AU780854B2 - Hormone-hormone receptor complexes and nucleic acid constructs and their use in gene therapy - Google Patents

Description

Hormone -Hormone Receptor Complexes and Nucleic Acid Constructs and Their Use in Gene Therapy Background of the Invention 1. Field of the Invention The invention relates to the use of a nucleic acid construct comprising at least one hormone responsive element and a transqene 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 t6 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, plasmlds, and compositions of matter of the invention 20 have applications in gene therapy, particularly in the treatment of human blood clotting dis6rders, such as hemophilia. They niay also be used to up- or down-regulate target genes and for the delivery of vaccines.

25 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 9* WO 00/49147 PCT/EP00/01368 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 o0 therapeutical strategies, retroviral vectors and adenoviral vectors are presently the most widely used vehicles (Salmons, B. and Gunzburg, W. Hum. Gene Ther., Vol. 4, 129, 1993; Kasahara, N. et al., Science, Vol. 266, 1373, 1994; Ali, 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, 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/EPOO/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 io 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, et al., Proc. Natl. Acad. Sci.

USA, Vol. 84, 7413, 1987; Behr, Bioconjugate Chemistry, Vol. 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. et al., Science, Vol. 247, 1990; Lin, et al., Circulation, Vol. 82, 2217, 1990; Schwartz, 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 4 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. JAMA, Vol.271, 1994; Hoeben, R. 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 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 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 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 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 WO 00/49147 PCT/EP00/01368 intracellular receptor, located in the cytoplasm or the nucleus (Evans, 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 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, Cell, Vol.

56, 335, 1989; O'Malley, 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 hormoneinduced gene, 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 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 liganddependent transcription factors characterized by a unique molecular structure. The centrally located highly conserved DNA-binding domain 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 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 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 present invention is directed at overcoming 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 resp6nsive 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 the use'of a nucleic acid construct comprising at least one 20 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); a preferred embodiment of above, wherein the agent S• further comprises a hormone-hormone receptor complex; a nucleic acid construct comprising at least one HRE and a transgene, wherein one of said at least one HREs is not functionally S* linked to the transgene; a vector comprising the nucleic acid construct of above; *9 9 log• l*l l *lo' WO 00/49147 PCT/EP00/01368 7 a transformed cell or transgenic organism comprising the nucleic acid construct as defined in above or the vector as defined in above; a composition of matter comprising a nucleic acid construct comprising at least one HRE and a transgene as defined in above and/or a vector as defined in above, said at least one HRE being coupled to a hormone-hormone receptor complex; a preferred embodiment of above, wherein the transgene is a gene encoding a blood clotting factor; 1o a preferred embodiment of above, wherein the blood clotting factor is factor IX; a preferred embodiment of above, wherein the blood clotting factor is factor VIII; a pharmaceutical composition comprising the nucleic acid construct as defined in above and/or the composition of matter as defined in to above; (11) a method for preparing the composition of matter as defined in 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 and or the composition of matter as defined in to 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 cells of the organism or the cells of the cellular system, which method comprises administering an agent as defined in above or composition of matter as defined in to 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 WO 00/49147 PCT/EP00/01368 8 and transport of the nucleic acid that is coupled to the hormonehormone 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 above to an organism or to a cellular system; a method of treating hemophilia B, comprising administering a therapeutically effective amount of the composition of matter as defined in above to an organism or to a cellular system; (16) method of treating hemophilia A, comprising administering io a therapeutically effective amount of the composition of matter as defined in 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 to (16) above the hormone responsive element is a steroid responsive element (SRE), most preferably a progesterone responsive element (PRE). In embodiments and 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/EPOO/01 368 9 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 io 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, 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 pTGFG1.

Figure 3 is a diagram of the vector Figure 4 is a diagram of the vector pTGFG2O.

Figure 5 is a diagram of the vector pTGFG33.

WO 00/49147 PCT/EPOO01368 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 o0 d) micrographs of HeLa cells transfected with pTGFG5 (a and b) and (c and 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 cobalt 2 affinity chromatography as described in Example 5. The final preparation (85pg protein) was separated on a denaturing 7,5% SDSpolyacrylamid 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.

WO 00/49147 PCT/EP00/01368

II

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: 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 io Figures 20 and 21 are taken from Genbank, accession number AF016381.

Detailed Description of the Invention 1. Definitions "Nucleic acid" means DNA, cDNA, mRNA, tRNA, rRNA. The nucleic acid may be linear or circular, double-stranded or singlestranded.

"Nucleic acid construct" refers to a composite of nucleic acid 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 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 3o 40 nucleotides in length, and more usually, about 13-20 nucleotides in WO 00/49147 PCT/EP00/01368 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.

WO 00/49147 PCTIEP00/01368 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 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, primary cell io 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 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 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 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.

WO 00/49147 PCT/EPOO/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 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.

Potential HREs for use in the present invention have been previously described. For example, GREs (Scheidereit, et al., Nature, Vol. 304, 749, 1983; von der Ahe, et al., Proc. Natl. Acad.

Sci. USA, Vol. 83, 2817, 1986), EREs or PREs (Chambon, et al., Rec. Prog. Horm. Res., Vol., 40, 1, 1984; Klock, 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, 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/EP00/01368 glucocorticoid receptors, retinoic acid receptors, androgen, calcitriol, thyroid hormone or progesterone receptors and orphan receptors.

Such receptors have been previously described. (Green, et al., Nature, Vol. 320, 134, 1986; Green, G. L.,et al., Science, Vol. 231, 1150, 1986; Arriza, J. et al., Science, Vol. 237, 268, 1987; Hollenberg, S. et al., Nature, Vol. 318, 635, 1985; Petkovitch, M., et al., Nature, Vol. 330, 444, 1987; Giguere, et al., Nature Vol.

330, 624, 1987; Tilley, et al., Proc. Natl. Acad. Sci. USA, Vol. 86, 327, 1989; Baker, A. et al., Proc. Natl. Acad. Sci. USA, Vol. io 3294, 1988; Weinberger, et al., Nature, Vol. 324, 641, 1986; Sap, et al., Nature, Vol. 324, 635, 1086; Misrahi, et al., Biochem.

Biophys. Res. Commun., Vol. 143, 740, 1987; Kastner, 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 1: M11050; retinoic acid receptor a: AF088888 (exon 1), AF088889 (exon AF088890 (exon AF088891 (exon 4), AF088892 (exon 5 and AF088893 (exon AF088894 (exon 8), AF088895 (exon 9 and complete cDNA); retinoic acid receptor y: M24857; androgen receptor: M27423 (exon M27424 (exon 2), M27425 (exon M27436 (exon M27427 (exon M27428 (exon M27429 (exon M27430 (exon thyroid hormone receptor a l M24748, thyroid hormone receptor a 2 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 PCRcloning 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, 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 cytosol 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 Sepharoseo 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 WO 00/49147 PCT/EP00/01368 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 The structure of the hPR polypeptide is depicted in Fig. 16. The hPR 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 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 frugiperda (Sf9) cells as biological fully active polypeptides (Christensen et al., 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 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 sites at the chromosomal DNA (PREs, Progesterone Responsive Elements) is known to be mediated by the DBD. The remaining Nterminal 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 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 io 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 and 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.

WO 00/49147 PCT/EPOO/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 include synthetic and natural hormones, preferably steroid hormones, such as estrogen, 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 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 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. et al., Fertil. Steril., Vol. 46, 402, 1986). Later on, the levels declined substantially, although they 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. et al., Fertil., Steril., Vol., 60, 26, 1993).

WO 00/49147 PCT/EPOO/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, J. et al., Am. J. Obstet. Gynecol., Vol. 156, 66, 1987) nor carbohydrate 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 HDLcholesterol 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, 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 and in the composition of matter of embodiment 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 and and the pharmaceutical composition of embodiment 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 WO 00/49147 PCT/EP00/01368 21 the hormone component thereof, may contain the following matrix compounds: glucose and related compounds (such as D-sorbitol, Dmannitol); solubilizing adjuvants (such as alcohols, 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 0o 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 B-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 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 ai-antitrypsin (further promoters).

WO 00/49147 PCT/EP00/01368 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, 6galactosidase 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 io insect promoters.

A preferred nucleic acid construct contains sequentially from the to the 3' end: a PRE, a CMV promoter, a gene of interest, Intron and SV40 poly A enhancer sequence, and an ampicillin resistant gene. Further PREs are evenly distributed on the vector backbone.

The nucleic acid construct may further contain origin of replication sequences (especially eukariotic origin of replication sequences), elements for gene targeting, integrational sequences AAV-ITR, transposon IS), 3'-UTR, "switch" systems TET 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 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 more than one transgene.

