TAD (amino acids 1 to 556) might be expressed as structurally stable and fully soluble dimers in the presence of progesterone. Complexes with such a truncated hPR (provided that said truncated hPR exhibits DNA-binding activity as well as progesterone-binding activity) may to functionally replace the complexes with the full length form of the described recombinant hPR-A or hPR-B proteins, since still mediating the contact between the plasmid DNA and the progesterone. Thus, the hPR in embodiments (2) and (6) to (16) of the invention preferably is a PR comprising nucleic acids 557 to 933 of natural hPR shown in SEQ
Is ID NO: 18.
Effective expression of such a truncated version of hPR is possible in the baculovirus system but also in other eukaryotic expression systems, such as cultivated mammalian cells or yeast cells.
Furthermore, also an E, coli overexpression strain is a possible 2o system for the production of those polypeptides. In this case, the fusion of such a truncated hPR-version to a suitable polypeptide sequence, e.g. a histidine containing sequence or the GST (glutathion S- transferase) protein, might be helpful to overcome insolubility problems as well as to facilitate the isolation and purification of the Zs expressed protein.
Mutated versions of these receptors and derivatives thereof, that still retain the function of the receptors to bind a ligand and thereby become activated and bind DNA and regulate transcription, may also be employed in the invention. Such derivative may be a 3o chemical derivative, variant, chimera, hybrid, analog, or fusion.

The third component of the gene transfer system of the invention is the hormone. The hormone in the agent of embodiment 2 and in the composition of matter of embodiment (6) include synthetic and natural hormones, preferably steroid hormones, such as estrogen, s testosterone, glucocorticoid, androgen, thyroid hormone, and progesterone or derivatives thereof. These are widely available.
Progesterone is most preferred. For example, natural micronized progesterone is the preferred progesterone from which has been marketed in France since 1980 under the trademark of UTROGESTAN~
~o and is still available in Germany under the trademark UTROGEST°. Its properties are similar to the endogenous progesterone, in particular, it has antiestrogen, gestagen, slightly antiandrogen and antimineralocorticoid properties. The natural micronized progesterone in said marketed products is dispersed in a matrix as described is hereinbelow.
The above micronized progesterone has advantages that make it a suitable carrier for genes or nucleic acid constructs to target cells.
Specifically, the synergistic effect of the double process of micronization and suspension in long-chain fatty acids residues of an Zo oii results in increasing progesterone absorption. It has been demonstrated that after oral administration of 100 mg of UTROGESTAN~, peak plasma progesterone levels were obtained after 1-4 hours in most cases (Padwick, M. L., et al., Fertil. Steril., Vol. 46, 402, 1986). Later on, the levels declined substantially, although they 2s were still elevated at 12 hours. Even at 84 hours the levels were slightly higher than baseline. A U.S. kinetic study confirmed earlier work demonstrating the bioavailability of oral micronized progesterone. They showed a peak effect at 2 hours followed by rapid decrease in plasma progesterone level (Simon, J. A., et al., Fertil., 3o Sterih, Vol., 60, 26, 1993).

A further advantage of using progesterone as a carrier is the low level of disadvantageous side effects. Orally administered progesterone adversely affects neither plasma lipids (Jensen, J. et al., Am. J. Obstet. Gynecol., Vol. 156, 66, 1987) nor carbohydrate s metabolism (Mosnier-Pudar, H. et al., Arch. Mal. Coeur, Vol 84, 1111, 1991). Further, progesterone does not affect liver enzymes (ASAT, ALAT, AFOS), sex-hormone binding-globulin (SHBG) synthesis or HDL-cholesterol levels at daily doses of 200 mg and 300 mg. Although the plasma levels of deoxycorticosterone may increase substantially Io during UTROGESTAN~ treatment, there are strong indications that the mineralocorticoid effects of this progesterone metabolite are completely counteracted by the anti-mineralocorticoid effects of progesterone itself. This is apparent from a comparative study (Corvol, P., et al., In: Progesterone and progestins. Raven Press, New ~s York, 179, 1983) in which oral UTROGESTAN~ was capable of antagonizing the mineralocorticoid effects of 9-a-fluorohydrocortisone.
In the agent of embodiment (2) and in the composition of matter of embodiment (6) of the invention the molar ratio of HRE (or SRE/or PRE) within the nucleic acid construct to hormone receptor is Zo preferably from 1:1 to 1:10, more preferably from 1:2 to 1:5. On the other hand, the molar ratio of hormone to hormone receptor is preferably at least 1000:1, more preferably at least 10000:1. Thus, the hormone is present in a large excess relative to the hormone receptor and the HRE, which is desirable in view of the ability of the Zs hormones to transfer nucleic acid constructs through cell membranes.
The skilled artisan will appreciate that the agent of embodiments (1) and (2) and the pharmaceutical composition of embodiment (10) may contain other components capable of assisting in introducing the nucleic acid into a cell for the purpose of gene therapy (matrix 3o compounds). Specifically, the agent and the composition, especially the hormone component thereof, may contain the following matrix compounds: glucose and related compounds (such as D-sorbitol, D-mannitol); solubilizing adjuvants (such as alcohols, e.g., ethanol);
polyhydric compounds such as glycerine, polyethylene glycol and s polypropylene glycol; nonionic surface active compounds, ionic surface active compounds such as lecithin; oily compounds such as sesame oil, peanut oil soybean oil, corn oil, etc.; starches and their derivatives such as cyclodextrines and hydroxyalkylated starches; stabilizers such as human serum albumin, preservatives such as benzyl alcohol and io phenol; and the like. The preferred matrix contains f3-cyclodextrine, glycerine, lecithin and/or corn oil. For example, the pharmaceutical composition of hormone-hormone receptor nucleic acid complex of the invention may be provided orally to humans or animals as a gelatin capsule. Progesterone therein (preferably in micronized form) could Is be present in a concentration of 50 to 1000 mg, preferably 200 -300 mg dissolved in a 35 % or 40 % f3-cyclodextrin solution or in cornoil or gycerol with peanut oil together with lecithin.
Alternatively, when - due to the selection of appropriate matrix components - the pharmaceutical composition is in a pasty, gel-like zo form, it may be provided topically.
The nucleic acid construct of embodiments (1) to (15) of the present invention may - aside from the transgene and the HREs, SREs, or PREs already disclosed above - further contain promoter, enhancer, and/or silencer sequences. The promoter may be ubiquitous Zs or tissue-specific. Of the ubiquitous promoters, the CMV promoter is most preferred. However, a tissue-specific promoter is preferred over a ubiquitous promoter. For example, the tissue-specific promoters envisioned for the instant invention include al-antitrypsin (further promoters).

The nucleic acid construct may further comprise additional sequences such as the ampicillin resistance gene. Other reporter sequences known to the skilled artisan may also be included, such as, for example, the green fluorescent protein (GFP), luciferase, f3-s galactosidase or chloramphenicolacetyltransferase (CAT). As an enhancer sequence, the SV40 intron and SV40 Poly A are most preferred. The nucleic acid construct may further contain inducible promoters such as, for example, a MMTV (Mouse Mammary Tumor Virus) promoters inducible via glucocorticoides and Ecdyson-inducible ~o insect promoters.
A preferred nucleic acid construct contains sequentially from the 5' to the 3' end: a PRE, a CMV promoter, a gene of interest, SV40 Intron and SV40 poly A enhancer sequence, and an ampicillin resistant gene. Further PREs are evenly distributed on the vector Is backbone.
The nucleic acid construct may further contain origin of replication sequences (especially eukariotic origin of replication sequences), elements for gene targeting, integrational sequences (e.g., AAV-ITR, transposon IS), 3'-UTR, "switch" systems (e.g., TET
Zo system, Cre/IoxP or Flp/ftr system).
The transgene may be chosen from those encoding proteins lacking in a variety of genetic disorders or involved in conditions related to inappropriate responses to hormones, for example, hormone-dependent cancers such as breast, ovarian, and endometrial Zs cancers and prostate cancer. The transgene may also be used to replace a defective gene resulting in such genetic disorders as hemophilia, von Willebrand disease, and cystic fibrosis. The transgene includes mutations of such gene or a gene encoding a fusion product.
The nucleic acid construct of the present invention may comprise ~o more than one transgene.

In particular, the transgene may replace genes for a blood clotting factor, and preferably a human blood-clotting factor. The genes encoding factor VIII and factor IX (sown in Fig. 2, SEQ ID NO:
2), involved in hemophilia A and B, respectively, are good candidates s for the invention. Other candidates include the gene encoding von Willebrand factor, factor IV, factor X, or protein C.
Other useful transgenes include, but are not limited to, hormone genes such as the genes encoding for insulin, parathyroid hormone, luteinizing hormone releasing factor (LHRH), a and f3 seminal inhibins to 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; enzyme genes; genes encoding cytokines or is lymphokines such as interferons, granulocytic macrophage colony stimulating factor (GM-CSF), colony stimulating factor-1 (CSF-1), tumor necrosis factor (TNF), and erythropoietin (EPO); genes encoding inhibitor substances such as al-antitrypsin, and genes encoding substances that function as drugs, e. g., genes encoding the Zo diphteria and cholera toxins, ricin or cobra venom factor. Also, antisense sequences may be administered as genetic material.
Another aspect of the present invention is vectors comprising the nucleic acid constructs of embodiment (3) of the present invention. These vectors may be used in the composition matter of 2s embodiment (6) of the present invention. Preferably, however, the nucleic acid sequence for use in the invention is circular rather than linear. The vectors may be capable of expressing the nucleic acid in the nucleic acid construct transiently or permanently (including episomally). As noted above, the nucleic acid construct therein may 3o further contain additional elements.

