NZ513595A - Hormone-hormone receptor complexes and nucleic acid constructs and their use in gene therapy - Google Patents

Description

513 5 95 1 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 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 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 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. 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 2 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 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 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.f et al., Gene Ther., Vol. 1, 367, 1994. ). Still, these systems have major disadvantages, such as 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. 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 they place an additional burden on the patient (Dai, Y., et al., Proc. Natl. 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 introducing several small gene sequences into host cells, gene WO 00/49147 PCT/EP00/01368 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., 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 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 (Feigner, P., et al., Proc. Natl. Acad. Sci. USA, Vol. 84, 7413, 1987; Behr, J.-P., Bioconjugate Chemistry, Vol. 5, 382, 1994).

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., 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 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 certain threshold.

WO 00/49147 PCT/EP00/01368 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, Vol.271, 1994; Hoeben, R. C., Biologicals, 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 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 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 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 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 is based on the concept of binding interaction of the hormone to an PCT/E P00/01368 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 5 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'Malley, B., et al., Biol. Reprod., Vol. 46, 163, 1992). The hormone responsive elements are enhancer elements 10 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 20 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 25 properties both at the molecular and the systemic level. Besides classical effects on the uterus, antiepiieptic, anxiolytic, hypnotic and anesthetic properties have been attributed to progesterone according to numerous studies.

Methods have been proposed for the use of mutant hormone 30 receptors, including mutant steroid receptors for gene therapy. For 6 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.

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 ability to drag a nucleic acid construct having one or more hormone responsive element(s) 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 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 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; (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; PCT/EPO0/O1368 7 (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 5 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; io (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 15 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; (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 25 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 8 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 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 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 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 responsive element is a steroid responsive element (5RE), 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.

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 hormone receptor as a link (or binding compound) between the WO 00/49147 PCT/EP00/01368 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 be an additional effect when regulation of the 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 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.

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 response.

Brief Description of the Drawings Figure 1 shows the concept of gene transfer of the present invention (with HRE = hormone responsive element, HR = hormone receptor, H = hormone, blank circles = lipophilic matrix).

Figure 2 is a diagram of the vector pTGFGl.

Figure 3 is a diagram of the vector pTGFG5.

Figure 4 is a diagram of the vector pTGFG20.

Figure 5 is a diagram of the vector pTGFG33.

Figure 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.

Figure 9 is the DNA sequence of vector pTGFG36 (SEQ ID NO: 1).

Figure 10 shows the protein sequence of factor IX encoded by vector pTGFG36 (SEQ ID NO: 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 d) micrographs of HeLa 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 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 cobalt2+ affinity chromatography as described in Example 5. The final preparation (85pg protein) was separated on a denaturing 7,5% SDS-polyacrylamid gel, followed by staining with coomassie® R250 (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 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.

Figure 18 shows the clotting time detected in Example 9.

Figure 19 shows the activity of human progesterone receptor as determined in Example 8.

Figure 20: shows the amino acid sequence of the hPR B-Form. The start methionine 165 of the hPR A-Form is underlined (SEQ ID NO: 5 18).

Figure 21 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 10 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 20 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 25 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 PCT/E P00/01368 12 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 "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.

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.

"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 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, 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 the control of transcription of a gene to which RNA polymerase binds.

The promoter forms an initiation complex with RNA polymerase 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 5 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 10 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 15 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 20 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 25 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.

PCT /E P00/01368 14 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 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 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.

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, 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. 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 are, for example, estrogen receptors, mineralocorticoid receptors, WO 00/49147 PCT/EPO0/O1368 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, 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 a!., Proc. Natl. Acad. Sci. USA, Vol. 86, 327, 1989; Baker, A. R., et al., Proc. Natl. Acad. Sci. USA, Vol. 85, 3294, 1988; Weinberger, C., et al., Nature, Vol. 324, 641, 1986; Sap, J., et al., Nature, Vol. 324, 635, 1086; Misrahi, M., et al., Biochem. Biophys. Res. Commun., Vol. 143, 740, 1987; Kastner, P., et al., Cell, Vol. 83, 859, 1995). These receptors may be from human or other mammalian sources, although human is preferred. Nucleotide and/or amino acid sequences of human steroid receptors are available in the GenBank: mineralocorticoid receptor: M16801; glucocorticoid receptor a: M10901; glucocorticoid receptor a2: U01351; glucocorticoid receptor B: M11050; retinoic acid receptor a: AF088888 (exon 1), AF088889 (exon 2), AF088890 (exon 3), AF088891 (exon 4), AF088892 (exon 5 and 6), AF088893 (exon 7), AF088894 (exon 8), AF088895 (exon 9 and 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 ai: 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-cloning of the known cDNAs from cDNA libraries and overexpression of WO 00/49147 PCT/EP00/01368 16 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 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, 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 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 cytosoi using receptor specific antibodies. In addition, US 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 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 17 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 5 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 10 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 al., Mol. Endocrinol. 5, 1755ff (1991); Eiiiston 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 20 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 25 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 30 the homodimerization of hPR after progesterone binding, it has been WO 00/49147 PCT/EP00/01368 18 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 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 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 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 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 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 chemical derivative, variant, chimera, hybrid, analog, or fusion.