WO 00/49147 PCT/EPOO/01368 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: involved in hemophilia A and B, respectively, are good candidates 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 B seminal inhibins 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 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 xa-antitrypsin, and genes encoding substances that function as drugs, e. genes encoding the 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 of the present invention. These vectors may be used in the composition matter of embodiment 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 further contain additional elements.

WO 00/49147 PCT/EP00/01368 24 The composition of matter of embodiment 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.

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, CaPO 4 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 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 WO 00/49147 PCT/EPOO/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, 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.

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/EP00/01368 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 pTGFG1: The vector pUC19 (MBI Fermentas) 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 SacI, treated with T4-polymerase, dephosphorylated with alkaline 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 pUC19/X.

WO 00/49147 PCT/EPOO/01368 27 In order to destroy the XbaI-site present in the vector phGFP- (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 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 pTGFG1 (Figure 2).

Starting with this vector all the vectors described in Table 1 were obtained. At the restriction sites for PstI, KpnI, Ehel, EcoO109 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 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'; 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'; SEQID NO:4) were hybridized and phosphorylated by kinase reaction, resulting in the insert PRE(ds).

Production of the vector pTGFG5: The vector pTGFG1 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE(ds) insert, WO 00/49147 PCT/EP00/01368 28 resulting in the vector pTGFG5 (Figure a vector which carries a PRE at position C of Fig. 2.

Production of the vector pTGFG20: The vector pTGFG1 was digested with KpnI, 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 to the vector pTGFG11. Subsequently, pTGFG11 was digested with EcoO109I, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was then ligated with the PRE(ds) insert, resulting in the vector pTGFG20 (Figure 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, KpnI, Ehel, EcoO109 and SapI in vector pTGFG1 giving rise to the plasmid pTGFG33 (Figure 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 SalI, treated with Klenow enzyme and dephosphorylated with alkaline phosphatase. It was ligated to the NotI-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 was inserted into the PstI-site of the vector pUC19/N which was digested with PstI, treated with T4-polymerase and WO 00/49147 PCT/EP00/01368 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 NotI. This fragment was ligated to the 4.3 kb fragment of the HindIII and NotI double-digested vector 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 (SEQ. ID NO: 5) and ALLG2 (5'-GGC CGC TTG CTC GAG GTC (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 Trans- PRE Plasmid Trans- PRE Plasmid Trans- PRE gene gene gene pTGFGO pTGFG 18 GFP BDE pTGFG34 FIX E pTGFGI GFP pTGFG19 GFP BCD pTGFG35 FIX A pTGFG2 FIX DTGFG20 GFP ABD pTGFG36 FIX D pTGFG3 GFP E pTGFG21 GFP CDE pTGFG37 FIX C WO 00/49147 PCT/EP00/01368 pTGFG4 GFP A pTGFG22 GFP ACD pTGFG38 FIX B GFP D pTGFG23 GFP ABC pTGFG53 FIX ABD pTGFG6 GFP C pTGFG24 GFP ABE pTGFG64 FIX ABCDE pTGFG7 GFP B pTGFG25 GFP ACE pTGFG66 A pTGFG8 GFP BC pTGFG26 GFP ADE pTGFG67 D pTGFG9 GFP BE pTGFG27 GFP BCE pTGFG68 C pTGFGIO GFP BD pTGFG28 GFP BCDE pTGFG69 B pTGFGII GFP AB pTGFG29 GFP ACDE pTGFG82 ABD pTGFG13 GFP CD pTGFG30 GFP ABCE pTGFG95 ABCDE pTGFG 14 GFP AC pTGFG3 GFP ABDE GFP DE pTGFG32 GFP ABCD pTGFG6 GFP AD pTGFG33 GFP ABCDE For the DNA sequence of pTGFG 36, pTGFG pTGFG 82 and pTGFG 95, see SEQ ID respectively.

53, pTGFG 64, pTGFG 67, NOs: 1 and 13 to 17, 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 mM Tris HCI pH 8.85, 25 mM KCI, 5 mM (NH 4 2 S0 4 2 mM MgSO 4 with 30 incubation cycles at 96 0 C for 1 min, 60 0 C for 1 min, 72 0 C for 2 min, followed by a final extension step at 72 0 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 WO 00/49147 PCT/EP00/01368 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) CGCGGATCCATTAAGTGAGCTTTG 1TI 1 T CCTTAATCC (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 colorimetric 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 3o 5.2 mg GFP/ml for pTGFG20 (3PREs) was found.

WO 00/49147 PCT/EPOO/01368 32 Figures 12 a-d show micrographs of HeLa cell cultures transfected with pTGFG5 (Fig. 12 a and b) and pTGFG20 (Fig. 12 c and 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 io 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 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 WO 00/49147 PCT/EP00/01368 33 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 3ng/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 bases added to introduce restriction sites are in capital letters and restriction sites used for cloning are underlined.

CGA GGA tcc agt cgt cat gac tga gc (SEQ ID NO: 9); hPGR-3'-primer: GCA GAA TT cat tat aaa aac tca aga cct cat aat cct gac (SEQ ID NO: hPGR-internal primer (Sal I) 1: ctc ctc ggg qtc gac cct gg (SEQ ID NO: 11); hPGR-internal primer (Sal I) 2: cca ggq tcq acc ccg agg ag (SEQ ID NO: 12).

WO 00/49147 PCT/EP00/01368 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 pl 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 0 C for 10 min, followed by 10 min at 72 0 C and 10 min at 42 0

C.

SuperScript II RT was added at 42 0 C and reaction was continued for min at 42 0 C, 15 min at 50 0 C and 1 h at 58 0

C.

The cDNA obtained from this synthesis reaction was used to amplify the hPGR coding sequence in two fragments by PCR. One fragment with 5'-primer and internal primer 2 and one fragment with 3' primer and internal primer 1. Reaction setup in 50 pl 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 0 C followed by cycles of 1 min 96 0 C, 2 min at 59 0 C, 2 min 72 0 C and a final extension step at 72 0 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 pTGhPR1.

WO 00/49147 PCT/EP00/01368 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 (BACto-BAC Baculovirus 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 io from the expressed protein. The N-terminal region of the expression cassettes is shown below.

MSYYHHHHHHDYDIPTTENLYFQ**GAMGIRNST-hPR-gen 6x His 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 WO 00/49147 PCT/EP00/01368 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 by immobilized cobalt 2 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 0

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 NaCI, 10 mM imidazol, 5% glycerol and a cocktail of proteinase inhibitors (Complete TM 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 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, 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.

et al., Nature 227, 680-685 (1970) and subsequent staining with WO 00/49147 PCT/EP00/01368 37 coomassiee 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 108 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 WO 00/49147 PCT/EPOO/01368 38 assay using Cephalin (phosphatidyl ethanolamine) activation with a manual coagulation instrument (ML-2, Instrumentation Laboratories).

For the study, 100 ul undiluted supernatant from transfected 293 Tcells, 100 pl deficiency plasma (Progen) and 100 pl Cephalin (Instrumentation Laboratories) were incubated for 5 minutes at 37 0

C.

Coagulation was started by adding 100 pl CaCI 2 Sample coagulation time was compared to normal plasma.

Number of cells Factor IX-concentration Clotting time [ng/ml] [s] 2,1 x 10 s 36 8,7 x 10 20 79 to 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 pl mouse blood, 20 pl deficiency plasma (Progen) ad 100 pl physiological NaCI and 100 pl DaPPTin (Progen) were incubated for 2 minutes at 37 0 C. Coagulation was started by adding 100 pl CaCl 2 To analyse the additive effect, human clotting factor IX (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 WO 00/49147 PCT/EP00/01368 39 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 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 added nm 1. Mouse Citrate Plasma 7 0,204 2. Mouse Citrate Plasma 2 0,130 3. Mouse Citrate Plasma 0,099 4. Control: Antibody 0,096 without antigen Control: 1. Antibody -0,072 without antigen 6. Control: 2. Antibody -0,085 without antigen 7. Substrate (ABTS) 0,072 0o 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 mouse coagulation factor IX.

WO 00/49147 PCT/EP00/01368 Example 8: Cloning and Activity Testing of the Human Progesterone Receptor (hPR) 2. Activity Testing: The human progesterone receptor encoded in plasmid pTGhPR1 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.

io 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 pSGhPR1 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 (pCMVluc).

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 MTVdriven luciferase gene (PR+MTV+R5020). The error bars give the standard deviation of a threefold experiment, the readout is relative light units (RLU).

WO 00/49147 PCT/EP00/01368 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 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 fOr Experimentelle Onkologie und Therapieforschung der Technischen Universitat M0nchen.

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 0 C and a humidity of 55 The room is additionally provided with an automatic light supply which maintains a 12 hours rhythm.

The test animals are supervised by specialized staff.

Mixture of substances: Group Hormone Hormone Plasmid Aqua dest. Route of receptor administration 2. 100 pl 10 g oral 3. 10 pg 100 pl oral -10 pg 50 pl i.m.