The composition of matter of embodiment (6) of the invention can be prepared by admixing the nucleic acid construct with the hormone receptor and the hormone. Preferably, an aqueous solution of nucleic acid construct was added to the oily suspension containing s the hormone at ambient temperature under stirring.
Embodiments) of the invention relates to transfected and transformed cells or transgenic organism comprising these vectors and/or nucleic acid constructs. Within the scope of this invention, a transfected cell is one in which foreign DNA has been incorporated.
~o Methods of transfection may include microinjection, CaP04 precipitation, electroporation, liposome fusion, or gene gun.
Transformation refers to introducing genetic material into a cell, such as the vectors or nucleic acid constructs of the invention, rendering the cell transiently or permanently altered so that the cell is expresses a specific gene product or is otherwise altered in its expression. Transformation may be achieved by in vivo or in vitro techniques, although in vivo transformation is preferred.
A further embodiment of the present invention is pharmaceutical compositions comprising a therapeutically effective dose of the nucleic Zo acid constructs of the invention and a hormone. The hormone is preferably a steroid, and most preferably, progesterone, as described above. The dose is dependent on the condition to be treated, the characteristics of the patient, and the result sought to be achieved.
Determining dosage is within the realm of the skilled artisan.
Zs The pharmaceutical composition (or, alternatively, the composition of matter, the nucleic acid construct, or the vector) of the present invention may be administered orally, intravenously, intramuscularly, subcutaneously, topically, through mucosa (including buccal, nasal spray) or by gene gun. Oral administration (of a micronized hormone dispersion) is preferred. Delivery may be systemic or directed at certain tissue.
The invention further includes a method of introducing into a cell a nucleic acid construct encoding a gene of interest, e.g., a human s blood-clotting factor, to express the blood-clotting factor in the cell. In this method, the nucleic acid encoding a human blood-clotting factor is combined with a nucleic acid construct comprising at least one hormone responsive element (HRE), preferably a progesterone responsive element.
~o The mixture of nucleic acid bound to the hormone-hormone receptor complex together with an excess of hormone, preferably progesterone, will be used to introduce the nucleic acid into a cell by various methods known to the skilled artisan and outlined above. The cell-uptake will be stimulated by the interaction of the hormone with Is the cell membrane. The hormone or steroid interacts with the lipid bilayer of the cell membrane not only through membrane perturbation but also through activation of certain hormone- or steroid-sensitive membrane receptors. This has been demonstrated for progesterone and other steroids. Last but not least, it is known that hormones are 2o able to cross the cell membrane by diffusion. In the present invention, the nucleic acid bound to the hormone-hormone receptor complex should be transported through the membrane during the process of diffusion or uptake.
Another aspect of the invention is a method of treating a blood 2s clotting disorder by administering a therapeutically effective amount of the composition of matter of the invention to an organism. This method involves the administration and dosage considerations already discussed.
Embodiments (17) and (18) of the invention pertain to the use of a steroid hormone for preparing an agent for gene therapy and/or gene transfer and to method for gene therapy and/or gene transfer which comprises administering a nucleic acid construct to an organism or to a cellular system, wherein the nucleic acid construct contains a transgene and is encapsulated in a steroid hormone. Suitable steroid s hormones are enumerated hereinafter. The preferred steroid hormone in said embodiments of the invention is a natural micronized steroid hormone, in particular a natural micronized progesterone. In a preferred embodiment, the micronized hormone is solubilized/dispersed in a lipophilic matrix as described hereinafter.
to Experiments have been performed to illustrate the technical aspects of the present invention. These experiments are described in examples 1 to 9 below. The skilled artisan will be readily recognize that the invention is not limited to these examples.
Is Examples Example 1: Construction of Vectors Production of the vector pTGFGi: The vector pUCl9 (MBI Fermentas) Zo was digested with XbaI, treated with Klenow enzyme and religated.
This XbaI deleted vector was then digested with EcoRI, treated with Klenow enzyme and religated in order to delete the EcoRI site. For insertion of a XbaI site in the SacI site of this vector it was digested with Sacl, treated with T4-polymerase, dephosphorylated with alkaline Zs phosphatase and ligated with the XbaI-linker CTCTAGAG (Biolabs #1032). Another XbaI-site was inserted by digesting the newly produced vector with HindIII, treating it with Klenow, dephosphorylating it with alkaline phosphatase and ligating it with the XbaI-linker CTCTAGAG (Biolabs #1032). This vector was named 3o pUCl9/X.

In order to destroy the XbaI-site present in the vector phGFP-S65T (Clontech) this vector was digested with XbaI, treated with Klenow enzyme and religated resulting in the vector pGFP/0. A 2.3 kb fragment containing the GFP-Gene was isolated after digesting pGFP/0 s with MIuI, treating it with Klenow enzyme and digesting it with BamHI.
This fragment was inserted into the multiple cloning site of the vector pUCl9/X which was digested with SaII, treated with Klenow enzyme and digested with BamHI. The resulting vector was named pTGFGI
(Figure 2).
~o Starting with this vector all the vectors described in Table 1 were obtained. At the restriction sites for PstI, KpnI, Ehel, Eco0109 and/or SapI a PRE(ds) was inserted giving rise to plasmids carrying the GFP gene and up to five PREs. By exchanging the GFP gene with a FIX gene a set of FIX expression plasmids were obtained. By excising is the GFP gene the cloning vectors without a transgene were obtained.
Production of the insert PRE(ds): The oligonucleotides (Metabion) 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';
Zo 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 N0: 4) were hybridized and phosphorylated by kinase reaction, resulting in 2s the insert PRE(ds).
Production of the vector pTGFGS: The vector pTGFGI was digested with Eco0109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE(ds) insert, resulting in the vector pTGFG5 (Figure 3), i.e., a vector which carries a PRE at position C of Fig. 2.
Production of the vector pTGFG20: The vector pTGFGI was digested s with KpnI, treated with T4-polymerise and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE(ds) insert, resulting in the vector pTGFG7. This vector pTGFG7 was digested with PstI, treated with T4-polymerise and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE(ds) insert, resulting in ~o the vector pTGFGI1. Subsequently, pTGFGli was digested with Eco0109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE(ds) insert, resulting in the vector pTGFG20 (Figure 4). This vector carries a PRE
at positions A, B and D of Fig. 2.
is Production of the vector pTGFG33: In a similar manner PRE(ds) were inserted at the restriction sites for PstI, KpnI, EheI, Eco0109 and SapI
in vector pTGFGi giving rise to the plasmid pTGFG33 (Figure 5), which is a vector that carries the GFP gene and five PREs, one each in Zo position A, B, C, D, E (Figure 2).
Production of the vectors hTGFG36, pTGFG53 and pTGFG64: The vector pUCl9 (MBI Fermentas) was digested with SaII, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It 2s was ligated to the NotI-linker GCGGCCGC (Biolabs # 1045), resulting in the vector pUCl9/N.
A 1.4 kb fragment containing the open reading frame of the human clotting factor IX, isolated from a human cDNA library (see example 2), was inserted into the PstI-site of the vector pUCl9/N
3o which was digested with PstI, treated with T4-polymerise and ?9 dephosphorylated with alkaline phosphatase. From the resulting vector pUCl9/N-FIX a 1.4 kb fragment containing the open reading frame of the human clotting factor IX was cut out by double-digestion with Hind III and NotI. This fragment was ligated to the 4.3 kb s fragment of the HindIII and NotI double-digested vector pTGFG5 resulting in the vector pTGFG36 shown in Figure 6. This vector is a preferred one for delivery of Factor IX into the cell, and its DNA
sequence is provided in Figure 9 (SEQ ID NO: 1).
In a similar manner plasmids pTGFG53 and pTGFG64 (shown in to Figures 7 and 8) were obtained by exchanging the GFP gene in plasmids pTGFG20 and pTGFG33 by the FIX gene.
Production of the insert ALLG(ds): The oligonucleotides (Metabion) ALLG1/1 (5'-AGC TTG ACC TCG AGC AAG C-3') (SEQ. ID NO: 5) and ALLG2 (5'-GGC CGC TTG CTC GAG GTC A-3') (SEQ. ID N0: 6) were is hybridized and phosphorylated by kinase reaction, resulting in the inserts ALLG(ds). The insert ALLG (ds) was constructed to introduce into the vector of choice a sequence with a multiple cloning site for the possible introduction of other transgenes.
Table 1 gives an overview of the available vectors with different ao transgenes and a different number of PREs in various positions. The positions of the PREs are given according to Figure 2. For the underlined vectors a map is provided (Figures 3 to 8).
Table 1: Vectors of the invention PlasmidTrans-PRE Plasmid Trans-PRE Plasmid Trans- PRE
gene gene gene pTGFGO -_ -- pTGFG GFP BDE pTGFG34 FIX E

~TGFGI GFP -- pTGFGI9 GFP BCD pTGFG3s FIX A

pTGFG2 FIX -- 1~TGFG20GFP ABD pTGFG36 FIX D

pTGFG3 GFP E pTGFG21 GFP CDE pTGFG37 FIX C

pTGFG4 GFP A pTGFG22 GFP ACD pTGFG38 FIX B

pTGFGS GFP D pTGFG23 GFP ABC pTGFG53 FIX ABD

pTGFGG GFP C pTGFG24 GFP ABE pTGFGG4 F1X ABCDE

pTGFG7 GFP B pTGFG25 GFP ACE pTGFGGG -- A

pTGFG8 GFP BC pTGFG2G GFP ADE pTGFG67 -- D

pTGFG9 GFP BE pTGFG27 GFP BCE pTGFGG8 -- C

pTGFGIO GFP BD pTGFG28 GFP BCDE pTGFGG9 -- B

pTGFGII GFP AB pTGFG29 GFP ACDE pTGFG82 -- ABD

pTGFGI3 GFP CD pTGFG30 GFP ABCE pTGFG95 -- ABCDE

pTGFGl4 GFP AC pTGFG31 GFP ABDE

pTGFG GFP DE pTGFG32 GFP ABCD

pTGFG GFP AD pTGFG33 GFP ABCDE

For the DNA sequence of pTGFG 36, pTGFG 53, pTGFG 64, pTGFG 67, pTGFG 82 and pTGFG 95, see SEQ ID NOs: i and 13 to 17, respectively.

Example 2: Isolation of Human Factor IX cDNA
Factor IX cDNA was amplified from human liver cDNA (Clontech) using two primers overlapping the start and termination codon of the to factor IX open reading frame resulting in a 1387 by fragment containing the entire open reading frame. Restriction sites for EcoRI
(upstream) and BamHI (downstream) were included at the end of each primer to facilitate cloning. Amplification was performed with Pwo polymerise (Boehringer Mannheim) in 50 ml reaction volume [10 Is mM Tris HCI pH 8.85, 25 mM KCI, 5 mM (NH4)2504, 2 mM MgS04]
with 30 incubation cycles at 96°C for 1 min, 60°C for i min, 72°C for 2 min, followed by a final extension step at 72°C for 10 min.
Reaction products were ligated into the EcoRI- and BamHI-sites of pUCi9 and transformed into E, coli DH5-a. Positive clones were selected. Sequences were confirmed by cycle sequencing (Amersham) from both ends with labeled primers (IR-700) and automated analysis on the LiCor sequencing system (MWG, Biotech).
The following primers were used s GGAATTCCGCAAAGGTTATGCAGCGCGTGAACATGATCATGGC
(upstream; SEQ. ID N0: 7) CGCGGATCCATTAAGTGAGCTTTGTTTTTTCCTTAATCC (downstream;
SEQ. ID NO: 8) ~o Example 3: Expression and Quantification of the Marker Protein GFP
Method 1: HeLa cells were transfected by electroporation with plasmids pTGFGS or pTGFG20. Transfected cells were harvested and Is the cell pellets were homogenized and lysed in a buffer containing phosphate buffered saline (pH 7.5) and 10 mM PMSF. The concentration of green fluorescent protein (GFP) in the cell homogenate was determined by competitive ELISA.
For this purpose, GFP was coated in a defined concentration on Zo microtiter plates. Then, GFP samples were added in the presence of anti-GFP antibody. After several washing steps a labeled secondary antibody was added in order to trace the first antibody. The colorimetric reaction was measured photometrically (extinction).
Generally, the more GFP was added the less antibody was left to bind Zs the coated GFP. Thus, reduction of extinction corresponded to higher GFP concentration in the sample.
A concentration curve of GFP was determined by linear regression (Figure 11) using bovine serum albumin (BSA) as a reference. A mean value of 2.4 mg GFP/ml for pTGFG5 (1 PRE) and 30 5.2 mg GFP/ml for pTGFG20 (3PREs) was found.