PCT/E P00/01368 19 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, 5 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® io 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 15 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 20 oil 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 25 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., 30 Steril., Vol., 60, 26, 1993).

WO 00/49147 PCT/E P00/01368 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, 3. et al., Am. J. Obstet. Gynecol., Vol. 156, 66, 1987) nor carbohydrate metabolism (Mosnier-Pudar, H. et al., Arch. Mai. Coeur, Vol 84, 1111, 1991). Further, progesterone does not affect iiver 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 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 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 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 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 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 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 phenol; and the like. The preferred matrix contains B-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 be present in a concentration of 50 to 1000 mg, preferably 200 -300 mg dissolved in a 35 % or 40 % G-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 form, it may be provided topically.

The nucleic acid construct of embodiments (1) to (16) 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 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 cxi-antitrypsin (further promoters). 22 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, 13-5 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 10 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 15 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 20 system, Cre/loxP 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 25 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 so more than one transgene. 23 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 5 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 6 seminal inhibins 10 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 ai-antitrypsin, and genes encoding substances that function as drugs, e. g., genes encoding the 20 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 25 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 30 further contain additional elements. 24 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 the hormone at ambient temperature under stirring.

Embodiment(s) 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. Methods of transfection may include microinjection, CaP04 precipitation, eiectroporation, 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 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 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.

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 PCT/E P00/01368 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 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.

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 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 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 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 WO 00/49147 PCT/E P00/01368 26 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 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.

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.

Examples Example 1: Construction of Vectors Production of the vector pTGFGl: The vector pUC19 (MBI Fermentas) was digested with Xbal, treated with Klenow enzyme and religated. This Xbal deleted vector was then digested with EcoRI, treated with Klenow enzyme and religated in order to delete the EcoRI site. For insertion of a Xbal site in the SacI site of this vector it was digested with SacI, treated with T4-polymerase, dephosphorylated with alkaline phosphatase and iigated with the Xbal-linker CTCTAGAG (Biolabs #1032). Another Xbal-site was inserted by digesting the newly produced vector with Hindlll, treating it with Klenow, dephosphorylating it with alkaline phosphatase and ligating it with the Xbal-linker CTCTAGAG (Biolabs #1032). This vector was named PUC19/X. 27 In order to destroy the Xbal-site present in the vector phGFP-S65T (Clontech) this vector was digested with Xbal, 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 with Mlul, treating it with Klenow enzyme and digesting it with BamHI. This fragment was inserted into the multiple cloning site of the vector pUC19/X which was digested with Sail, treated with Klenow enzyme and digested with BamHI. The resulting vector was named pTGFGl (Figure 2).

Starting with this vector all the vectors described in Table 1 were obtained. At the restriction sites for PstI, Kpnl, Ehel, Eco0109 and/or Sapl 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 the GFP gene the cloning vectors without a transgene were obtained.

Production of the insert PREfds^: 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'; SEQ ID NO: 3) and PRE-AS (5'-GGG GTA CCA GAA CAT GAT GTT CTA GCT ACG AAG CTG ATA TCC CAG AAC ATG ATG TTC TAG CTA CGA AGC TGG TAC CCC-3'; SEQ ID NO: 4) were hybridized and phosphorylated by kinase reaction, resulting in the insert PRE(ds).

Production of the vector PTGFG5: The vector pTGFGl was digested with Eco0109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE(ds) insert, PCT/E P00/01368 28 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 pTGFGl was digested with Kpnl, treated with T4-polymerase 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-polymerase and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE(ds) insert, resulting in the vector pTGFGl 1. Subsequently, pTGFGll 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.

Production of the vector PTGFG33: In a similar manner PRE(ds) were inserted at the restriction sites for PstI, Kpnl, Ehel, Eco0109 and Sapl in vector pTGFGl giving rise to the plasmid pTGFG33 (Figure 5), which is a vector that carries the GFP gene and five PREs, one each in position A, B, C, D, E (Figure 2).