100 pl 4.35 pg 10 pg oral Plasmid and hPR: pTGFG 64 (shown in Figure 8; production see Example 1) Theragene GmbH Hormone: Utrogest® by Dr. Kade/Besins Pharma GmbH, Rigistr. 2, D-12277 Berlin Aqua dest.: 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, 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 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 pl 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 25 esophageal 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 S 30 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. Without further delay, the blood coagulation time in seconds was WO 00/49147 PCT/EPOO/01368 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 io 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, 0.75 ml of whole blood was collected intracardially using U-40 insulin syringes (Mikro-Fine 12.4 mm) filled with 50-75 pl of sodium citrate 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 0 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 0

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 WO 00/49147 PCT/EP00/01368 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 io a subcutaneous administration of 1 ml of 5% glucose solution (Delta Pharma GmbH, 72793 Pfuilingen) 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 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 WO 00/49147 PCT/EP00/01368 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 s water (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 0io -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 standard error of deviation 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 Test for independent random samples Levene test for T test for equal mean values equal variance F Signifi T df sig. 12-sided) mean standard error 95% confidence interval cance difference of difference of 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 Vaianes -2.357 11.461 0.037 -67.3714 28.5869 -129.9833 -4.7596 are not equal Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

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EDITORIAL NOTE APPLICATION NUMBER 28061/00 The following Sequence Listing pages 1-20 are part of the description. The claims pages follow on pages 47-54 WO 00/49147 WO 0049147PCT/EPOO/01368 SEQUENCE LISTING <110> Theragene Biomedical Laboratories GmnbH <120> Hormone-Hormone Receptor Complexes and Nucleic Acid Constructs and Their Use in Gene Therapy <130> 000065wo/JH/ml <140> <141> <160> <170> <210> <211> <212> <213> <220> <223> 19 Patentln Ver. 2.1 1 5753

DNA

Artificial Sequence Description of Artificial Sequence: vector pTGFG36 <220> <221> CDS <222> (2071) <400> 1 cgcgttgaca atagcccata cqcccaacga tagggacttt tacatcaagt ccgcctggca acgtattagt gatagcggtt tgttttggca cgcaaatggg ctagagaacc aagcttgcat.

ttgattattq tatggagttc cccccgccca ccattgacgt gtatcatatg ttatgcccag catcqctatt tgactcacgg ccaaaatcaa cggtaggcgt cactgcttac gccaattccg actagttatt aatagtaatc aattacggqg tcattagttc cgcqttacat ttgacgtcaa caatgggtgg ccaagtacgc tacatgacct accatqgtga ggatttccaa cgggactttc gtacggtqggg aacttacggt taatqacgta agtatttacg cccctattga tatgggactt tgcggttttg gtctccaccc caaaatgtcg aggtctatat aaatggcccg tgttcccata gtaaactgcc cgtcaatgac tcctacttgg gcagtacatc cat tga cgt c taacaactcc aagcagagct cctggctqac qtaacgccaa cacttggcag ggtaaatgqc cagtacatct aatgggcgtg a at ggga gt t gccccattga ctctggctaa aggqagaccc atc atg Ile Met 120 180 240 300 360 420 480 540 600 660 712 760 tggcttatcg aaattaatac qactcactat.

caaaggtt atg cag cgc gtg aac atg Met Gin Arg Val Asn Met gca gaa tca cca ggc ctc atc acc atc tgc ctt tta gga tat cta ctc Ala Giu Ser Pro Gly Leu Ile Thr Ile Cys Leu Leu Giy Tyr Leu Leu 15 WO 00/49147 WO 0049147PCT/EPOO/01368 agt gct Ser Ala ctg aat Leu Asn caa ggg Gin Gly gaa gca Glu Ala aag cag Lys Gin ggC ggC Gly Gly 105 ttt gga Phe Gly aag aat Lys Asn gtg gtt Val Val tcc tgt Ser Cys 170 caa act Gin Thr 185 tat gta Tyr Val agc acc Ser Thr gcc aaa Ala Lys gat gca.

Asp Ala 250 gaa Giu cgg Arg aac As n cga Arg 75 tat Tyr ag t Ser ttt Phe ggc Gly tgC Cys 155 gaa Glu tct Ser aat Asn caa Gin cca Pro 235 ttc Phe tgt aca Cys Thr cca aag Pro Lys ctt gag Leu Giu gaa gtt Giu Val gtt gat Val Asp tgc aag Cys Lys gaa gga Giu Gly 125 aga tgc Arg Cys 140 tcc tgt Ser Cys cca gca Pro Ala aag ctc Lys Leu tct act Ser Thr 205 tca ttt Ser Phe 220 ggt caa Gly Gin tgt gga Cys Gly gtt Val1 30 agq Arg aga Arg ttt Phe gga Ci y g atr Asp 110 aag Lys gag Giu act Thr gtg Val1 acc Thr 190 ga a Giu aat Asn ttc Phe ggc Gly ttt Phe tat Tyr gaa Giu gaa Giu gat Asp 95 gac Asp aac Asn cag Gin gag Giu cca Pro 175 cgt Arg gct Ala gac Asp cct Pro tct Ser 255 ctt Leu aat Asn tgt Cys aac Asn 80 cag Gin at t Ile tgt Cys ttt Phe gga Giy 160 ttt Phe gct Ala gaa Giu ttc Phe tgg Trp 240 at c Ile gat cat gaa Asp His Giu tca ggt aaa Ser Gly Lys 50 atg gaa gaa Met Giu Giu 65 act gaa aga Thr Giu Arg tgt gag tcc Cys Giu Ser aat tcc tat Asn Ser Tyr 115 gaa tta gat Giu Leu Asp 130 tgt aaa aat Cys Lys Asn 145 tat cga ctt Tyr Arg Leu cca. tgt gga Pro Cys Gly gag act gtt Glu Thr Val 195 acc att ttg Thr Ile Leu 210 act cgg gtt Thr Arg Val 225 cag gtt gtt Gin Val Val gtt aat gaa Val Asn Giu aac gcc aac aaa att Asn Ala Asn Lys Ile ttg Leu aag Lys aca Thr aat Asn 100 gaa Giu gta.