Figures 12 a-d show micrographs of HeLa cell cultures transfected with pTGFG5 (Fig. 12 a and b) and pTGFG20 (Fig. 12 c and d), respectively. Figures 12 a and c represent light microscopic views as controls, and Fig. 12 b and d show the corresponding cell s patches in the fluorescent mode. Routinely, more than 50% of the cells expressed GFP, indicating very efficient transfection, the presence of only one PRE showing more efficient expression.
Method 2: 293 T cells were transfected with pTGFG 5, 20 and 33 ~o using calcium phosphate method and fluorescence was detected with a fluorimeter (Labsystems, Extinction: 485 nm Emission: 520 nm). In the case of the mock transfection, non GFP-expressing DNA was used.
Background indicates the fluorescence of the empty plate (96-well plate, Dynex, Immulon-4). The results are summarized in Fig. 13.
is Again the vector with just one PRE (pTGFGS) shows the highest expression.
Example 4: Human Factor IX Quantification by ELISA Assay Zo HeLa cells were transfected either by electroporation or using liposome reagent DOTAP (Boehringer Mannheim) with plasmids pTGFG36, pTGFG53 and pTGFG64. These plasmids contain the cDNA
of human clotting factor IX. Recombinant human factor IX was secreted into the supernatant of the cell culture and quantified using a as sandwich ELISA method.
0.11 M sodium citrate and 10 mM PMSF were added in order to prevent degradation of human factor IX. The enzyme-immunological in vitro assay "Asserachrom IX:AG" from Boehringer-Mannheim was used in order to determine the concentration of expressed human ~J
clotting factor IX. The factor IX-standard from Octapharma AG was used as a standard in aqueous solutions of 28 IU/ml.
In six different transfection experiments, in which HeLa cells with plasmids containing human factor IX-cDNA (pTGFG36, 53 and s 64) were transfected using either electroporation or lipid-transfection reagent (DOTAP, Boehringer Mannheim), a concentration range of 3-25 ng/ml human clotting factor IX was reached.
Example 5: Production and Purification of hPR (A Form) ~o 1. Cloning of the human ~rogesterone receptor' The cloning was performed as follows: Total human RNA was isolated from human white blood cells or liver cells using cell lysis in guanidinium hydrochloride buffer and CsCI-density centrifugation.
is For cloning of the hPR coding sequence, hPR specific cDNA was-prepared and used for amplification of the hPR coding sequence in two fragments by PCR.
The following oligonucleotide primers were selected based on the published mRNA sequence (Genbank: NM_000926 and X51730).
ao Oligonucleotides used were obtained from MWG, Ebersberg or Metabion, Munchen. All primers used are listed 5' to 3', bases added to introduce restriction sites are in capital letters and restriction sites used for cloning are underlined.
hPGR-5'-primer: CGA GGA tcc agt cgt cat gac tga gc (SEQ ID NO: 9);
2s hPGR-3'-primer: GCA GAA TT cat tat aaa aac tca aga cct cat aat cct gac (SEQ ID N0: 10);
hPGR-internal primer (Sal I) 1: ctc ctc ggg tc ac cct gg (SEQ ID NO:
11);
hPGR-internal primer (Sal I) 2: cca ggcLtc_g acc ccg agg ag (SEQ ID
3o NO: 12).

Synthesis of cDNA was perfomed using 3 pg of total RNA and 200 pmol of the 3'-primer with Superscript II reverse transcriptase (Gibco BRL). Reaction volume was 50 pl and buffer was used as s recommended, supplemented with RNase Inhibitor and 10 mM DTT
and 1 mM dNTPs. Before adding the enzyme, samples were heated to 80°C for 10 min, followed by. 10 min at 72°C and 10 min at 42°C.
Superscript II RT was added at 42°C and reaction was continued for 15 min at 42°C, 15 min at 50°C and 1 h at 58°C.
~o The cDNA obtained from this synthesis reaction was used to amplify the hPGR coding sequence in two fragments by PCR. One fragment (5') with 5'-primer and internal primer 2 and one fragment (3') with 3' primer and internal primer 1. Reaction setup in 50 pl was . Pwo is polymerase (Roche Diagnostics), buffer as supplied by Roche Diagnostics, supplemented with DMSO, 50 pmol of each primer and 0.2 mM dNTPs. Reaction conditions were: 10 min 96°C followed by 35 cycles of 1 min 96°C, 2 min at 59°C, 2 min 72°C and a final extension step at 72°C for 10 min.
PCR-products were purified by gel electrophoresis and digested with Sal I. The BamHI and Hind III sites introduced in the primer were not used to avoid cutting at two internal restriction sites of the hPR coding sequence. Both fragments were ligated into pBluescript SK+ vector 2s cut with EcoRV through blunt end ligation into the vector and sticky end ligation through the internal Sal I site. Vectors containing the appropriate insert were identified by mini-prep, restriction digest and sequencing. The obtained vector was designated pTGhPRI.

2. Production of hPR (A-form: Initially, the gene for hPR-B inclusive its 3 ~-UTR was cut out from pTGh PR1 and cloned in frame in the multiple cloning site of the expression plasmid pFASTBAC HTc (BAC-to-BAC Baculovirus Expression System, Life Technologies). This s resulted in an expression casette of a N-terminally histidine-tagged version of hPR-B under expression control of the viral polyhedrin promotor as shown below. A rTEV protease cleavage site is located between the six histidine residues and the initial methionine of the hPR-B reading frame, which allows removal of the histidine residues to from the expressed protein. The N-terminal region of the expression cassettes is shown below.
MSYYHHHHHHDYDIPTTENLYFQ**GAMGIRNST-hPR-gen 6xHis is spacer rTEV cleavage site Amino acids are presented in the single letter code. The cleavage site of the rTEV protease is represented by **
In order to generate the expression cassette for the truncated hPR-A
form, the DNA sequence encoding for the amino acids between Met 1 and Met 165 of the hPR-B form was removed using a PCR-based strategy. Two primer pairs were designed which allowed amplification 2s of either a DNA fragment just downstream of the start AUG of the hPR-B gene and a DNA-fragment just upstream of the AUG coding for Met 165, respectively. In a subsequent PCR reaction these two DNA
fragments were annealed to each other at their homologous 3'-ends, and amplified using the outermost amplification primers. The resulting 3o DNA-fragment was digested by EcoRI and Mlu I and the cleavage product was exchanged against the corresponding fragment of the hPR-B expression cassette in the pFASTBAC HTc vector. Thereby the reading frame coding for an N-terminal histidine tagged version of the hPR-A polypeptide (94kDA) was restored.
This 6xHis-tag was utilised for affinity purification of the protein s by immobilized cobalt2+ affinity chromatography on a TALON° resin (Clontech). The procedure, following the method of Boonyaratanakornkit et al. Mol. Cell. Biol.l8, 4471 (1998), was as follows (all steps were carried out at 0 to 8°C):
Sf9 cells were cultivated in monolayer culture in serum free SF900 io medium. Viral infection of the cells was done at a multiplicity of infection (MOI) of 5-8.
The harvesting was done 48 hours after infection with baculovirus containing the hPR expression cassette and lysed mechanically by homogenising in buffer A containing 20 m~1 Tris-CI pH 8.0, 350 mM
is NaCI, 10 mM imidazol, 5% glycerol and a cocktail of proteinase inhibitors (CompIeteTM EDTA-free, Roche Diagnostics, Penzberg, Germany). After a 10 min centrifugation at 10000 x g, supernatant originating from 10$ cells was incubated for 1 h with 0,5 ml settled TALON° resin equilibrated in buffer A. TALON° was washed with 20 2o volumes of buffer A. hPR-A was eluted with 10 Vol buffer B, containing all ingredients of buffer A, but 100 mM imidazol. The eluate was concentrated 50-fold and dialysed against 100 volumes buffer C (PBS
+ 100 nM progesteron) by centrifugal ultrafltration at a molecular exclusion size of 10 kDa (Centricon Plus-20 PL-10, Millipore, Eschborn, 2s Germany).

3. Determination of identity, purity and yield of hPR-A' Purity and yield of the product were determined by application on denaturing reducing polyacrylamid- gelelectrophoresis according to Laemmli, U.
3o et al., Nature 227, 680-685 (1970) and subsequent staining with coomassie° blue 8250. By this one-step procedure hPR-A was enriched to a final specific hPR content of 0.2 - 0.5 mg hPR/mg protein. As depicted in Figure 15, lane A, the final preparation consisted predominantly of two distinct protein species displaying s apparent molecular masses of 94 and 74 kDa (Fig. 15, arrows).
Yield was estimated by parallel separation of standardised protein preparations. Data taken from a set of three separate experiments hint at a typical yield of 30 pg enriched hPR A-receptor per 10$ cells.
~o Identity of hPR was determined by immunodetection of the product transferred to nitrocellulose by western blotting with mouse monoclonal antibodies directed against recombinant hPR (PR Ab-1, Oncogene, Cambridge, MA, USA).
The final product was transferred to nitrocellulose BA-83 and Is immunostained as described above. As presented in Figure 15, lane C, three major protein bands were detected, including the two dominant protein species described above. The smaller sized bands may display copurified proteolytic fragments of hPR.
Intracellular GFP from adherent cells was detected by a 2o fluorimeter after media was taken off and PBS (colourless) was added.
The results are summarized in Fig. 13.
Example 6: Clotting Activity of Human Clotting Factor IX from Transfected 293 T Cells A concentration range of 55 - 95 ng/ml human clotting factor IX has been reached by transfection of 293 T-cells with plasmids containing human factor IX-cDNA (pTGFG 36, 53, 64 and 2) in 11 different experiments using ELISA (Example 4).
3o Clotting activity was deterined with a partial thromboplastin time assay using Cephalin (phosphatidyl ethanolamine) activation with a manual coagulation instrument (ML-2, Instrumentation Laboratories).
For the study, 100 pl undiluted supernatant from transfected 293 T-cells, 100 pl deficiency plasma (Progeny and 100 pl Cephalin s (Instrumentation Laboratories) were incubated for 5 minutes at 37°C.
Coagulation was started by adding 100 NI CaCl2. Sample coagulation time was compared to normal plasma.
Number of cells Factor IX-concentrationClotting time /ml n /ml s 2,1 x 105 36 45 8,7 x 105 20 79 ~o Normal plasma: 37 - 39 s Factor IX deficient plasma: 137 - 140 s Example 7: Analysis of an Additive Effect of Human Clotting Factor IX on the Clotting Time of Mice Blood IS
1. Clotting time: Clotting activity was determined with a partial thromboplastin time assay using Cephalin (phosphatidyl ethanolamine) activation with a manual coagulation instrument (KC 4 A, Amelung).
2o For the study, 5 NI mouse blood, 20 pl deficiency plasma (Progeny ad 100 III physiological NaCI and 100 pl DaPPTin (Progeny were incubated for 2 minutes at 37°C. Coagulation was started by adding 100 pl CaCl2.
To analyse the additive effect, human clotting factor IX
Zs (housestandard, Octapharma) was added to the mouse blood and diluted 1:10 within the system. As it is shown in Figure 15, the additive effect of human clotting factor IX on clotting activity can be detected up to a limit concentration of 0,07 mIU hFIX/ml (= 31,5 ng/ml).
2. ELISA: The addition of human clotting factor IX to the mouse blood ~s was monitored by ELISA as described in Example 4. Citrate plasma was made out of mouse blood and human clotting factor IX was added in different concentrations.
NO. Description Concentration Extinction at 405 [mIU/ml] hFIX addednm [-]

1. Mouse Citrate Plasma 7 0,204 2. Mouse Citrate Plasma 2 0,130 3. Mouse Citrate Plasma - 0,099 4. Control: 1.+2. Antibody - 0,096 without antigen 5. Control: 1. Antibody - 0,072 without antigen 6. Control: 2. Antibody - 0,085 without antigen 7. Substrate (ABTS) - 0,072 Io Mouse plasma without the addition of human clotting factor IX showed an extinction of 0,099 at 405 nm background. When added human factor IX in a concentration of 2 mIU/ml (= 9 ng/ml human factor IX) the detection limit is reached. It can be deduced that the antihuman factor IX antibodies used in the ELISA are not cross-reactive with is mouse coagulation factor IX.