Production of the vectors PTGFG36. PTGFG53 and PTGFG64: The vector pUC19 (MBI Fermentas) was digested with Sail, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was ligated to the Notl-linker GCGGCCGC (Biolabs # 1045), resulting in the vector pUC19/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 Pstl-site of the vector pUC19/N which was digested with PstI, treated with T4-polymerase and 29 dephosphorylated with alkaline phosphatase. From the resulting vector pUC19/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 Notl. This fragment was ligated to the 4.3 kb fragment of the Hindlll and Notl 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 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 NO: 6) were 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 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 Plasmid Transgene PRE Plasmid T rans-gene PRE Plasmid Transgene PRE pTGFGO - ~ pTGFGl8 GFP BDE pTGFG34 FIX E DTGFGI GFP - pTGFGl9 GFP BCD pTGFG35 FIX A pTGFG2 FIX -- dTGFG20 GFP ABD DTGFG36 FIX D pTGFG3 GFP E pTGFG21 GFP CDE pTGFG37 FIX C pTGFG4 GFP A pTGFG22 GFP ACD pTGFG38 FIX B dTGFGS GFP D pTGFG23 GFP ABC DTGFG53 FIX ABD pTGFG6 GFP C pTGFG24 GFP ABE PTGFG64 FIX ABCDE pTGFG7 GFP B pTGFG25 GFP ACE pTGFG66 -- A pTGFG 8 GFP BC pTGFG26 GFP ADE pTGFG67 -- D pTGFG9 GFP BE pTGFG27 GFP BCE pTGFG68 -- C pTGFG10 GFP BD pTGFG28 GFP BCDE pTGFG69 -- B pTGFG11 GFP AB pTGFG29 GFP ACDE pTGFG82 -- ABD pTGFG13 GFP CD pTGFG30 GFP ABCE pTGFG95 -- ABCDE pTGFG14 GFP AC pTGFG31 GFP ABDE pTGFG 15 GFP DE pTGFG32 GFP ABCD pTGFG 16 GFP AD DTGFG33 GFP ABCDE For the DNA sequence of pTGFG 36, pTGFG 53, pTGFG 64, pTGFG 67, pTGFG 82 and pTGFG 95, see SEQ ID NOs: 1 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 io factor IX open reading frame resulting in a 1387 bp 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 polymerase (Boehringer Mannheim) in 50 ml reaction volume [10 is mM Tris HC1 pH 8.85, 25 mM KCI, 5 mM (NH4)2S04, 2 mM MgS04] with 30 incubation cycles at 96°C for 1 min, 60°C for 1 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 pUC19 and transformed into E. coli DH5-a. Positive clones were 31 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 : GGAATTCCGCAAAGGTTATGCAGCGCGTGAACATGATCATGGC (upstream; SEQ. ID NO: 7) CGCGGATCCATTAAGTGAGCTTTG11 I I I ICCTTAATCC (downstream; SEQ. ID NO: 8) Example 3: Expression and Quantification of the Marker Protein GFP Method 1: HeLa cells were transfected by electroporation with plasmids pTGFG5 or pTGFG20. Transfected cells were harvested and 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 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 coiorimetric reaction was measured photometrically (extinction). Generally, the more GFP was added the less antibody was left to bind 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 5.2 mg GFP/ml for pTGFG20 (3PREs) was found.

WO 00/49147 PCT/EP00/01368 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 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 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. Again the vector with just one PRE (pTGFG5) shows the highest expression.

Example 4: Human Factor IX Quantification by ELISA Assay 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 sandwich EUSA 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 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 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) 1. Cloning of the human progesterone 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.

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). 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); hPGR-3'-primer: GCA GAA TT cat tat aaa aac tea aga cct cat aat cct gac (SEQ ID NO: 10); hPGR-internai primer (Sal I) 1: ctc ctc ggg qtc oac cct gg (SEQ ID NO: 11); hPGR-internal primer (Sal I) 2: cca ggg teg acc ccg agg ag (SEQ ID NO: 12). 34 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 pi and buffer was used as 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.

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 (31) with 3' primer and internal primer 1. Reaction setup in 50 pi was : Pwo 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 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 pTGhPRl.

WO 00/49147 PCT/EPOO/01368 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 Bacuiovirus Expression System, Life Technologies). This 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 from the expressed protein. The N-terminal region of the expression cassettes is shown below.