Val1 agt Ser gca Al a aga Arg 180 ttt Phe gat Asp gtt Val1 ttg Leu aaa Lys 260 qaa Giu tgt Cys act Thr cca Pro tgt Cys aca Thr gct Ala gaa Giu 165 gtt Val1 cct Pro aac As n ggt Giy aat Asn 245 tgg T rp gag Giu agt Ser gaa Giu tgt Cys tgg Trp tgt Cys gat Asp 150 aac As n tct Ser gat Asp atc Ile gga Ci y 230 ggt Gly att Ile ttt Ph a ttt Phe ttt Phe tta Le u tgt Cys aac Asn 135 aac Asn cag Gin gtt Val1 gtg Val1 act Thr 215 gaa Giu aaa Lys gta Val1 gt t Val1 gaa Giu tgg T rp aat Asn ccc Pro 120 att Ile aag Lys aag Lys t ca Ser gac Asp 200 caa Gin gat Asp gtt Vai act Thr 856 904 952 1000 1048 1096 1144 1192 1240 1288 1336 1384 1432 1480 WO 00/49147 WO 0049147PCT/EPOO/01368 gct Ala 265 gcc cac tgt gtt Ala His Cys Val cat aat att gag His Asn Ile Glu 285 gaa Giu 270 act ggt gtt aaa Thr Gly Val Lys att Ile 275 aca gtt gtc gca Thr Val Val Ala ggt Gly 280 gaa Giu gag aca gaa cat Giu Thr Glu His gag caa aag cga Giu Gin Lys Arg aat gtg Asn Val 295 att cga att Ile Arg Ile att Ile 300 cct cac cac aac Pro His His Asn tac Tyr 305 aat gca gct att Asn Ala Ala Ile aat aag tac Asn Lys Tyr 310 gtg cta aac Val Leu Asn aac cat qac Asn His Asp 315 att gcc ctt ctg Ile Ala Leu Leu gaa Glu 320 ctg gac gaa ccc Leu Asp Giu Pro agc tac Ser Tyr 330 gtt aca cct att Vai Thr Pro Ile t gc Gys 335 att gct gac aag Ile Ala Asp Lys gaa Giu 340 tac acg aac atc Tyr Thr Asn Ile Phe 345 ctc aaa ttt gga Leu Lys Phe Gly ggc tat gta agt Gly Tyr Vai Ser tgg gga aga gtc Trp Gly Arg Val ttc Phe 360 cac aaa ggg aga His Lys Gly Arg tca Ser 365 gct tta gtt ctt Ala Leu Val Leu tac ctt aga gtt Tyr Leu Arg Val cca ctt Pro Leu 375 1528 1576 1624 1672 1720 1768 1816 1864 1912 1960 2008 2056 2111 gtt qac cga Val Asp Arg aac atg ttc Asn Met Phe 395 gcc Ala 380 aca tgt ctt cga Thr Cys Leu Arg tct Ser 385 aca aag ttc acc Thr Lys Phe Thr atc tat aac Ile Tyr Asn 390 tca tqt caa Ser Cys Gin tgt gct ggc ttc Cys Ala Gly Phe cat His 400 gaa gga ggt aga Glu Gly Gly Arg ga t Asp 405 gga gat Gly Asp 410 agt gqg gga ccc Ser Gly Gly Pro gtt act gaa gtg Val Thr Giu Val ggg acc agt ttc Gly Thr Ser Phe tta Leu 425 act gga att att Thr Gly Ile Ile agc Ser 430 tgg ggt gaa gag Trp Gly Glu Glu tgt Cys 435 gca atg aaa ggc Ala Met Lys Gly aaa Lys 440 tat gga ata tat Tyr Gly Ile Tyr acc Thr 445 aag gta tcc cgg Lys Val Ser Arg tat Tyr 450 gtc aac tgg att Val Asn Trp Ile aag gaa Lys Giu 455 aaa aca aag Lys Thr Lys ctc act Leu Thr 460 taatgggatc ggtcgagcgg ccgcgactct actagaggat ctttgtgaag gaaccttact tctgtggtgt gacataattg gacaaactac ctacagagat 2171 ttaaagctct aaggtaaata taaaattttt aagtgtataa tgtgttaaac tactgattct 2231 aattgtttgt gtattttaga ttccaaccta tgqaactqat gaatgggagc agtggtggaa 2291 tgcctttaat gaggaaaacc tgttttgctc agaagaaatg ccatctagtg atgatgaggc 2351 tactgctgac tctcaacatt ctactcctcc aaaaaagaag agaaaggtag aagaccccaa 2411 WO 00/49147 WO 0049147PCT/EPOO/01368 qgactttcct tcagaattgc taagtttttt tqcttqcttt qgaaaaatat ttttcttact tacctttagc I0 gactagagat tcccacacct ttattgcagc catttttttc tctggatccc gtgactggga ccagctggcq tgaatggcga accgcatatg gacacccgcc acagacaagc cgaaacgcgc atatcagctt tataggttaa atgtgcgcgg tgagacaata aacatttccg acccagaaac acatcgaact ttccaatgat ccgggcaaga caccagtcac ccataaccat aggagctaac aaccggagct tggcaacaac gctatttaca tctgtaacct ccacacaggc tttttaattt cataatcagc ccccctgaac ttataatqgt actgcattct cqggt accct aaaccctggc taatagcgaa atggcgcctg gtgcactctc aacacccgct tgtgaccgtc gagacgaaag cgtagctaga tgtcatgata aacccctatt accctgataa tgtcgccctt gctggtgaaa ggatctcaac gagcactttt gcaactcggt agaaaagcat gagtgataac cgcttttttg qaatgaagcc qttgcgcaaa ccacaaagga ttataagtag atagagtgtc gtaaaggggt cataccacat ctgaaacata tacaaataaa agttgtggtt ctagagcgaa gttacccaac gaggcccgca atgcggtatt agtacaatct gacgcgccct tccggqagct ggggggtacc acatcatqtt ataatggttt tgtttatttt atgcttcaat attccctttt gtaaaagatg agcggtaaga aaagttctgc cgccgcatac cttacggatg actgcggcca cacaacatgg ataccaaacg ctattaactg gagtcatgct aaaagctqca gcataacagt tgctattaat taataaggaa ttgtagaggt aaatgaatgc gcaatagcat tgtccaaact ttaattcact ttaatcgcct ccqatcgccc ttctccttac gctctgatgc gacgggcttg gcatgtgtca agcttcgtag ctggtacccc cttagacgtc tctaaataca aatattgaaa ttgcggcatt ctgaagatca tccttgagag tatgtggcgc actattctca gcatgacagt acttacttct gggatcatgt acqagcqtga gcqaactact qtqtttagta ctgctataca tataatcata aactatgctc tatttgatgt tttacttgct aattgttgtt cacaaatttc catcaatgta ggccgtcgtt tgcagcacat ttcccaacag gcatctgtgc cgcatagtta tctgctcccg gaggttttca ctagaacatc cctcgtgata aggtggcact ttcaaatatg aaggaagagt ttgccttcct gttgggtgca ttttcgcccc ggtattatcc gaatgacttq aagagaatta gacaacgatc aactcgcctt caccacgatg tactctagct atagaactct 2471 agaaaattat 2531 acatactgtt 2591 aaaaattgtg 2651 atagtgcctt 2711 ttaaaaaacc 2771 gttaacttgt 2831 acaaataaag 2891 tcttatcatg 2951 ttacaacgtc 3011 ccccctttcg 3071 ttgcgcagcc 3131 ggtatttcac 3191 agccagcccc 3251 gcatccgctt 3311 ccgtcatcac 3371 atgttctggg 3431 cgcctatttt 3491 tttcggggaa 3551 tatccgctca 3611 atgagtattc 3671 gtttttgctc 3731 cgagtgggtt 3791 gaagaacgtt 3851 cgtattqacg 3911 gttgaqtact 3971 tgcagtgctg 4031 ggaggaccga 4091 gatcgttggg 4151 cctgtagcaa 4211 tcccggcaac 4271 WO 00/49147 WO 0049147PCT/EPOO/01368 aattaataga cggctggctg ttgcagcact gtcaqgcaac aqca t tggt a atttttaatt cttaacqtqa cttqagatcc cagcggtgqt t cagcagaqc tcaagaactc ctgccagtgq aggcgcagcg cctacaccqa ggagaaagqc agcttccagg ttgagcgtcg acgcggcctt cgttatcccc qccgcagccg tacgcaaacc ttcccgactg aggcacccca gataacaatt tagagctcta ctggatggag gtttattgct ggggccagat tatggatgaa actqtcagac taaaaggatc gttttcgttc tttttttctg ttgtttgccq gcagatacca tgtagcaccg cgataagtcg gtcgggctga actgagatac ggacaggtat gggaaacgcc atttttgtga tttacggttc tgattctgtg aacgaccgag gcctctcccc gaaagcgggc ggctttacac tcacacagga gagctctaga gcggataaaq qataaatctq ggtaagccct cgaaatagac caagtttact tagqtgaaga cactgagcgt cgcgtaatct gatcaagagc aatactgttc cctacatacc tgtcttaccg acggggggtt ctacagcgtq ccggtaagcg tggtatcttt tgctcgtcag ctggcctttt qataaccgta cgcagcgagt gcgcgttggc agtgaqcgca tttatgcttc aacagctatg gagcttgcat ttgcaggacc acttctgcgc tcggcccttc 4331 gagccggtga cccgtatcgt agatcgctga catatatact tcctttttga cagaccccgt gctgcttgca taccaactct ttctagtgta tcgctctgct ggttggactc cqtgcacaca agctatgaga gcagggtcqg ataqtcctgt ggggqcggag gctggccttt ttaccqcctt cagtgagcga cgattcatta acgcaattaa cggctcgtat accatgatta gcctgcaggt gcgtgggtct agttatctac qataggtgcc ttagattgat taatctcatg agaaaagatc aacaaaaaaa ttttccgaag gccgtagtta aatcctgtta aagacqataq gcccagcttg aagcgccacg aacaggagag cgggtttcqc cctatggaaa tgctcacatg tgagtgagct ggaagcggaa atgcagctgg tgtgagttag gttgtgtgga cgccaagctc cg cgcggtatca 4391 acgacgggga 4451 tcactgatta 4511 ttaaaacttc 4571 accaaaatcc 4631 aaaggatctt 4691 ccaccgctac 4751 gtaactggct 4811 ggccaccact 4871 ccagtggctg 4931 ttaccggata 4991 gagcgaacga 5051 cttcccgaaq 5111 cgcacgaggg 5171 cacctctgac 5231 aacgccagca 5291 ttctttcctq 5351 gataccgctc 5411 gagcgcccaa 5471 cacgacaqgt 5531 ctcactcatt 5591 attgtgagcq 5651 tctagagctc 5711 5753 <210> 2 <211> 461 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: vector pTGFG36 <400> 2 Met Gin Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr 1 5 10 WO 00/49147 WO 0049147PCTIEPOO/0 1368 6 Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Giu Cys Thr Val Phe Leu 25 Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 Ser Gly Lys Leu Giu Giu Phe Val Gin Gly Asn Leu Giu Arg Giu Cys 55 Met Giu Giu Lys Cys Ser Phe Giu Giu Ala Arq Giu Val Phe Giu Asn 70 75 Thr Giu Arq Thr Thr Giu Phe Trp Lys Gin Tyr Vai Asp Giy Asp Gin 90 Cys Giu Ser Asn Pro Cys Leu Asn Gly Giy Ser Gys Lys Asp Asp Ile 100 105 .110 Asn Ser Tyr Giu Cys Trp Cys Pro Phe Gly Phe Giu Gly Lys Asn Cys 115 120 125 Giu Leu Asp Vai Thr Cys Asn Ile Lys Asn Gly Arg Cys Giu Gin Phe 130 135 140 Cys Lys Asn Ser Ala Asp Asn Lys Val Vai Cys Ser Cys Thr Giu Gly 145 150 155 160 Tyr Arg Leu Ala Glu Asn Gin Lys Ser Cys Giu Pro Ala Val Pro Phe 165 170 175 Pro Cys Gly Arq Val Ser Val Ser Gin Thr Ser Lys Leu Thr Arg Ala 180 185 190 Giu Thr Val Phe Pro Asp Val Asp Tyr Vai Asn Ser Thr Glu Ala Giu 195 200 205 Thr Ile Leu Asp Asn Ile Thr Gin Ser Thr Gin Ser Phe Asn Asp Phe 210 215 220 Thr Arg Val Vai Gly Gly Giu Asp Ala Lys Pro Gly Gin Phe Pro Trp 225 230 235 240 Gin Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Giy Giy Ser Ile 245 250 255 Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Vai Giu Thr Gly 260 265 270 Val Lys Ile Thr Vai Val Ala Gly Giu His Asn Ile Giu Giu Thr Giu 275 280 285 His Thr Giu Gin Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn 290 295 300 Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Giu 305 310 315 320 Leu Asp Giu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile 325 330 335 Ala Asp Lys Giu Tyr Thr Asn Ile Phe Leu Lys Phe Giy Ser Gly Tyr 340 345 350 WO 00/49147 WO 0049147PCT/EPOO/01368 Val Ser Gly Trp Gi 355 Leu Gin Tyr Leu Ar 370 Ser Thr Lys Phe Th 385 Giu Gly Gly Arg As Thr Glu Val Giu GI 420 Giu Glu Cys Ala Me 435 Arg Tyr Vai Asn Tr 450 <210> 3 <211> 78 <212> DNA <213> Homo sapiens <400> 3 ggggtaccag cttcgta atcatgttct ggtaccc <210> 4 <211> 78 <212> DNA <213> Homo sapiens <400> 4 ggggtaccag aacatgal ctacgaagct ggtaccc <210> <211> 19 <212> DNA <213> Homo sapiens <400> agcttgacct cgagcaac <210> 6 <211> 19 <212> DNA <213> Homo sapiens <400> 6 ggccgcttgc tcgaggtc