Example 8: Cloning and Activity Testing of the Human Progesterone Receptor (hPR) 2. Activity Testing: The human progesterone receptor encoded in s plasmid pTGhPRi (s. Example 8.1 above) was tested for its phyiological activity. In a functional form and after activation with a progestin like 85020 the receptor should be able to induce the expression of luciferase from a Mouse Mammary Tumor Virus (MTV) promoter.
~o To test this 293T cells were grown in phenol red-free DMEM
supplemented with 10% charcoal-filtrated fetal calf serum and with or without 10 nM of 85020 (NEN) in 6 well plates. Transfections were performed by the calcium phosphate method using 2 pg of a pSG-hPRi constructt and pMTV-luc (Hollenberg et al., 1985, Cell 55, p899-~s 906) per well. One day after transfection the cells were washed in PBS
and the luciferase expression assayed with the Berthold luciferase kit according to the manufacturer's directions in a fluorimeter (Labsystems). The controls were as follows: 85020 was omitted (PR+MTV) and both plasmids alone were transfected with (PR+85020, 2o MTV+85020) and without 85020 (PR, MTV). As positive control a plasmid with a CMV-driven luciferase gene was transfected (pCMV-luc).
As can be seen in Figure 19, there is a clear induction of luciferase expression when all the necessary elements are present, 2s that is human progesterone receptor, progestin 85020 and the MTV-driven luciferase gene (PR+MTV+85020). The error bars give the standard deviation of a threefold experiment, the readout is relative light units (RLU).

Example 9: Oral Gene Transfer in in vivo Animal Experiment Purpose of experiment: The object of this pilot study is to prove oral gene transfer in an in vivo animal experiment. Successful gene transfer is established by coagulation measurement: an additive effect of s expressed human factor IX on the coagulation time of healthy murine whole blood is expected. The presence of expression of human factor IX
in mouse blood is quantitated by ELISA.
Animals: The animals employed are 35 male C57BL/6J mice from Iffa ~o Credo, France, with an initial age of 9 weeks and a weight of 23-33 g.
The mice are kept in groups of 7 animals each in conventional test animal cages with wooden chips in the Institut fur Experimentelle Onkologie and Therapieforschung der Technischen Universitat Munchen.
~s The animals are fed ad libitum with "Altrum Ratten and Mause Haltung" and are given tap water, also ad libitum.
The test animal cages are kept at an ambient temperature of 19-24°C and a humidity of 55 5%. The room is additionally provided with an automatic light supply which maintains a 12 hours rhythm.
Zo The test animals are supervised by specialized staff.
Mixture of substances:
Group Hormone Hormone Plasmid Aqua dest.Route of receptor administration 1.-2. - 100 NI 10 pg oral 3. - 10 pg 100 NI oral 4. - 10 Ng 50 pl i.m.

5. - 100 pl 4.35 Ng 10 pg oral Plasmid and hPR: Theragene GmbH
Hormone: Utrogest° by Dr. Kade/Besins Pharma GmbH, Rigistr. 2, D-12277 Berlin Aqua dest.: Aqua ad injectabilia Delta-Pharma GmbH, 72793 s Pfullingen Esophageal sound: Vein catheter, diam. 0.5 x 0.9 mm, Lot 7077 62221, B. Braun Melsungen AG, Western Germany i.m. injection: Micro-Fine 12.7 mm, Becton Dickinson GmbH, ~o Tullastr. 8-12, D-69126 Heidelberg Course of experiment: The 35 mice were divided into 5 groups of 7 mice each. One group serves as a control, the second group was daily administered a total of 100 pl of hormone and plasmid via the gastro-intestinal tract orally with an esophageal sound, the third group was ~s daily administered a total of 100 pl of plasmid with aqua dest. orally with an esophageal sound, the fourth group was administered a total of 50 NI of plasmid with aqua dest. i.m. into the musculus quadriceps femoris, the fifth group was daily administered a total of 100 pl of hormone, hormone receptor and plasmid orally with an esophageal ao sound.
About 2-3 hours before the manipulation, the mice were prewarmed under a red light. Immediately before, during and after the manipulation, the mice were examined and supervised by a veterinarian.
2s Blood sampling from the mice was performed daily from the caudal artery of animals slightly sedated by inhalation anesthesia. For this purpuse the artery was punctured with a disposable injection cannula (0.90 x 40 mm). Whole blood welling out of the puncture site (5 pl of blood) was immediately collected with an Eppendorf pipette.
3o Without further delay, the blood coagulation time in seconds was determined using an Amelung-Koagulometer KC 4A by means of an aPTT assay (activated partial thromboplastin time). The blood coagulation analysis was always performed by the same person.
Immediately after the blood sampling, the bleeding was stopped by s compression.
Sedation of the mice was achieved by inhalation anesthesia (active substance: isoflurane: Forene , Abbott GmbH, 65205 Wiesbaden, Western Germany) in a whole body chamber.
The daily manipulation was performed through an overall period of 7 to days. This was followed by a day (day 8 of experiment) without any manipulation, and at day 9 of experiment, again 5 pl of whole blood was withdrawn from the ventral caudal artery under anesthesia, and the coagulation time established as described above. Further, 0.5 0.75 ml of whole blood was collected intracardially using U-40 insulin is syringes (Mikro-Fine 12.4.mm) filled with 50-75 pl of sodium citrate-(3.1%), transferred into Eppendorf cuvettes, and about 100 pl of whole blood with citrate was reserved for PCR examination and stored in a cool environment. The remaining citrate blood was centrifuged for min using a centrifuge 6000 rpm, 4°C, at 5000 rpm, and the plasma was recovered for the ELISA determination of the factor IX
concentration.
Then, the animals were sacrificed using 0.5 ml Narkoren i.p.
Immediately after the sacrificing, the animal bodies were dissected.
The following organs were removed from the mice for an 2s immunohistochemical examination: brain, spleen, liver, kidneys, testes, lungs, m. quadriceps femoris, heart, appendix; and frozen at -80°C.
Deviation from the scheduled experimental course' Due to the poor general condition of the mice in the course of the long-term 3o administration series, the administration had to be interrupted at days 3 (except one mouse) and 5 for test group 2 (hormone and plasmid), at days 3 and 5 for group 5 (hormone, hormone receptor and plasmid), and two mice were additionally spared the administration of the reagents at days 2 and 7 of the experiment.
s The poor general condition is accounted for by the hypnotic effect of the hormone progesterone. It causes the mice to sleep for about 24 hours without eating and drinking. This again has an adverse effect on the water balance of the mice, resulting in exsiccotic phenomena and apathic behavior. Therefore, the mice were prophylactically treated with ~o a subcutaneous administration of 1 ml of 5% glucose solution (Delta Pharma GmbH, 72793 Pfullingen) and 1 ml of Ringer solution (Delta Pharma GmbH, 72793 Pfullingen) when the hormone was administered orally. Among the group which was orally administered hormone, hormone receptor and plasmid, two mice died at days 3 and 6, is respectively; they were dissected.
Among the group which was orally administered hormone with plasmid, one mouse was found dead in its cage on day 8 of the experiment; it was also dissected.
The results are summarized in Figures 17 and 18. The statistical Zo evaluations were performed according to the generalized linear model with repeated measurements (MANOVA with repeated measurements).
In none of the test groups a non-linear course was observed.
Therefore, the course was calculated by a simple representation of the linear increase or decrease, namely initial value minus final value per Zs mouse. The particularly interesting difference between the control and the group "plasmid in the hormone with hormone receptor" (group 5) was examined using a T test for independent random samples.
Figure 17 shows the mean values of the calculated differences: In the control, for example, this difference was about 50 seconds. The 3o vertical lines show plus and minus one standard deviation from these 4>
values. The T test is based both on the differences between the mean values and on the degree of overlapping which can be seen from these lines: The larger the overlapping, the less is the significance of the mean value differences. Thus, the groups "control" and "plasmid and water i.m." (groups 1 and 5, respectively) are distinguished in a purely numerical way in the mean value, but the degree of overlapping is so high that these groups are not significantly different.
The only significant difference was between group 1 and 5: The decrease of the tatter is significantly higher than that of the control (T =
~o -2.357; d.f. = 12; p < 0.05).
The following Tables contain the concluding statistics and the results of the statistical tests (T test) performed on the differences between the mean values obtained in the course of the test:
is Group statistics ADMIN N mean valuestandard standard error of deviationthe mean value DIF control 7 47.3857 58.9946 22.2978 Hormone, hormone receptor 7 114.7571 47.3300 17.8891 and plasmid orally ~'~'O 00/49147 PCT/EP00/01368 Test for independent random samples Levene T
test test for for equal mean values equal variance F SignifiT df sig. mean standard95%
(2-sided) error confidence interval cance differenceof differenceof difference lower upper DIF variances0.0260.874-2.35712 0.036 -67.371428.5869 -129.6570-5.0858 are equal Variances -2.35711.4610.037 -67.371428.5869 -129.9833-4.7596 are not equal The human F IX was also detectable in the treated mice of the "hormone-hormone reception and plasmid orally group using an Elisa s as described in Example 4.

FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
AL Albania ES Spain LS Lesotho SI Slovenia AM Armenia FI Finland LT Lithuania SK Slovakia AT Austria FR France LU Luxembourg SN Senegal AU Australia GA Gabon LV Latvia SZ Swaziland AZ Azerbaijan GB United KingdomMC Monaco TD Chad BA Bosnia and GE Georgia MD Republic of TG Togo Herzegovina Moldova BB Barbados GH Ghana MG Madagascar TJ Tajikistan BE Belgium GN Guinea MK The former TM Turkmenistan Yugoslav BF Burkina Faso GR Greece Republic of TR Turkey Macedonia BG Bulgaria HU Hungary ML Mali TT Trinidad and Tobago BJ Benin IE Ireland MN Mongolia UA Ukraine BR Brazil IL Israel MR Mauritania UG Uganda BY Belarus IS Iceland MW Malawi US United States of America CA Canada IT Ttaly MX Mexico UZ Uzbekistan CF Central AfricanJP Japan NE Niger VN Viet Nam Republic CG Congo KE Kenya NL Netherlands YU Yugoslavia CH Switzerland KG Kyrgyzstan NO Norway ZW Zimbabwe CI Cdte d'IvoireKP Democratic NZ New Zealand People's CM Cameroon Republic of PL Poland Korea CN China KR Republic of PT Portugal Korea CU Cuba KZ Kazakstan RO Romania CZ Czech RepublicLC Saint Lucia RU Russian Federation DE Germany LI LiechtensteinSD Sudan DK Denmark LK Sri Lanka SE Sweden EE Estonia LR Liberia SG Singapore SEQUENCE LISTING