MSYYHHHHHHDYDIPTTENLYFQ**GAMGIRNST-hPR-g-en 6xHis 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 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 DNA-fragment was digested by EcoRI and Mlu I and the cleavage product was exchanged against the corresponding fragment of the PCT/EP0O/O1368 36 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 5 by immobilized cobalt2+ affinity chromatography on a TALON® resin (Clontech). The procedure, following the method of Boonyaratanakornkit et al. Mol. Cell. Biol.18, 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 mM Tris-CI pH 8.0, 350 mM 15 NaCI, 10 mM imidazol, 5% glycerol and a cocktail of proteinase inhibitors (Complete™ EDTA-free, Roche Diagnostics, Penzberg, Germany). After a 10 min centrifugation at 10000 x g, supernatant originating from 108 cells was incubated for 1 h with 0,5 ml settled TALON® resin equilibrated in buffer A. TALON® was washed with 20 20 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, 25 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. 30 et al., Nature 227, 680-685 (1970) and subsequent staining with 37 coomassie® blue R250. 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 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 10s cells.

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 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 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).

Clotting activity was deterined with a partial thromboplastin time 38 assay using Cephalin (phosphatidyl ethanolamine) activation with a manual coagulation instrument (ML-2, Instrumentation Laboratories). For the study, 100 pi undiluted supernatant from transfected 293 T-cells, 100 pi deficiency plasma (Progen) and 100 |jl Cephalin 5 (Instrumentation Laboratories) were incubated for 5 minutes at 37°C. Coagulation was started by adding 100 pi CaCh. Sample coagulation time was compared to normal plasma.

Number of cells [/ml] Factor IX-concentration [ng/ml] Clotting time M 2,1 x 10s 36 45 8,7 x 105 79 io 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 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).

For the study, 5 pi mouse blood, 20 ijI deficiency plasma (Progen) ad 100 |jl physiological NaCI and 100 pi DaPPTin (Progen) were incubated for 2 minutes at 37°C. Coagulation was started by adding 100 pi CaCh.

To analyse the additive effect, human clotting factor IX 25 (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 PCT/E P00/01368 39 detected up to a limit concentration of 0,07 mil) hFIX/ml (= 31,5 ng/ml). 2. ELISA: The addition of human clotting factor IX to the mouse blood 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 [mlU/ml] hFIX added Extinction at 405 nm [-] 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 without antigen 0,096 .

Control: 1. Antibody without antigen 0,072 6.

Control: 2. Antibody without antigen 0,085 7.

Substrate (ABTS) - 0,072 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 mlU/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 mouse coagulation factor IX. 40 Example 8: Cloning and Activity Testing of the Human Progesterone Receptor (hPR) 2. Activity Testing: The human progesterone receptor encoded in plasmid pTGhPRl (s. Example 8.1 above) was tested for its phyiological activity. In a functional form and after activation with a progestin like R5020 the receptor should be able to induce the expression of luciferase from a Mouse Mammary Tumor Virus (MTV) promoter.

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 R5020 (NEN) in 6 well plates. Transfections were performed by the calcium phosphate method using 2 pg of a pSG-hPRl constructt and pMTV-luc (Hollenberg et al., 1985, Cell 55, p899-906) 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: R5020 was omitted (PR+MTV) and both plasmids alone were transfected with (PR+R5020, MTV+R5020) and without R5020 (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, that is human progesterone receptor, progestin R5020 and the MTV-driven luciferase gene (PR+MTV+R5020). The error bars give the standard deviation of a threefold experiment, the readout is relative light units (RLU). 41 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 5 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 io 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 und Therapieforschung der Technischen Universitat Munchen. is The animals are fed ad libitum with "Altrum Ratten und 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. 20 The test animals are supervised by specialized staff.

Mixture of substances: Group Hormone Hormone receptor Plasmid Aqua dest.

Route of administration 1. - 2. - 100 pi _ pg _ oral 3. - - - pg 100 pi oral 4. - - - pg 50 pi i.m. . - 100 pi 4.35 pg pg - oral 42 Plasmid and hPR: Theragene GmbH Hormone: Utrogest® by Dr. Kade/Besins Pharma GmbH, Rigistr. 2, D-12277 Berlin Aquadest.: Aqua ad injectabilia Delta-Pharma GmbH, 72793 Pfullingen Esophageal sound: Vein catheter, diam. 0.5 x 0.9 mm, Lot 7077 G2221, B. Braun Melsungen AG, Western Germany i.m. injection: Micro-Fine 12.7 mm, Becton Dickinson GmbH, io 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 pi of hormone and plasmid via the gastrointestinal tract orally with an esophageal sound, the third group was is daily administered a total of 100 pi of plasmid with aqua dest. orally with an esophageal sound, the fourth group was administered a total of 50 pi of plasmid with aqua dest. i.m. into the musculus quadriceps femoris, the fifth group was daily administered a total of 100 pi of hormone, hormone receptor and plasmid orally with an esophageal 20 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.