Y,

g r p 5 y t p Arg Val Ile 390 Ser Thr Lys Ile Val1 Pro 375 Tyr Cys Ser Gly Lys 4 55- Phe 360 Leu As n Gin Phe Lys 440 Giu His Val1 Asn Gly Leu 425 Tyr Lys Lys Asp Met Asp 410 Thr Gly Thr Gly Arg Phe 395 Ser Gi y Ile Lys Arg Al a 380 Gys Gly Ile Tyr Leu 4 Ser 365 Thr Al a Gi y Ile Thr 445 Thr Ala Cys Gi y Pro Ser 430 Lys Leu Leu Phe His 415 Trp Val Val1 Arg His 400 Vai Gly Ser gct agaacatcat. gttctqggat atcagcttcg tagctagaac c 78 tgt tctagctacg aagctgatat cccagaacat gatgttctag c 78 jc :a WO 00/49147 WO 0049147PCT/EPOO/01368 <210> 7 <211> 43 <212> DNA <213> Homo sapiens <400> 7 ggaattccqc aaagqttatq 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 qtcgtcatqa ctqagc 26 <210> <211> 41 <212> DNA <213> Homo sapiens <400> gcaqaattca ttataaaaac tcaagacctc ataatcctga c 41 <210> 11 <211> <212> DNA <213> Homo sapiens <400> 11 ctcctcgggg tcqaccctgg <210> 12 <211> <212> DNA <213> Homo sapiens <400> 12 ccagggtcga ccccgaqgag <210> 13 <211> 5905 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: vector pTGFG53 WO 00/49147 WO 0049147PCTEPOOO 1368 <400> 13 cgcgt tgaca atagcccata cgcccaacga taggqacttt tacatcaagt ccgcctggca acgtattagt gatagcggtt tgttttgqca cqcaaatggg ctagagaacc aagcttgcat accaggcctc tcttgatcat at tggaaqag tqaaqaaqca tgttqatgga cattaattcc tgtaacatgt caaqqtggtt accagcaqtg tgctgagact ggataacatc agatgccaaa ctgtggaggc tggtgttaaa gcaaaagcga gtacaaccat tacacctatt ctatgtaagt ccttagagtt taacaacatg tgggggaccc gqgtqaagag caactggatt tactagagga cctacagaga ctactgattc cagtggtgga gatgatgagg gaagacccca aatagaactc aagaaaatta aacatactgt caaaaattqt tatagtgcct tttaaaaaac tgttaacttg cacaaataaa atcttatcat ggatatcagc ttcactggcc tcgccttgca tcgcccttcc ccttacgcat tgatgccgca ggcttgtctg gtgtcagagg acatgatgtt gt accccggc ttagacgtca ttgattattg tatggagttc cccccgccca ccattgacgt gtatcatatg ttatgcccag catcgctatt tqactcacgg ccaaaatcaa cggtaggcgt cactgcttac gccaattccq atcaccatct gaaaacgcca tttgttcaag cgagaagttt gatcagtgtg tatgaatgtt aacattaaga tgctcctgta ccatttccat gtttttcctg actcaaagca ccaggt caat tctatcgtta attacagttg aatgtgattc gacattgccc tgcattgctg ggctggggaa ccacttgttg ttctgtgctg catgttactg tgtgcaatga aaggaaaaaa tctttgtgaa tttaaagctc taattgtttg atgcctttaa ctactgctga aggactttcc ttgcttgctt tggaaaaata tttttcttac gtacctttag tgactagaga ct cccacacc tttattgcag gcattttttt gtctggatcc ttcgtagcta qtcgttttac gcacatcccc caacagttgc ctgtgcggta tagttaagcc ctcccggcat ttttcaccgt ctagctacga ctcgtgatac ggtggcactt actagttatt cgcgttacat ttgacgtcaa caatgggtgg ccaagtacgc tacatgacct accatggtga ggatttccaa cgggactttc gtacqgtggg tggcttatcg caaaggttat gccttttagg acaaaattct ggaaccttqa ttgaaaacac agtccaatcc ggtgtccctt atggcagatg ctgagggata gtggaagagt atgtggacta cccaatcatt tcccttggca atgaaaaatg tcgcaggtga gaattattcc ttctggaact acaaggaat a gagtcttcca accgagccac gcttccatga aagtggaagg aaggcaaata caaagctcac ggaaccttac taaggtaaat tgtattttag tgaggaaaac ctctcaacat ttcagaattg tgctatttac ttctgtaacc tccacacagg ctttttaatt tcataatcag tccccctgaa cttataatgg cactgcattc ccggggggt a gaacatcatq aacgtcgtga ctttcgccag gcagcctgaa tttcacaccg agccccgaca ccgcttacag catcaccgaa agctgatatc gcctattttt ttcggggaaa aataqtaatc aacttacggt taatgacgta agtatttacg cccctattga tatgggactt tgcggttttg gtctccaccc caaaatgtcg aggtctatat aaattaatac gcagcgcgtg atatctactc gaatcqgcca gagagaatgt tgaaagaaca atgtttaaat tggatttgaa cgagcagtt t tcgacttgca ttctgtttca tgtaaattct taatgacttc ggttgttttg gattgtaact acataatatt tcaccacaac ggacgaaccc cacgaacatc caaaqggaga atgtcttcga aggagqtaga gaccagtttc tggaatatat ttaatgggat ttctgtggtg ataaaatttt attccaacct ctgttttgct tctactcctc ctaagttttt accacaaagg tttataagta catagagtgt tgtaaagggg ccataccaca cctgaaacat ttacaaataa tagttgtggt ccagcttcgt ttctggtacc ctgggaaaac ctggcgtaat tggcqaatgg catatggtgc cccgccaaca acaagctgtg acgcgcgaqa ccagaacatg ataggttaat tgtgcgcgga aattacgggg aaatggcccg tgttcccata gtaaactgcc cgtcaatgac tcctacttgg gcagtacatc cattgacqtc taacaactcc aagcagagct gactcactat aacatqatca agt gctgaat aaqaggtata atggaagaaa actgaatttt ggcgqcagtt ggaaagaact tqtaaaaata gaaaaccaga caaacttcta actqaagctg actcqggttg aatggtaaag gctgcccact qaggagacag tacaatgcag ttagtgctaa ttcctcaaat tcagctttag tctacaaagt gattcatgtc ttaactggaa accaaggtat cggtcqagcg tgacataatt taagtgtata atggaactga cagaagaaat caaaaaagaa tgagtcatgc aaaaagctgc ggcataacag ctgctattaa ttaataagga tttgtagagg aaaatgaatg agcaatagca ttgtccaaac agctagaaca cccgctctag cctggcgtta agcgaagagq cgcctgatgc actctcagta cccgctgacg accgtctccg cgaaagggcq atgttctagc gtcatgataa acccctattt tcattaqttc cctggctgac 120 gtaacgccaa 180 cacttggcag 240 ggtaaatggc 300 cagtacatct 360 aatgggcgtg 420 aatgggagtt 480 gccccattga 540 ctctggctaa 600 agggagaccc 660 tggcaqaatc 720 gtacagtttt 780 attcaggtaa 840 agtgtagttt 900 ggaagcaqta 960 gcaaggatqa 1020 gtgaattaga 1080 gtactaataa 1140 agtcctgtga 1200 agctcacccg 1260 aaaccatttt 1320 ttggtqqaga 1380 ttgatqcatt 1440 gtqttgaaac 1500 aacatacaga 1560 ctattaataa 1620 acagctacgt 1680 ttggatctgg 1-740 ttcttcagta 1800 tcaccatcta 1860 aagqagatag 1920 ttattagctg 1980 cccggtatgt 2040 gccgcgactc 2100 ggacaaacta 2160 atgtgttaaa 2220 tgaatgggag 2280 gccatctaqt 2340 gagaaaggta 2400 tgtgtttagt 2460 actqctatac 2520 ttataatcat 2580 taactatgct 2640 atatttgatq 2700 ttttacttgc 2760 caattgttgt 2820 tcacaaattt 2880 tcatcaatqt 2940 tcatgttctg 3000 agcgaattaa 3060 cccaacttaa 3120 cccgcaccga 3180 ggtattttct 3240 caatctqctc 3300 cgccctqacg 3360 qgagctgcat 3420 gggtaccaga 