<110> Theragene dical Laboratories Biome GmbH

<120> Hormone-HormoneReceptor cid Complexes and Nucleic A

Constructs and Their Use n Gene i Therapy <130> 000065wo/JH/ml 10<140>

<141>

<160> 19 15<170> PatentIn Ver..1 <210> 1 <211> 5753 20<212> DNA

<213> Artificial ence Sequ <220>

<223> Description ArtificialSequence:ector of v pTGFG36 <220>

<221> CDS

<222> (689)..(2071) 30<400> 1 cgcgttgaca ttgattattgactagttattaatagtaatcaattacggggtcattagttc60 atagcccata tatggagttccgcgttacataacttacggtaaatggcccgcctggctgac120 35cgcccaacga cccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa180 tagggacttt ccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcag240 tacatcaagt gtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggc300 ccgcctggca ttatgcccagtacatgaccttatgggactttcctacttggcagtacatct360 acgtattagt catcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtg420 45gatagcggtt tgactcacggggatttccaagtctccaccccattgacgtcaatgggagtt480 tgttttggca ccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattga540 cgcaaatggg cggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaa600 SO

ctagagaacc cactgcttactggcttatcgaaattaatacgactcactatagggagaccc660 aagcttgcat gccaattccgcaaaggtt tg cag gtg aac atc atg 712 a cgc atg M et Gln Val Asn Ile Met Arg Met gca gaa tca cca ggc ctc atc acc atc tgc ctt tta gga tat cta ctc 760 Ala Glu Ser Pro Gly Leu Ile Thr Ile Cys Leu Leu Gly Tyr Leu Leu agt get gaa tgt aca gtt ttt ctt gat cat gaa aac gcc aac aaa att 808 Ser Ala Glu Cys Thr Val Phe Leu Asp His Glu Asn Ala Asn Lys Ile ctg aat cgg cca aag agg tat aat tca ggt aaa ttg gaa gag ttt gtt 856 Leu Asn Arg Pro Lys Arg Tyr Asn Ser Gly Lys Leu Glu Glu Phe Va1 caa ggg aac ctt gag aga gaa tgt atg gaa gaa aag tgt agt ttt gaa 904 Gln Gly Asn Leu Glu Arg Glu Cys Met Glu Glu Lys Cys Ser Phe Glu gaa gca cga gaa gtt ttt gaa aac act gaa aga aca act gaa ttt tgg 952 Glu Ala Arg Glu Val Phe Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp aag cag tat gtt gat gga gat cag tgt gag tcc aat cca tgt tta aat 1000 Lys Gln Tyr Val Asp Gly Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn ggc ggc agt tgc aag gat gac att aat tcc tat gaa tgt tgg tgt ccc 1048 Gly Gly Ser Cys Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro ttt gga ttt gaa gga aag aac tgt gaa tta gat gta aca tgt aac att 1096 Phe Gly Phe Glu Gly Lys Asn Cys Glu Leu Asp Val Thr Cys Asn Ile aag aat ggc aga tgc gag cag ttt tgt aaa aat agt get gat aac aag 1144 Lys Asn Gly Arg Cys Glu Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys gtg gtt tgc tcc tgt act gag gga tat cga ctt gca gaa aac cag aag 1192 Val Val Cys Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys tcc tgt gaa cca gca gtg cca ttt cca tgt gga aga gtt tct gtt tca 1240 Ser Cys Glu Pro Ala Val Pro Phe Pro Cys Gly Arg Val Ser Val Ser caa act tct aag ctc acc cgt get gag act gtt ttt cct gat gtg gac 1288 Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp Val Asp tat gta aat tct act gaa get gaa acc att ttg gat aac atc act caa 1336 Tyr Val Asn Ser Thr Glu Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln agc acc caa tca ttt aat gac ttc act cgg gtt gtt ggt gga gaa gat 1384 Ser Thr Gln Ser Phe Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp gcc aaa cca ggt caa ttc cct tgg cag gtt gtt ttg aat ggt aaa gtt 1432 Ala Lys Pro Gly Gln Phe Pro Trp Gln Val Val Leu Asn Gly Lys Val gat gca ttc tgt gga. ggc tct atc gtt aat gaa aaa tgg att gta act 1480 Asp Ala Phe Cys Gly Gly Ser Ile Val Asn Glu Lys Trp Ile Val Thr get gcc cac tgt gtt gaa act ggt gtt aaa att aca gtt gtc gca ggt 1528 Ala Ala His Cys Val Glu Thr Gly Val Lys Ile Thr Val Val Ala Gly gaa cat aat att gag gag aca gaa cat aca gag caa aag cga aat gtg 1576 Glu His Asn Ile Glu Glu Thr Glu His Thr Glu Gln Lys Arg Asn Val att cga att att cct cac cac aac tac aat gca get att aat aag tac 1624 Ile Arg Ile Ile Pro His His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr aac cat gac att gcc ctt ctg ga.a ctg gac gaa ccc tta gtg cta aac 1672 Asn His Asp Ile Ala Leu Leu Glu Leu Asp Glu Pro Leu Val Leu Asn agc tac gtt aca cct att tgc att get gac aag gaa tac acg aac atc 1720 Ser Tyr Val Thr Pro Ile Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile ttc ctc aaa ttt gga tct ggc tat gta agt ggc tgg gga aga gtc ttc 1768 Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser Gly Trp Gly Arg Val Phe cac aaa ggg aga tca get tta gtt ctt cag tac ctt aga gtt cca ctt 1816 His Lys Gly Arg Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro Leu gtt gac cga gcc aca tgt ctt cga tct aca aag ttc acc atc tat aac 1864 Val Asp Arg Ala Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn aac atg ttc tgt get ggc ttc cat gaa gga ggt aga gat tca tgt caa 1912 Asn Met Phe Cys Ala Gly Phe His Glu Gly Gly Arg Asp Ser Cys Gln gga gat agt ggg gga ccc cat gtt act gaa gtg gaa ggg acc agt ttc 1960 Gly Asp Ser Gly Gly Pro His Val Thr Glu Val Glu Gly Thr Ser Phe tta act gga att att agc tgg ggt gaa gag tgt gca atg aaa ggc aaa 2008 Leu Thr Gly Ile Ile Ser Trp Gly Glu Glu Cys Ala Met Lys Gly Lys tat gga ata tat acc aag gta tcc cgg tat gtc aac tgg att aag gaa 2056 Tyr Gly Ile Tyr Thr Lys Val Ser Arg Tyr Val Asn Trp Ile Lys Glu aaa aca aag ctc act taatgggatc ggtcgagcgg ccgcgactct actagaggat 2111 Lys Thr Lys Leu Thr ctttgtgaag gaaccttact tctgtggtgt gacataattg gacaaactac ctacagagat 2171 ttaaagctct aaggtaaata taaaattttt aagtgtataa tgtgttaaac tactgattct 2231 aattgtttgt gtattttaga ttccaaccta tggaactgat gaatgggagc agtggtggaa 2291 tgcctttaat gaggaaaacc tgttttgctc agaagaaatg ccatctagtg atgatgaggc 2351 tactgctgac tctcaacatt ctactcctcc aaaaaagaag agaaaggtag aagaccccaa 2411 ggactttcct tcagaattgc taagtttttt gagtcatgct gtgtttagta atagaactct 2471 tgcttgcttt gctatttaca ccacaaagga aaaagctgca ctgctataca agaaaattat 2531 S ggaaaaatat tctgtaacct ttataagtag gcataacagt tataatcata acatactgtt 2591 ttttcttact ccacacaggc atagagtgtc tgctattaat aactatgctc aaaaattgtg 2651 tacctttagc tttttaattt gtaaaggggt taataaggaa tatttgatgt atagtgcctt 2711 gactagagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc 2771 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt 2831 1S ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 2891 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 2951 tctggatccc cgggtaccct ctagagcgaa ttaattcact ggccgtcgtt ttacaacgtc 3011 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 3071 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 3131 2S tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 3191 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 3251 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 3311 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 3371 cgaaacgcgc gagacgaaag ggggggtacc agcttcgtag ctagaacatc atgttctggg 3431 3S atatcagctt cgtagctaga acatcatgtt ctggtacccc cctcgtgata cgcctatttt 3491 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 3551 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3611 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 3671 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 3731 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 3791 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 3851 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 3911 SO
ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 3971 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 4031 SS ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 4091 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 4151 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 4211 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 4271 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 4331 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 4391 5 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 4451 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 4511 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4571 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4631 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4691 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4751 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4811 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact 9871 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4931 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4991 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 5051 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 5111 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 5171 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 5231 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 5291 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 5351 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 5411 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 5471 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt 5531 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt 5591 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg 5651 gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc tctagagctc 5711 tagagctcta gagctctaga gagcttgcat gcctgcaggt cg 5753 <210> 2 <211> 461 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: vector pTGFG36 <400> 2 Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe Pro Cys GlyArgVal SerValSer GlnThrSer LysLeuThr ArgAla Glu Thr ValPhePro AspValAsp TyrValAsn SerThrGlu AlaGlu Thr Ile LeuAspAsn IleThrGln SerThrGln SerPheAsn AspPhe Thr Arg ValValGly GlyGluAsp AlaLysPro GlyGlnPhe ProTrp Gln Val ValLeuAsn GlyLysVal AspAlaPhe CysGlyGly SerIle Val Asn GluLysTrp IleValThr AlaAlaHis CysValGlu ThrGly Val Lys IleThrVal ValAlaGly GluHisAsn IleGluGlu ThrGlu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn SS Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser Trp Gly ValPheHisLys GlyArgSer AlaLeuVal Gly Arg Leu Gln Leu Arg ProLeuValAsp ArgAlaThr CysLeuArg Tyr Val Ser Thr Phe Thr TyrAsnAsnMet PheCysAla GlyPheHis Lys Ile 38.5 390 395 400 Glu Gly Arg Asp CysGlnGlyAsp SerGlyGly ProHisVal Gly Ser Thr Glu Glu Gly SerPheLeuThr GlyIleIle SerTrpGly Val Thr Glu Glu Ala Met GlyLysTyrGly IleTyrThr LysValSer Cys Lys 20Arg Tyr Asn Trp LysGluLysThr LysLeuThr Val Ile <210> 3 25<211> 78 <212> DNA

<213> Homo Sapiens <400> 3 30ggggtaccag cttcgtagct gttctgggat atcagcttcg tagctagaac agaacatcat atcatgttct ggtacccc 7g <210> 4 35<211> 78 <212> DNA

<213> Homo Sapiens <400> 4 40ggggtaccag aacatgatgt aagctgatat cccagaacat gatgttctag tctagctacg ctacgaagct ggtacccc 7g <210> 5 45<211> 19 <212> DNA

<213> Homo Sapiens <400> 5 50agcttgacct cgagcaagc 19 <210> 6 <211> 19 55<212> DNA