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 pi of blood) was immediately collected with an Eppendorf pipette. 30 Without further delay, the blood coagulation time in seconds was 43 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 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 days. This was followed by a day (day 8 of experiment) without any manipulation, and at day 9 of experiment, again 5 pi 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 syringes (Mikro-Fine 12.4 mm) filled with 50-75 pi of sodium citrate (3.1%), transferred into Eppendorf cuvettes, and about 100 pi of whole blood with citrate was reserved for PCR examination and stored in a cool environment. The remaining citrate blood was centrifuged for 10 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 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 administration series, the administration had to be interrupted at days 44 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.

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 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, 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 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 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 vertical lines show plus and minus one standard deviation from these PCT/EPO0/01368 <15 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 5 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 latter is significantly higher than that of the control (T = io -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: Group statistics ADMIN N mean value standard deviation standard error of the mean value DIF control 7 47.3857 58.9946 22.2978 Hormone, hormone receptor 7 114.7571 47.3300 17.8891 and plasmid orally 46 Test for independent random samples Levene test for T test for equal mean values equal variance F Signifi cance T df sig. (2-sided| mean difference standard error of difference 95% confidence interval al difference lower upper DIF variances are 0.026 0.874 -2.357 12 0.036 •67.3714 28.5869 -129.6570 ■5.0858 equal Variances are not equal ■2.357 11.461 0.037 ■67.3714 28.5869 ■129.9833 •4.7596 The human FIX was also detectable in the treated mice of the "hormone-hormone reception and plasmid orally group using an Elisa 5 as described in Example 4. -2001 ?00l 14:10 DOMPATENT VON KREISLER, KOELN NR. 6625 ' EP 00000136* PCT/EP00/01368 Octagene GmbH JH/ml May 25, 2001

Claims (45)

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 antlsense 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). EmPfansszeit 25■ Mai ■ 14:16SHEET 'DIM 14:1U UIWATENT VON KKiilSLER, KOELN NR. 6625 2001 EP 00000136E 2

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 a-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 fromT:l 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. 4 *;r i ■ .. ..AMENDED SHEET;Empfansszeit 25■Mai. 14:16;UU1 14:JU uuMmtHi vufi mibUK, iwuln m. 00^3 s * -2001 EP000001368 3

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 NO: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-teast 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. Empfansszei t 25.Mai. 14:1 (_AMENDED SHEET 50

24. A vector comprising the nucleic acid construct or 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 a cellular system, with the proviso that said cellular system does not constitute a living human being.

29. A method for delivering into a cellular system a nucleic acid encoding a transgene to be expressed in 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 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, with the proviso that said cellular system does not constitute a living human being.

30. The method of claim 29, wherein a nucleic acid encoding human factor VIII or factor IX is delivered into the cell! INTELLECTUAL PROPERTY OFFICE OF N.Z. 17 SEP 2003 RECEIVED 51

31. Use of the pharmaceutical composition of claim 18 for preparing a medicament for treating blood clotting disorders.

32. Use of the pharmaceutical composition of claim 19 for preparing a medicament for treating hemophilia B.

33. Use of the pharmaceutical composition of claim 20 for preparing a medicament for treating hemophilia A.

34. A use according to claim 1 substantially as herein described or exemplified.

35. A pharmaceutical composition according to claim 16 substantially as herein described or exemplified.

36. A nucleic acid construct according to claim 22 substantially as herein described or exemplified.

37. A vector according to claim 24 substantially as herein described or exemplified.

38. A transformed cell or transgenic organism according to claim 25 substantially as herein described or exemplified.

39. A composition according to claim 26 substantially as herein described exemplified.

40. A method according to claim 27 substantially as herein described or exemplified. 52

41. A method according to claim 28 substantially as herein described or exemplified.

42. A method according to claim 29 substantially as herein described or exemplified.

43. A use according to claim 31 substantially as herein described or exemplified.

44. A use according to claim 32 substantially as herein described or exemplified.

45. A use according to claim 33 substantially as herein described or exemplified.