3480 tacgaaqctg 3540 taatggtttc 3600 gtttattttt 3660 WO 00/49147 WO 0049147PCT/EPOO/01368 ctaaatacat atattgaaaa tgcggcattt tqaagatcag ccttgagagt atgtggcgcg ctattctcag catgacagta cttacttctg ggatcatgta cgagcgtgac cgaactactt tgcaggacca agccggtgag ccgtatcgta gatcgctgag atatatactt cctttttgat agaccccgta ctgcttgcaa accaactctt tctagtgtaq cqctctgcta gttggactca tgcacacagc ctatgagaaa agggt cggaa agtcctgtcg gggcgqagcc tggccttttg accgcctttg gtgagcgagq attcattaat gcaattaatg qctcgtatgt catgattacg cggggtacca catcatgttc tcaaatatgt aggaagagta tgccttcctg ttgggtgcac tttcgccccg qtattatccc aatgacttgg a gaga at tat acaacqatcg actcgccttg accacgatgc actctaqctt cttctgcgct cgtgggtctc gttatctaca ataggtgcct t agat tgat t aatctcatga qaaaagatca acaaaaaaac tttccgaagg ccgtagttag atcctgttac agacqatagt ccagcttgqa gcgccacgct caggagagcg ggtttcqcca tatggaaaaa ctcacatgtt agtgagctga aagcggaaga gcagctggca tgagttagct tqtgtggaat ccaagctctc gcttcgtagc tqgtaccccg atccgctcat tgagtattca tttttgctca gagtggqtta aagaacqttt gtattqacgc ttgagtactc gcaqtgctgc gaggaccgaa atcgttggga ctgtaqcaat cccggcaaca cggcccttcc gcggtatcat cgacggggaq cactgattaa taaaacttca ccaaaatccc aaggatcttc caccqctacc taactggctt qccaccact t cagtqgctgc tacggataag gcgaacgacc tcccgaaqgq cacgagggag cctctgactt cgccagcaac cttttctgcg taccgctcgc gcgcccaata cgacaggttt cactcattag tqtqagcgga tagagctcta tagaacatca gtcga qagacaataa acatttccgt cccagaaacg catcgaactg tccaatgatg cgggcaagag accagtcaca cataaccatg ggaqctaacc accgqagctg ggcaacaacg attaatagac ggctggctgg tgcagcactg tcaggcaact gcattggtaa tttttaattt ttaacgtgag ttgagatcct agcqqtggtt cagcagagcg caagaactct tgccaqtgqc gcgcagcggt tacaccgaac agaaaggcgg ct tccaggqg gagcgtcgat gcqgcctttt ttatcccctg cgcagccgaa cgcaaaccqc cccgactgga gcaccccagg taacaatttc gagctctaga tgttctggga ccctgataaa gtcgccctta ctqgtgaaag gatctcaaca aqca ct tt ta caactcggtc qaaaagcatc agtqataaca gcttttttgc aatgaagcca ttgcgcaaac tggatggagg tttattgctg gqgccaqatg atggatgaac ctgtcagacc a aaag~a t ct ttttcgttcc ttttttctgc tcgtttccci cagataccaa gtagcaccgc gataagtcqt cgggctgaac tgagatacct acaggtatcc gaaacqcctg ttttgtgatg tacggttcct attctgtgqa cgaccgagcg ctctccccgc aagcgggcag ctttacactt acacaggaaa qctctagaga tatcagcttc tgcttcaata 3720 ttcccttttt 3780 taaaaqatgc 3840 gcggtaaqat 3900 aagttctgct 3960 qccgcataca 4020 ttacggatgg 4080 ctgcggccaa 4140 acaacatggg 4200 taccaaacqa 4260 tattaactqg 4320 cggataaaqt 4380 ataaatctgg 4440 gtaagccctc 4500 qaaatagaca 4560 aagtttactc 4620 agqtgaagat 4680 actgagcgtc 4740 gcgtaatctg 4800 atcaagaact 4860 atactgtcct 4920 ctacatacct 4980 qtcttaccqg 5040 ggqgggttcg 5100 acaqcgtgag 5160 ggtaagcggc 5220 gtatctttat 5280 ctcgtcaqgg 5340 ggccttttqc 5400 taaccgjtatt 54-60 cagcgagtca 5520 gcgttggccq 5580 tgagcgcaac 5640 tatqcttccg 5700 cagctatgac 5760 gcttgcatgc 5820 gtagctagaa 5880 5905 <210> 14 <211> 6052 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: vector pTGFG64 <400> 14 cgcgttqaca atagcccata cgcccaacga tagggacttt tacatcaagt ccgcctggca acgtattagt gatagcggtt tgttttggca cgcaaatggg ctagagaacc aagcttgcat accaggcctc ttgattattg tatggagttc cccccgccca ccattgacgt gtatcatatg ttatgcccag catcgctatt tgactcacgg ccaaaatcaa cggtaggcgt cactgcttac gccaattccg atcaccatct act agt tat t cgcgttacat ttgacqtcaa caatgggtgg ccaagtacgc tacatgacct accatggtga ggatttccaa cgggactttc gtacggtggg tggcttatcg caaaggttat gccttttagg aatagtaatc aacttacggt taatgacqta agtatttacq cccctattqa tat qgga ct t tgcqgttttg gtctccaccc caaaatgtcg aggtctatat aaattaatac gcagcgcgtg a tat ct act c aattacgggg aaatqgcccg tgttcccata gtaaactgcc cgtcaatgac tcctacttgg gcagtacatc cattgacgtc taacaactcc aagcagagct gactcactat aacatgatca agtgctgaat tcattagttc cctggctgac gtaacgccaa cacttggcag ggtaaatggc cagtacatct aatggqcgtg aatqggagtt gccccattga ctctqgctaa agggagaccc tggcagaatc gtacagtttt 120 180 240 300 360 420 480 540 600 660 720 780 WO 00/49147 WO 0049147PCT/EPOO/01368 tcttgatcat attggaagag tqaagaagca tqttgatqga cattaattcc tqtaacatgt caaggtggtt accagcagtq tgctgagact ggataacatc agatgccaaa ctgtggaggc tqgtgttaaa gcaaaagcga qtacaaccat tacacctatt ctatgtaagt ccttagagtt taacaacatg tgggqgaccc gggtgaagag caactggatt tactagagga cctacagaga ctactgattc cagtggtgga gatgatgagg gaagacccca aatagaactc aagaaaatta aacatactgt caaaaattgt tatagtgcct tttaaaaaac tgttaacttg cacaaataaa atcttatcat ggatatcagc tcactggccg cgccttqcag cgcccttccc agaacatcat ctgatgcggt ctcagtacaa gctgacgcgc gtctccggga aagggcacca gctacgaagc ataatggttt tgtttatttt atgcttcaat attccctttt gtaaaagatg agcqgtaaga aaagttctgc cgccgcatac cttacggatg actgcggcca cacaacatgg ataccaaacg ctattaactg gcggataaag qaaaacgcca tttgttcaag cgagaagttt qatcaqtqtg tatgaatgtt aacattaaga tgctcctgta ccatttccat gtttttcctg actcaaagca ccaggtcaat tctatcqtta attacagttg aatgtgattc gacattgccc tgcattgctg ggctggggaa ccacttgttg ttctgtgctg catgttactg tgtgcaatga aaqqaaaaaa tctttgtgaa tttaaagctc taattgtttg atgcctttaa ctactgctga aggactttcc ttgcttgctt tggaaaaata tttttcttac gtacctttag tgactagaga ctcccacacc tttattqcag gcattttttt gtctggatcc ttcgtagcta tcgttttaca cacatccccc aacagttgcg gttctgggat attttctcct tctgctctga cctgacgggc gctgcatgtg gaacatgatg tggtaccccg cttagacgtc tctaaataca aatattgaaa ttgcggcatt ctgaagatca tccttgagag tatgtggcgc actattctca gcatgacagt acttacttct gggatcatgt acgagcgtga gcgaactact ttgcaggacc acaaaattct ggaaccttqa ttgaaaacac agtccaatcc qgtgtccctt atggcagatg ctgaqqgata gtgqaagagt atgtgqacta cccaatcatt tccct tggca atgaaaaatg tcgcaqgtga gaattattcc