<213> Homo sapiens <400> 6 ggccgcttgc tcgaggtca lg <210> 7 <211> 43 <212> DNA
<213> Homo sapiens <400> 7 ggaattccgc aaaggttatg cagcgcgtga acatgatcat ggc 43 <210> 8 <211> 39 <212> DNA
<213> Homo Sapiens <400> 8 cgcggatcca ttaagtgagc tttgtttttt ccttaatcc 39 <210> 9 <211> 26 <212> DNA
<213> Homo Sapiens <400> 9 cgaggatcca gtcgtcatga ctgagc 26 <210> 10 <211> 41 <212> DNA
<213> Homo sapiens <400> 10 gcagaattca ttataaaaac tcaagacctc ataatcctga c 41 <210> 11 <211> 20 <212> DNA
<213> Homo Sapiens <400> 11 ctcctcgggg tcgaccctgg 20 <210> 12 <211> 20 <212> DNA
<213> Homo sapiens <400> 12 ccagggtcga ccccgaggag 20 <210> 13 <211> 5905 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: vector pTGFG53 <400> 13 cgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttc60 atagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgac120 cgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa180 tagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcag240 tacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggc300 ccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct360 acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtg420 gatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtt480 tgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattga540 cgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaa600 ctagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagaccc660 aagcttgcatgccaattccgcaaaggt.tatgcagcgcgtgaacatgatcatggcagaatc720 accaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtacagtttt780 tcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaa840 attggaagagtttgttcaagggaaccttgagagagaatgtatggaagaaaagtgtagttt900 tgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagta960 tgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatga1020 cattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattaga1080 tgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataa1140 caaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtga1200 accagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccg1260 tgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccatttt1320 ggataacatcactcaaagcacccaatcatttaatgacttcactcgggttgttggtggaga1380 agatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcatt1440 ctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaac1500 tggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacaga1560 gcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataa1620 gtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgt1680 tacacctatttgcattgctgacaaggaatac-acgaacatcttcctcaaatttggatctgg1-7-40 ctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagta1800 ccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatcta1860 taacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatag1920 tgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctg1980 gggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgt2040 caactggattaaggaaaaaacaaagctcacttaatgggatcggtcgagcggccgcgactc2100 tactagaggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaacta2160 cctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaa2220 ctactgattctaattgtttgtgtattttagattccaacctatggaactgatgaatgggag2280 cagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagt2340 gatgatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggta2400 gaagaccccaaggactttccttcagaattgctaagttttttgagtcatgctgtgtttagt2460 ' aatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatac2520 aagaaaattatggaaaaatattctgtaacctttataagtaggcataacagttataatcat2580 aacatactgttttttcttactccacacaggcatagagtgtctgctattaataactatgct2640 caaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatg2700 tatagtgccttgactagagatcataatcagccataccacatttgtagaggttttacttgc2760 tttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgt2820 tgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaattt2880 cacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgt2940 atcttatcatgtctggatccccggggggtaccagcttcgtagctagaacatcatgttctg3000 ggatatcagcttcgtagctagaacatcatgttctggtacccccgctctagagcgaattaa3060 ttcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaa3120 tcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccga3180 tcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttct3240 ccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctc3300 tgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacg3360 ggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcat3420 gtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcggggtaccaga3480 acatgatgttctagctacgaagctgatatcccagaacatgatgttctagctacgaagctg3540 gtaccccggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttc3600 ttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttattttt3660 ctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaata3720 atattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttt3780 tgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgc3840 tgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagat3900 S ccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgct3960 atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcataca4020 ctattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatgg4080 catgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaa4140 cttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggg4200 10 ggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacga4260 cgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactgg4320 cgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagt4380 tgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctgg4440 agccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctc4500 IS ccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagaca4560 gatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactc4620 atatatactttagattgatttaaaacttcatttttaatttaaaagg.atctaggtgaagat4680 cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtc4740 agaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctg4800 ctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagct4860 accaactctttttccgaa.ggtaactggcttcagcagagcgcagataccaaatactgtcct4920 tctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacct4980 cgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgg5040 gttggactcaagacgatagttacggataaggcgcagcggtcgggctgaacggggggttcg5100 tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgag5160 ctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc5220 agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat5280 agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg5340 gggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgc5400 tggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtatt54-60 accgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtca5520 gtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccg5580 attcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac5640 gcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccg5700 3S gctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgac5760 catgattacgccaagctctctagagctctagagctctagagctctagagagcttgcatgc5820 cggggtaccagcttcgtagctagaacatcatgttctgggatatcagcttcgtagctagaa5880 catcatgttctggtaccccggtcga 5905 <210> 14 <211> 6052 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: vector pTGFG64 <400> 14 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300 SS ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660 aagcttgcat gccaattccg caaaggttat gcagcgcgtg aacatgatca tggcagaatc 720 accaggcctc atcaccatct gccttttagg atatctactc agtgctgaat gtacagtttt 780 tcttgatcat gaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaa840 attggaagag tttgttcaagggaaccttgagagagaatgtatggaagaaaagtgtagttt900 tgaagaagca cgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagta960 tgttgatgga gatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatga1020 cattaattcc tatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattaga1080 tgtaacatgt aacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataa1140 caaggtggtt tgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtga1200 accagcagtg ccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccg1260 tgctgagact gtttttcctgatgtggactatgtaaattctactgaagctgaaaccatttt1320 10ggataacatc actcaaagcacccaatcatttaatgacttcactcgggttgttggtggaga1380 agatgccaaa ccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcatt1440 ctgtggaggc tctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaac1500 tggtgttaaa attacagttgtcgcaggtgaacataatattgaggagacagaacatacaga1560 gcaaaagcga aatgtgattcgaattattcctcaccacaactacaatgcagctattaataa1620 15gtacaaccat gacattgcccttctggaactggacgaacccttagtgctaaacagctacgt1680 tacacctatt tgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctgg1740 ctatgtaagt ggctggggaagagtcttccacaaagggagatcagctttagttcttcagta1800 ccttagagtt ccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatcta1860 taacaacatg ttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatag1920 20tgggggaccc catgttactgaagtggaagggaccagtttcttaactggaattattagctg1980 gggtgaagag tgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgt2040 caactggatt aaggaaaaaacaaagctcacttaatgggatcggtcgagcggccgcgactc2100 tactagagga tctttgtgaaggaaccttacttctgtggtgtgacataattggacaaacta2160 cctacagaga tttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaa2220 25ctactgattc taattgtttgtgtattttagattccaacctatggaactgatgaatgggag2280 cagtggtgga atgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagt2340 gatgatgagg ctactgctgactctcaacattctactcctccaaaaaagaagagaaaggta2400 gaagacccca aggactttccttcagaattgctaagttttttgagtcatgctgtgtttagt2460 aatagaactc ttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatac2520 30aagaaaatta tggaaaaatattctgtaacctttataagtaggcataacagttataatcat2580 aacatactg t tttttcttactccacacaggcatagagtgtctgctattaataactatgct2640 caaaaattgt gtacctttagctttttaatttgtaaaggggttaataaggaatatttgatg2700 tatagtgcct tgactagagatcataatcagccataccacatttgtagaggttttacttgc2760 tttaaaaaac ctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgt2820 35tgttaacttg tttattgcagcttataatggttacaaataaagcaatagcatcacaaattt2880 cacaaataaa gcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgt2940 atcttatcat gtctggatccccggggggtaccagcttcgtagctagaacatcatgttctg3000 ggatatcagc ttcgtagctagaacatcatgttctggtacccccctctagagcgaattaat3060 tcactggccg tcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaat3120 40cgccttgcag cacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgat3180 cgcccttccc aacagttgcgcagcctgaatggcgaatggcggggtaccagcttcgtagct3240 agaacatcat gttctgggatatcagcttcgtagctagaacatcatgttctggtaccccgc3300 ctgatgcggt attttctcct-tacgcatctgtgcggtatttcacaccgcatatggtgcact3360 ctcagtacaa tctgctctgatgccgcatagttaagccagccccgacacccgccaacaccc3420 45gctgacgcgc cctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgacc3480 gtctccggga gctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacga3540 aagggcacca gaacatgatgttctagctacgaagctgatatcccagaacatgatgttcta3600 gctacgaagc tggtaccccgcctcgtgatacgcctatttttataggttaatgtcatgata3660 ataatggttt cttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatt3720 SOtgtttatttt tctaaatacattcaaatatgtatccgctcatgagacaataaccctgataa3780 atgcttcaat aatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccctt3840 attccctttt ttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaa3900 gtaaaagatg ctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac3960 agcggtaaga tccttgagagttttcgccccgaagaacgttttccaatgatgagcactttt4020 55aaagttctgc tatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggt9080 cgccgcatac actattctcagaatgacttggttgagtactcaccagtcacagaaaagcat4140 cttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataac4200 actgcggcca acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttg4260 cacaacatgg gggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagcc4320 60ataccaaacg acgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaa4380 ctattaactg gcgaactacttactctagcttcccggcaacaattaatagactggatggag4440 gcggataaag ttgcaggaccacttctgcgctcggcccttccggctggctggtttattgct4500 gataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagat4560 ggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaa4620 cgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagac4680 caagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatc4740 taggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttc4800 cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctg4860 cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccg4920 gatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatacca4980 aatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccg5040 cctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcg5100 tgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga5160 acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatac5220 ctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtat5280 ccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcc5340 tggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtga5400 tgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc5460 ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg5520 gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgag5580 cgcagcgagtcagtgagcgaggggtaccagaacatgatgttctagctacgaagctgatat5640 cccagaacatgatgttctagctacgaagctggtaccccagcggaagagcgcccaatacgc5700 aaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttccc5760 gactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggca5820 ccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataa5880 caatttcacacaggaaacagctatgaccatgattacgccaagctctctagagctctagag5940 ctctagagctctagagagcttgcatgccggggtaccagcttcgtagctagaacatcatgt6000 tctgggatatcagcttcgtagctagaacatcatgttctggtaccccggtcga 6052 <210> 15 <211> 4344 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: vector pTGFG67 <400> 15 cgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttc60 atagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgac120 cgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa180 tagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcag240 tacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggc300 ccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct360 acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtg420 gatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtt480 tgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattga540 cgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaa600 ctagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagaccc660 aagcttgacctcgagcaagcggccgcgactctactagaggatctttgtgaaggaacctta720 cttctgtggtgtgacataattggacaaactacctacagagatttaaagctctaaggtaaa780 tataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtatttta840 gattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaa900 cctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaaca960 ttctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaatt1020 gctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctattta1080 caccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaac1140 ctttataagtaggcataacagttataatcataacatactgttttttcttactccacacag1200 gcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaat1260 ttgtaaaggggttaataaggaatatttgatgtatagtgccttgactagagatcataatca1320 gccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctga1380 acctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatg1440 gttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcatt1500 ctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatccccgggtacc1560 ctctagagcgaattaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctg1620 gcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcg1680 aagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcc1740 tgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactc1800 tcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccg1860 ctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccg1920 tctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaa1980 agggggggtaccagcttcgtagctagaacatcatgttctgggatatcagcttcgtagcta2040 gaacatcatgttctggtacccccctcgtgatacgcctatttttataggttaatgtcatga2100 taataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaaccccta2160 tttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgat2220 aaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccc2280 ttattccct t ttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtga2340 aagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctca2400 acagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactt2460 ttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcg2520 gtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagc2580 atcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgata2640 acactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttt2700 tgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaag2760 ccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgca2820 aactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgg2880 aggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattg2940 ctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccag3000 atggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatg3060 aacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcag3120 accaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaagga3180 tctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgt3240 tccactgagcgtcagaccccgtagaaaagat~aaaggatcttcttgagatcctttttttc33D0 tgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgc3360 cggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatac3420 caaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcac3480 cgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagt3540 cgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggct3600 gaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagat3660 acctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggt3720 atccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacg3780 cctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgt3840 gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggt3900 tcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctg3960 tggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccg4020 agcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctcc4080 ccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg4140 gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttac4200 actttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacag4260 gaaacagctatgaccatgattacgccaagctctctagagctctagagctctagagctcta4320 gagagcttgcatgcctgcaggtcg 4344 <210> 16 <211> 4496 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: vector pTGFG82 <400> 16 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180 tagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcag240 tacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggc300 ccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct360 acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtg420 gatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtt480 tgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattga540 cgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaa600 ctagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagaccc660 aagcttgacctcgagcaagcggccgcgactctactagaggatctttgtgaaggaacctta720 cttctgtggtgtgacataattggacaaactacctacagagatttaaagctctaaggtaaa780 tataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtatttta840 gattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaa900 cctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaaca960 ttctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaatt1020 gctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctattta1080 caccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaac1140 ctttataagtaggcataacagttataatcataacatactgttttttcttactccacacag1200 gcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaat1260 ttgtaaaggggttaataaggaatatttgatgtatagtgccttgactagagatcataatca1320 gccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctga1380 acctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatg1440 gttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcatt1500 ctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatccccggggggt1560 accagcttcgtagctagaacatcatgttctgggatatcagcttcgtagctagaacatcat1620 gttctggtacccccctctagagcgaattaattcactggccgtcgttttacaacgtcgtga1680 ctgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccag1740 ctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaa1800 tggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccg1860 catatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgaca1920 cccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacag1980 acaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaa2040 acgcgcgagacgaaagggcggggtaccagaacatgatgttctagctacgaagctgatatc2100 ccagaacatgatgttctagctacgaagctggtaccccggcctcgtgatacgcctattttt2160 ataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaa2220 tgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcat2280 gagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattca2340 acatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctca2400 cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggtta2460 catcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttt2520 tccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgc2580 cgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactc2640 accagtcacagaaaagcatc,ttacggatggcatgacagtaagagaattatgcagtgctgc2700 cataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaa2760 ggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttggga2820 accggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaat2880 ggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaaca2940 attaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttcc3000 ggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcat3060 tgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggag3120 tcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaa3180 gcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttca3240 tttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccc3300 ttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc3360 ttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctacc3420 agcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggctt3480 cagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccactt3540 caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgc3600 tgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataa3660 ggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgac3720 ctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagg3780 gagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggga3840 gcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgact3900 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 3960 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 4020 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 4080 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 4140 5 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt 4200 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta 4260 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 4320 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct ctagagctct 4380 agagctctag agctctagag agcttgcatg ccggggtacc agcttcgtag ctagaacatc 4440 10 atgttctggg atatcagctt cgtagctaga acatcatgtt ctggtacccc ggtcga 4496 <210> 17 <211> 4644 15 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: vector pTGFG95 <400> 17 cgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttc60 atagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgac120 cgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa180 tagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcag240 tacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggc300 ccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct360 acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtg420 gatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtt480 tgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattga54.0 cgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaa600 ctagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagaccc660 aagcttgacctcgagcaagcggccgcgactctactagaggatctttgtgaaggaacctta720 cttctgtggtgtgacataattggacaaactacctacagagatttaaagctctaaggtaaa780 tataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtatttta840 gattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaa900 cctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaaca960 ttctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaatt1020 gctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctattta1080 caccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaac1140 ctttataagtaggcataacagttataatcataacatactgttttttcttactccacacag1200 gcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaat1260 ttgtaaaggggttaataagg-aatatttgatgtatagtgccttgactagagatcataatca1320 gccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctga1380 acctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatg1440 gttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcatt1500 ctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatccccggggggt1560 accagcttcgtagctagaacatcatgttctgggatatcagcttcgtagctagaacatcat1620 gttctggtacccccctctagagcgaattaattcactggccgtcgttttacaacgtcgtga1680 ctgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccag1740 ctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaa1800 tggcgaatggcggggtaccagcttcgtagctagaacatcatgttctgggatatcagcttc1860 gtagctagaacatcatgttctggtaccccgcctgatgcggtattttctccttacgcatct1920 gtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcata1980 gttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgct2040 cccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggtt2100 ttcaccgtcatcaccgaaacgcgcgagacgaaagggctaccagaacatgatgttctagct2160 acgaagctgatatcccagaacatgatgttctagctacgaagctggtaccccgcctcgtga2220 tacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggca2280 cttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaata2340 tgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaaga2400 gtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttc2460 ctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtg2520 cacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgcc2580 ccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattat2640 cccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgact2700 tggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaat2760 tatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacga2820 tcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcc2880 ttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacga2940 tgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctag3000 cttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgc3060 gctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggt3120 ctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatct3180 acacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtg3240 cctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattg3300 atttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctca3360 tgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaaga3420 tcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaa3480 aaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccga3540 aggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagt3600 taggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgt3660 taccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgat3720 agttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagct3780 tggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgcca3840 cgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggag3900 agcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttc3960 gccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatgga4020 aaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcaca4080 tgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgag4140 ctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggggtacc4200 agaacatgatgttctagctacgaagctgatatcccagaacatgatgttctagctacgaag42-60 ctggtaccccagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccga4320 ttcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacg4380 caattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccgg4440 ctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc4500 atgattacgccaagctctctagagctctagagctctagagctctagagagcttgcatgcc4560 ggggtaccagcttcgtagctagaacatcatgttctgggatatcagcttcgtagctagaac4620 atcatgttctggtaccccggtcga 4694 <210> 18 <211> 933 <212> PRT
<213> Homo Sapiens <400> 18 Met Thr Glu Leu Lys Ala Lys Gly Pro Arg Ala Pro His Val Ala Gly Gly Pro Pro Ser Pro Glu Val Gly Ser Pro Leu Leu Cys Arg Pro Ala Ala Gly Pro Phe Pro Gly Ser Gln Thr Ser Asp Thr Leu Pro Glu Val Ser Ala Ile Pro Ile Ser Leu Asp Gly Leu Leu Phe Pro Arg Pro Cys Gln Gly Gln Asp Pro Ser Asp Glu Lys Thr Gln Asp Gln Gln Ser Leu Ser Asp Val Glu Gly Ala Tyr Ser Arg Ala Glu Ala Thr Arg Gly Ala Gly Gly Ser Ser Ser Ser Pro Pro Glu Lys Asp Ser Gly Leu Leu Asp Ser ValLeuAsp ThrLeuLeu AlaProSer GlyProGly GlnSerGln Pro SerProPro AlaCysGlu ValThrSer SerTrpCys LeuPheGly Pro GluLeuPro GluAspPro ProAlaAla ProAlaThr GlnArgVal Leu SerProLeu MetSerArg SerGlyCys LysValGly AspSerSer Gly ThrAlaAla AlaHisLys ValLeuPro ArgGlyLeu SerProAla 20Arg GlnLeuLeu LeuProAla SerGluSer ProHisTrp SerGlyAla Pro ValLysPro SerProGln AlaAlaAla ValGluVal GluGluGlu Asp GlySerGlu SerGluGlu SerAlaGly ProLeuLeu LysGlyLys Pro ArgAlaLeu GlyGlyAla AlaAlaGly GlyGlyAla AlaAlaVal Pro ProGlyAla AlaAlaGly GlyValAla LeuValPro LysGluAsp 35Ser ArgPheSer AlaProArg ValAlaLeu ValGluGln AspAlaPro Met AlaProGly ArgSerPro LeuAlaThr ThrValMet AspPheIle His ValProIle LeuProLeu AsnHisAla LeuLeuAla AlaArgThr Arg GlnLeuLeu GluAspGlu SerTyrAsp GlyGlyAla GlyAlaAla Ser AlaPheAla ProProArg SerSerPro CysAlaSer SerThrPro 50Val AlaValGly AspPhePro AspCysAla TyrProPro AspAlaGlu Pro LysAspAsp AlaTyrPro LeuTyrSer AspPheGln ProProAla Leu LysIleLys GluGluGlu GluGlyAla GluAlaSer AlaArgSer Pro ArgSerTyr LeuValAla GlyAlaAsn ProAlaAla PheProAsp Phe Pro Leu Gly Pro Pro Pro Pro Leu Pro Pro Arg Ala Thr Pro Ser Arg ProGly GluAlaAla ValThrAla AlaProAla SerAlaSer Val Ser SerAla SerSerSer GlySerThr LeuGluCys IleLeuTyr Lys Ala GluGly AlaProPro GlnGlnGly ProPheAla ProProPro Cys Lys AlaPro GlyAlaSer GlyCysLeu LeuProArg AspGlyLeu Pro Ser ThrSer AlaSerAla AlaAlaA1a GlyAlaAla ProAlaLeu Tyr Pro AlaLeu GlyLeuAsn GlyLeuPro GlnLeuGly TyrGlnAla Ala Val LeuLys GluGlyLeu ProGlnVal TyrProPro TyrLeuAsn Tyr Leu ArgPro AspSerGlu AlaSerGln SerProGln TyrSerPhe Glu Ser LeuPro GlnLysIle CysLeuIle CysGlyAsp GluAlaSer Gly Cys HisTyr GlyValLeu ThrCysGly SerCysLys ValPhePhe Lys Arg AlaMet GluGlyGln HisAsnTyr LeuCysAla GlyArgAsn Asp Cys IleVal AspLysIle ArgArgLys AsnCysPro AlaCysArg Leu Arg LysCys CysGlnAla GlyMetVal LeuGlyGly ArgLysPhe Lys Lys PheAsn LysValArg ValValArg AlaLeuAsp AlaValAla Leu r15 645 650 655 Pro Gln Pro Leu Gly Val Pro Asn Glu Ser Gln Ala Leu Ser Gln Arg Phe Thr Phe Ser Pro Gly Gln Asp Ile Gln Leu Ile Pro Pro Leu Ile Asn Leu Leu Met Ser Ile Glu Pro Asp Val Ile Tyr Ala Gly His Asp Asn Thr Lys Pro Asp Thr Ser Ser Ser Leu Leu Thr Ser Leu Asn Gln Leu Gly Glu Arg Gln Leu Leu Ser Val Val Lys Trp Ser Lys Ser Leu Pro Gly Phe Arg Asn Leu His Ile Asp Asp Gln Ile Thr Leu Ile Gln Tyr Ser Trp Met Ser Leu Met Val Phe Gly Leu Gly Trp Arg Ser Tyr Lys His Val Ser Gly Gln Met Leu Tyr Phe Ala Pro asp Leu Ile Leu Asn Glu Gln Arg Met Lys Glu Ser Ser Phe Tyr Ser ~eu Cys Leu Thr Met Trp Gln Ile Pro Gln Glu Phe Val Lys Leu Gln '.'al Ser Gln Glu Glu Phe Leu Cys Met Lys Val Leu Leu Leu Leu Asn ~hr Ile Pro Leu Glu Gly Leu Arg Ser Gln Thr Gln Phe Glu Glu Met Arg Ser Ser Tyr Ile Arg Glu Leu Ile Lys Ala Ile Gly Leu Arg Gln _ys Gly Val Val Ser Ser Ser Gln Arg Phe Tyr Gln Leu Thr Lys Leu ~eu Asp Asn Leu His Asp Leu Val Lys Gln Leu His Leu Tyr Cys Leu Asn Thr Phe Ile Gln Ser Arg Ala Leu Ser Val Glu Phe Pro Glu Met Met Ser Glu Val Ile Ala Ala Gln Leu Pro Lys Ile Leu Ala Gly Met Val Lys Pro Leu Leu Phe His Lys Lys <210> 19 <211> 2970 <212> DNA
<213> Homo sapiens <400> 19 ctgaccagcg ccgccctccc ccgcccccga cccaggaggt ggagatccct ccggtccagc 60 cacattcaac acccactttc tcctccctct gcccctatat tcccgaaacc ccctcctcct 120 tcccttttcc ctcctccctg gagacggggg aggagaaaag gggagtccag tcgtcatgac 180 tgagctgaag gcaaagggtc cccgggctcc ccacgtggcg ggcggcccgc cctcccccga 240 ggtcggatcc ccactgctgt gtcgcccagc cgcaggtccg ttcccgggga gccagacctc 300 ggacaccttg cctgaagttt cggccatacc tatctccctg gacgggctac tcttccctcg 360 gccctgccag ggacaggacc cctccgacga aaagacgcag gaccagcagt cgctgtcgga 420 cgtggagggc gcatattcca gagctgaagc tacaaggggt gctggaggca gcagttctag 480 tcccccagaa aaggacagcg gactgctgga cagtgtcttg gacactctgt tggcgccctc 540 aggtcccggg cagagccaac ccagccctcc cgcctgcgag gtcaccagct cttggtgcct 600 gtttggcccc gaacttcccg aagatccacc ggctgccccc gccacccagc gggtgttgtc 660 cccgctcatg agccggtccg ggtgcaaggt tggagacagc tccgggacgg cagctgccca 720 taaagtgctg ccccggggcc tgtcaccagc ccggcagctg ctgctcccgg cctctgagag 780 ccctcactgg tccggggccc cagtgaagcc gtctccgcag gccgctgcgg tggaggttga 840 ggaggaggat ggctctgagt ccgaggagtc tgcgggtccg cttctgaagg gcaaacctcg 900 ggctctgggt ggcgcggcgg ctggaggagg agccgcggct gtcccgccgg gggcggcagc 960 aggaggcgtcgccctggtccccaaggaagattcccgcttctcagcgcccagggtcgccct1020 ggtggagcaggacgcgccgatggcgcccgggcgctccccgctggccaccacggtgatgga1080 tttcatccacgtgcctatcctgcctctcaatcacgccttattggcagcccgcactcggca1140 getgctggaagacgaaagttacgacggcggggccggggctgccagcgcctttgccccgcc1200 5 gcggagttcaccctgtgcctcgtccaccccggtcgctgtaggcgacttccccgactgcgc1260 gtacccgcccgacgccgagcccaaggacgacgcgtaccctctctatagcgacttccagcc1320 gcccgctctaaagataaaggaggaggaggaaggcgcggaggcctccgcgcgctccccgcg1380 ttcctaccttgtggccggtgccaac.cccgcagccttcccggatttcccgttggggccacc1440 gcccccgctgccgccgcgagcgaccccatccagacccggggaagcggcggtgacggccgc1500 10 acccgccagtgcctcagtctcgtctgcgtcctcctcggggtcgaccctggagtgcatcct1560 gtacaaagcggagggcgcgccgccccagcagggcccgttcgcgccgccgccctgcaaggc1620 gccgggcgcgagcggctgcctgctcccgcgggacggcctgccctccacctccgcctctgc1680 cgccgccgccggggcggcccccgcgctctaccctgcactcggcctcaacgggctcccgca1740 gctcggctaccaggccgccgtgctcaaggagggcctgccgcaggtctacccgccctatct1800 IS caactacctgaggccggattcagaagccagccagagcccacaatacagcttcgagtcatt1860 acctcagaagatttgtttaatctgtggggatgaagcatcaggctgtcattatggtgtcct1920 tacctgtgggagctgtaaggtcttctttaagagggcaatggaagggcagcacaactactt1980 atgtgctggaagaaatgactgcatcgttgataaaatccgcagaaaaaactgcccagcatg2040 tcgccttagaaagtgctgtcaggctggcatggtccttggaggtcgaaaatttaaaaagtt2100 20 caataaagtcagagttgtgagagcactggatgctgttgctctcccacagccattgggcgt2160 tccaaatgaaagccaagccctaagccagagattcactttttcaccaggtcaagacataca2220 gttgattccaccactgatcaacctgttaatgagcattgaaccagatgtgatctatgcagg2280 acatgacaacacaaaacctgacacctccagttctttgctgacaagtcttaatcaactagg2340 cgagaggcaacttctttcagtagtcaagtggtctaaatcattgccaggttttcgaaactt2400 acatattgatgaccagataactctcattcagtattcttggatgagcttaatggtgtttgg2460 tctaggatggagatcctacaaacatgtcagtgggcagatgctgtattttgcacctgatct2520 aatactaaatgaacagcggatgaaagaatcatcattctattcattatgccttaccatgtg2580 gcagatcccacaggagtttgtcaagcttcaagttagccaagaagagttcctctgtatgaa2640 agtattgttacttcttaatacaattcctttggaagggctacgaagtcaaacccagtttga2700 ggagatgaggtcaagctacattagagagctcatcaaggcaattggtttgaggcaaaaagg27-60 agttgtgtcgagctcacagcgtttctatcaacttacaaaacttcttgataacttgcatga2820 tcttgtcaaacaacttcatctgtactgcttgaatacatttatccagtcccgggcactgag2880 tgttgaatttccagaaatgatgtctgaagttattgctgcacaattacccaagatattggc2940 agggatggtgaaaccccttctctttcataa 2970