ttctggaact acaaggaata gagtcttcca accgaqccac gcttccatga aagtggaagq aaggcaaata caaagctcac ggaaccttac taaggtaaat tgtattttag tgaggaaaac ctctcaacat ttcagaattg tgctatttac ttctgtaacc tccacacagg ctttttaatt tcataatcag tccccctgaa cttataatgg cactgcattc ccggggggta gaacatcatg acgtcgtgac tttcgccagc cagcctgaat atcagcttcg tacqcatctg tgccgcatag ttgtctgctc tcagaggttt ttctagctac cctcgtgata aggtggcact ttcaaatatg aaggaagagt ttgccttcct gttgggtgca ttttcgcccc ggtattatcc gaatgacttg aagagaatta gacaacgatc aactcgcctt caccacgatg tactctagct acttctgcgc gaatcggcca gagagaatqt tgaaagaaca atgtttaaat tggatttgaa cgaqcagttt tcgacttqca ttctgtttca tgtaaattct taatgacttc ggttgttttg gattgtaact acataatatt tcaccacaac ggacgaaccc cacgaacatc caaagggaqa atgtcttcga agqaggtaga gaccagtttc tggaatatat ttaatgggat ttctgtggtg ataaaatttt attccaacct ctgttttgct tctactcctc ctaagttttt accacaaagg tttataagta catagagtgt tgtaaagggg ccataccaca cctgaaacat ttacaaataa tagttgtggt ccagcttcgt ttctggtacc tgqgaaaacc tggcgtaata ggcgaatqgc tagctagaac tgcgqtattt ttaaqccagc ccggcatccg tcaccgtcat gaagctgata cgcctatttt tttcggggaa tatccgctca atgagtattc gtttttgctc cgagtgggtt gaagaacgtt cgtattgacg gttgagtact tgcagtgctg ggaggaccga gatcgttggg cctgtagcaa tcccggcaac tcggcccttc aaqaggtata atggaagaaa actgaatttt ggcggcagtt ggaaaqaact tqtaaaaata gaaaaccaga caaacttcta actgaagctg actcgggttg aatqgtaaag gctgcccact gaggaqacag tacaatgcaq ttagtgctaa ttcctcaaat tcagctttag tctacaaagt gattcatgtc ttaactgqaa accaaggtat cggtcgagcg tgacataatt taagtgtata atggaactga cagaagaaat caaaaaagaa tgagtcatgc aaaaagctgc ggcataacag ctgctattaa ttaataagga tttgtaqagg aaaatgaatg agcaatagca ttgtccaaac agctagaaca cccctctaga ctggcgttac gcgaagaggc ggggtaccag atcatgttct cacaccgcat cccgacaccc cttacagaca caccgaaacg tcccagaaca tatagqttaa atgtgcgcgg tgagacaata aacatttccg acccagaaac acatcgaact ttccaatgat ccgggcaaga caccagtcac ccataaccat aggagctaac aaccggagct tggcaacaac aattaataga cggctggctg attcaggtaa 840 agtgtagttt 900 ggaagcagta 960 gcaaggatga 1020 gtgaattaga 1080 gtgctgataa 1140 aqtcctgtga 1200 agctcacccg 1260 aaaccatttt 1320 ttggtgqaga 1380 ttgatgcatt 1440 qtgttqaaac 1500 aacatacaga 1560 ctattaataa 1620 acagctacgt 1680 ttggatctgg 1740 ttcttcagta 1800 tcaccatcta 1860 aaggagatag 1920 ttattagctg 1980 cccqgtatgt 2040 gccgcgactc 2100 ggacaaacta 2160 atgtgttaaa 2220 tgaatgggag 2280 gccatctagt 2340 gagaaaggta 2400 tgtgtttagt 2460 actgctatac 2520 ttataatcat 2580 taactatgct 2640 atatttgatg 2700 ttttacttgc 2760 caattgttgt 2820 tcacaaattt 2880 tcatcaatgt 2940 tcatgttctg 3000 gcgaattaat 3060 ccaacttaat 3120 ccgcaccgat 3180 cttcgtagct 3240 ggtaccccgc 3300 atgqtgcact 3360 gccaacaccc 3420 agctgtgacc 3480 cgcgaqacga 3540 tgatgttcta 3600 tgtcatqata 3660 aacccctatt 3720 accctgataa 3780 tgtcgccctt 3840 gctggtgaaa 3900 ggatctcaac 3960 gagcactttt 4020 gcaactcggt 4080 agaaaagcat 4140 gagtgataac 4200 cgcttttttg 4260 gaatgaagcc 4320 gttgcgcaaa 4380 ctggatgqaq 4440 gtttattqct 4500 WO 00/49147 WO 0049147PCT/EPOO/01368 qataaatctg ggtaagccct cgaaatagac caagtttact taggtgaaga cactgagcqt cgcgtaat ct gatcaagagc aatactgtcc cctacatacc tgtcttaccg acqqggggtt ctacagcgtg ccggtaagcg tggtatcttt tgctcgtcag ctggcctttt gataaccgta cgcagcgagt cccaqaacat aaaccgcctc qactggaaag ccccaggctt caatttcaca ctctagagct tctgggatat gagccggtga cccqtatcgt agatcgctga catatatact tcctttttga cagaccccgt gctgcttgca taccaactct ttctagtgta tcgctctgct ggttgqactc cgtgcacaca agctatgaga gcagggtcgg atagtcctgt gggggcggag gctggccttt ttaccgcctt cagtgagcga aatattctaa tccccgcgcg cgggcagtga tacactttat caggaaacag ctagagaqct cagcttcgta gcgtgggtct agttatctac gataggtgcc ttagattgat taatctcatg agaaaagatc aacaaaaaaa ttttccgaag gccgtagtta aatcctgtta aagacgatag gcccagcttg aagcgccacg aacaggagag cqggtttcqc cctatggaaa tgctcacatq tgagtgagct ggggtaccag ctacaaoct ttggccgatt gcgcaacgca gcttccggct ctatgaccat tgcatqccqq gctagaacat cqcggtatca acgacgqgga tcactgatta ttaaaacttc accaaaatcc aaaggatctt ccaccgctac gtaactggct ggccaccact ccagtggctq ttaccggata gagcgaacga cttcccgaag cgcacgaggg cacctctqac aacgccaqca ttctttcctg gataccgctc aacatgatgt ggtaccccag cattaatgca attaatgtga cgtatgttgt gattacgcca ggtaccagct cat gtt ct gg ttqcagcact gtcaggcaac agcattggta at t tt taat t cttaacgtqa cttgagatcc caqcggt ggt tcagcaqagc tcaagaactc ctgccaqtgg aggcqcaqcq cctacaccga ggagaaagqc agcttccagg t tgagcgtcg acgcggcctt cgttatcccc gccgcagccg tctagctacg cggaagagcg gctggcacga gttagctcac gtqgaattgt agctctctaq tcgtagctag taccccggtc ggggccagat tatggatqaa actgtcagac taaaaggatc gttttcgttc tttttttctg ttgtttgccg gcagatacca tgtagcaccg cgataagtcg gt cqggctga actgagatac ggacaggtat qggaaacqcc atttttqtga tttacggttc tgattctgtg aacgaccgag aagctqatat cccaat.acgc caggtttccc tcattaggca gagcggataa agctctagag aacatcatqt ga 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6052 <210> <211> 4344 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: vector pTGFG67 <400> cgcgttgaca atagcccata cgcccaacga tagggacttt tacatcaagt ccgcctggca acgtattagt gatagcggtt tgttttggca cgcaaatggg ctagagaacc aagcttgacc cttctgtggt tataaaattt gattccaacc cctgttttgc ttctactcct gctaagtttt caccacaaag ctttataagt gcatagagtg ttgtaaaggg gccataccac acctgaaaca gttacaaata ttgattattg tatggagttc cccccgccca ccattgacgt gtatcatatg ttatgcccag catcgctatt tgactcacgg ccaaaatcaa cggtaggcgt cactgcttac tcqagcaagc gtgacataat ttaagtgtat tatqqaactg tcagaagaaa ccaaaaaaga ttgagtcatg gaaaaagctg aggcataaca tctgctatta gttaataagg atttgtagag taaaatgaat aaqcaatagc