Claims (49)

1. Use of (i) a nucleic acid construct comprising at least one hormone responsive element (HRE) and a transgene, said at least one HRE being not functionally linked to the transgene, and (ii) a hormone-hormone receptor complex for preparing an agent for gene transfer.

2. The use of claim 1, wherein the transgene is selected from the group consisting of genes encoding a blood clotting factor, hormone genes, hormone receptor genes, growth factors, enzyme genes, genes encoding cytokines or lymphokines, genes encoding inhibitor substances, genes encoding substances that function as drugs or vaccines, and antisense sequences.

3. The use of claim 2, wherein the transgene is a gene encoding a blood clotting factor and the agent is suitable for treating hemophilia.

4. The of claim 3, wherein the blood clotting factor is a human blood clotting factor and preferably is selected from the group consisting of factor VIII, factor IX, and von Willebrand Factor (vWF).

5. The use of any one of claims 1 to 4, wherein the nucleic acid construct comprises 1 to 20, preferably 3 to 10 HRE(s).

6. The use of any one of claims 1 to 5, wherein the at least one HRE is a steroid responsive element, preferably a progesterone responsive element (PRE).

7. The use of claim 4, wherein the HRE is a PRE and the blood clotting factor is factor IX, preferably the factor IX has a nucleotide sequence of 689 to 2071 of SEQ ID NO: 1.

8. The use of claim 5, wherein the HRE is a PRE and the blood clotting factor is factor VIII.

9. The use of any one of claims 6 to 8, wherein the PRE has the double stranded DNA sequence comprised of the DNA sequences of SEQ ID NOs:
3 and 4.

10. The use of any one of claims 1 to 9, wherein the construct further comprises functional DNA sequences selected from the group consisting of promoter sequences, enhancer sequences, silencer sequences, origin of replication sequences, integrational sequences, marker genes and switch sequences.

11. The use of claim 10, wherein the construct further comprises a tissue-specific promoter, preferably an .alpha.-antitrypsin promoter.

12. The use according to any one of claims 1 to 11, wherein the hormone-hormone receptor complex is a steroid-steroid receptor complex.

13. The use of claim 12, wherein the molar ratio of HRE within the nucleic acid construct to hormone receptor is from 1:1 to 1:10, preferably 1:2 to 1:5, and/or the molar ratio of hormone to hormone receptor is at least 1000:1, preferably at least 10000:1.

14. The use of claim 12 or 13, wherein the receptor is a progesterone receptor and the steroid is progesterone or a progesterone derivative.

15. The use of claim 14, wherein the progesterone is natural micronized progesterone solubilized in a liphophilic matrix system and/or the progesterone receptor is hPR-A, hPR-B or comprises the nucleotide sequence of 557 to 933 SEQ ID N0:18.

16. A pharmaceutical composition comprising a nucleic acid construct comprising at least one HRE and a transgene as defined in claims 1 to 11 and/or a vector comprising said nucleic acid construct, said at least one HRE being coupled to a hormone-hormone receptor complex.

17. The pharmaceutical composition of claim 16, wherein the hormone-hormone receptor complex is as defined in claims 12 to 15.

18. The pharmaceutical composition of claim 16, wherein the transgene is a gene encoding a blood clotting factor.

19. The pharmaceutical composition of claim 18 wherein the blood clotting factor is factor IX.

20. The pharmaceutical composition of claim 18 wherein the blood clotting factor is factor VIII.

21. The pharmaceutical composition of any one of claims 18 to 20, which is suitable for gene transfer, preferably for treating hemophilia.

22. A nucleic acid construct comprising at-least one HRE and a transgene being a gene encoding a blood clotting factor, wherein one of said at least one HREs is not functionally linked to the transgene.

23. The nucleic acid construct of claim 22, which is as defined in claims 4 to 11.

24. A vector comprising the nucleic acid construct of claim 22 or 23.

25. A transformed cell or transgenic organism comprising the nucleic acid construct as defined in claims 22 or 23 or the vector as defined in claim 24.

26. A composition of matter comprising - the nucleic acid construct comprising at least one HRE and a transgene as defined in of claim 22 or 23, and/or - a vector comprising said nucleic acid construct, said at least one HRE
being coupled to a hormone-hormone receptor complex.

27. A method for preparing the composition of matter as defined in claim 26, which method comprises admixing the nucleic acid construct with the hormone receptor and the hormone.

28. A method for gene transfer which comprises administering the agent as defined in claims 1 to 15, or the pharmaceutical composition as defined in claims 16 to 20 to an organism or to a cellular system.

29. A method for delivering into an organism or into a cellular system a, nucleic acid encoding a transgene to be expressed in the cells of the organism or the cells of the cellular system, which method comprises administering an agent as defined in claims 1 to 15 or a pharmaceutical composition as defined in claims 16 to 20 to the organism or to the cellular system so that the hormone in the-composition interacts with the cell membrane and therewith enhances diffusion and transport of the nucleic acid that is coupled to the hormone-hormone receptor complex across the membrane and into the cell.

30. The method of claim 29, wherein a nucleic acid encoding human factor VIII or factor IX is delivered into the cell.

31. A method of treating blood clotting disorders comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 18 to an organism or to a cellular system.

32. A method of treating hemophilia B, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 19 to an organism or to a cellular system.

33. A method of treating hemophilia A, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 20 to an organism or to a cellular system.

34. Use of (i) a nucleic acid construct comprising at least one hormone responsive element (HRE) and a transgene wherein the transgene is a gene encoding a blood clotting factor and the at least one HRE is functionally linked to the transgene, and (ii) a hormone-hormone receptor complex for preparing an agent for treating heomphilia.

35. The of claim 34, wherein the blood clotting factor is a human blood clotting factor and preferably is selected from the group consisting of factor VIII, factor IX, and von Wiilebrand Factor (vWF).

36. The use of claims 34 or 35, wherein the nucleic acid construct comprises 1 to 20, preferably 3 to 10 HRE(s).

37. The use of claim 34 to 36, wherein the at least one HRE is a steroid responsive element, preferably a progesterone responsive element (PRE).

38. The use of claim 35, wherein the HRE is a PRE and the blood clotting factor is factor IX, preferably the factor IX has a nucleotide sequence of 689 to 2071 of SEQ ID NO: 1.

39. The use of claim 35, wherein the HRE is a PRE and the blood clotting factor is factor VIII.

40. The use of claim 37 to 39, wherein the PRE has the double stranded DNA sequence comprised of the DNA sequences of SEQ ID NOs: 3 and 4.

41. The use of claims 34 to 40, wherein the construct further comprises functional DNA sequences selected from the group consisting of promoter sequences, enhancer sequences, silencer sequences, origin of replication sequences, Integrational sequences, marker genes and switch sequences.

42. The use of claim 41, wherein the construct further comprises a tissue-specific promoter, preferably an a-antitrypsin promoter.

43. The use according to any one of claims 34 to 42, wherein the hormone-hormone receptor is a steroid-steroid receptor complex.

44. The use of claim 43, wherein the molar ratio of HRE within the nucleic acid construct to hormone receptor is From 1:1 to 1:10, preferably 1:2 to 1:5, and/or the molar ratio of hormone to hormone receptor is at least 1000:1, preferably at least 10000:1.

45. The use of claim 43 or 44, wherein the receptor is a progesterone receptor and the steroid is progesterone or a progesterone derivative.

46. The use of claim 45, wherein the progesterone is natural micronized progesterone solubiiized in a liphophilic matrix system and/or the progesterone receptor is hPR-A, hPR-B or comprises the nucleotide sequence of 557 to 933 SEQ ID NO: 18.

47. A method for gene transfer which comprises administering the agent as defined in claims 34 to 46 to an organism or to a cellular system.

48. A method for delivering into an organism or into a cellular system a nucleic acid encoding a transgene to be expressed in the cells of the organism or the cells of the cellular system, which method comprises administering an agent as defined in claims 34 to 46 to the organism or to the cellular system so that the hormone in the composition interacts with the cell membrane and therewith enhances diffusion and transport of the nucleic acid that is coupled to the hormone-hormone receptor complex across the membrane and into the cell.

49. The method of claim 48, wherein a nucleic acid encoding human factor VIII or factor IX is delivered into the cell.