actagttatt cgcgttacat ttgacgtcaa caatgggtgg ccaagtacgc tacatgacct accatggtga ggatttccaa cqggactttc gtacgqtgqg tggcttatcg ggccgcgact tggacaaact aatgtgttaa atgaatggga tgccatctag agagaaaggt ctgtgtttag cactgctata gttataatca ataactatgc aatatttgat gttttacttg gcaattgttg atcacaaatt aataqtaatc aacttacggt taatqacqta agtatttacg cccctattga tatgqgactt tgcggttttg gtctccaccc caaaatgtcg aggtctatat aaattaatac ctactagagg acctacagag act act gat t gcagtggtgg tgatqatgag agaagacccc taatagaact caagaaaatt taacatactg tcaaaaattg gtataqtgcc ctttaaaaaa ttgttaactt tcacaaataa aattacgggg aaatggcccg tgttcccata gtaaactqcc cgtcaatqac tcctacttgg gcagtacatc cattqacgtc taacaactcc aaqcagagct qactcactat atctttgtga atttaaagct ctaattqttt aatgccttta gctactgctq aaggactttc cttgcttgct atqqaaaaat ttttttctta tgtaccttta ttqactagag cctcccacac qtttattgca agcatttttt tcattagttc cctggctqac gtaacgccaa cacttggcag ggtaaatggc cagtacatct aatggqcgtg aatqggagtt qccccattqa ctctggctaa agggagaccc agqaacctta ctaaggtaaa qtgtatttta atqaggaaaa actctcaaca cttcagaatt ttgctattta attctgtaac ctccacacaq gctttttaat atcataatca ctccccctga gcttataatg tcactgcatt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 00/49147 WO 0049147PCTIEPOO/01368 ctagttgtgq ctctagagcq gcgttaccca aaqaggcccg tgatgcggta tcagtacaat ctgacgcqcc tctccgqgag agggqqta gaacatcatg taataatqgt tttgtttatt aaatgcttca ttattccctt aagtaaaaga acaqcqgtaa ttaaagttct qtcgccqcat atcttacgga acactgcggc tgcacaacat ccataccaaa aactattaac aggcggataa ctgataaatc atqgtaagcc aacgaaatag accaagttta tctaggtgaa tccactgagc tgcgcgtaat cggatcaaga caaatactgt cgcctacata cgtgtcttac gaacgqggggg acctacagcg atccggtaag cctggtatct qatgctcgtc tcctggcctt tggataaccg agcgcagcga ccgcgcgttg gcagtgagcg actttatgct gaaacagcta gagagcttgc tttgtccaaa aattaattca acttaatcgc caccgatcgc ttttctcctt ctgctctgat ctgacgggct ctgcatgtgt ccagcttcgt ttctgqtacc ttcttagacg tttctaaata ataatattga ttttgcggca tgctgaagat gatccttgag gctatqtggc acactattct tggcatgaca caacttactt gggggatcat cgacgagcgt tggcgaacta aqttgcagga tqgaqccggt ctcccgtatc acagatcgct ctcatatata gatccttttt gtcagacccc ctgctgcttg gctaccaact tcttctagtg cctcgctctg cgggttggac ttcgtgcaca tgagctatga cggcagggtc ttatagtcct aggggggcgg ttgctggcct tattaccgcc gtcagtgagc gccgattcat caacgcaatt tccggctcgt tgaccatgat atgcctgcag ctcatcaatg ctggccgtcg cttqcagcac ccttcccaac acgcatctgt gccgcatagt tqtctgctcC cagaggtttt agctagaaca cccctcqtga tcagqtggca cattcaaata aaaagqaaga ttttgccttc cagttgggtg agttttcgcc gcggtattat cagaatgact gtaagagaat ctgacaacqa gtaactcgcc gacaccacga cttactctag ccacttctgc qagcgtgggt gtagttatct gagataggtg ctttaqattg gataatctca gtagaaaaga caaacaaaaa ctttttccga tagccgtagt ctaatcctgt tcaagacgat cagcccagct gaaagcgcca ggaacaggag gtcgggtttc agcctatgga tttgctcaca tttgagtgag gaggaagcgg taatgcagct aatgtgagtt atgttgtgtg tacgccaagc gt cg tatcttatca ttttacaacg atcccccttt agttgcgcag gcggtatttc taaqccagcc cggcatccgc caccgtcatc tcatgttctg tacgcctatt Cttttcgggg tgtatccgct gtatgagtat ctgtttttgc cacgagt gg ccgaagaacg cccgtattga tggttgagta tatgcagtqc tcggaggacc ttgatcgttq tgcctgtagc cttcccggca gctcggccct ctcgcggtat acacgacggg cctcactgat atttaaaact tgaccaaaat t caaaggat c aaccaccgct aggtaactgg taggccacca taccaqtqgc agttaccgga tggagcgaac cgcttcccga agcgcacgag qccacct ctg aaaacgccag tgttctttcc ctgataccgc aagagcgccc ggcacgacag agctcactca gaattgtgag tctctagagc tgtctggatc tcgtgactqq cgccagct gq cctgaatqqc acaccgcata ccgacacccg ttacagacaa accgaaacgc gqatatcaqc tttataggtt aaatgtgcgc catgagacaa tcaacatttc tcacccagaa ttacatcgaa ttttccaatg cgccgggcaa ctcaccagtc tgccataacc qaaqgagcta ggaaccggag aatggcaaca acaattaata tccggctggc cattgcagca qagtcaggca taagcattgg tcatttttaa cccttaacgt ttcttgagat accagcggtg ctt cagcaga cttcaagaac tgctgccagt taaggcgcag gacctacacc aggqagaaag ggaqcttcca acttqagcgt caacgcggcc tgcgttatcc tcgccgcagc aatacgcaaa gtttcccqac ttaggcaccc cggataacaa tctagaqctc cccggqtacc 1560 gaaaaccctq 1620 cgtaataqcg 1680 gaatggcgcc 1740 tggtgcactc 1800 ccaacacccg 1860 gctgtgaccg 1920 qcgagacqaa 1980 ttcgtagcta 2040 aatqtcatga 2100 ggaaccccta 2160 taaccctgat 2220 cgtqtcgccc 2280 acgctggtga 2340 ctqgatctca 2400 atgagcactt 2460 gagcaactcg 2520 acagaaaagc 2580 atgaqtgata 2640 accgcttttt 2700 ctgaatgaag 2760 acqttgcgca 2820 gactggatqg 2880 tqgtttattg 2940 ctggggccag 3000 actatggatg 3060 taactgtcaq 3120 tttaaaagga 3180 gagttttcgt 3240 gtttgtttqc 3360 gcqcagatac 3420 tctgtagcac 3480 ggcgataagt 3540 cggtcgggct 3600 gaactqagat 3660 gcggacaggt 3720 gggggaaacg 3780 cgatttttqt 3840 tttttacggt 3900 cctgattctg 3960 cgaacgaccg 4020 ccgcctctcc 4080 tggaaagcqg 4140 caggctttac 4200 tttcacacag 4260 tagagctcta 4320 4344 <210> 16 <211> 4496 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: vector pTGFG82 <400> 16 cgcgttgaca ttgattattg actaqttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctgqctgac 120 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180 WO 00/49147 WO 0049147PCT[EPOO/01368 tagggacttt tacatcaaqt oogcctggca acgtattagt qatagoggtt tgttttgqca cgcaaatqggg ctagagaacc aagcttgacc ottotgtggt tataaaattt gattccaacc cctgttttgc ttctactcct gctaagtttt oaooacaaag ctttataagt gcatagagtg ttgtaaaggg gccataccac acctgaaaca gttacaaata ctagttgtgg accagcttcg gttctqgtac ctgggaaaac ctggcgtaat tggcgaatgg catatqgtgc cccgccaaca acaaqctgtg acgcqogaga coagaacatg ataggttaat tgtqcgcgga qagacaataa acatttccgt cccagaaacg catcgaactg tccaatgatg cgggcaagag accagtcaca cataaccatg ggagctaacc accggagctg ggcaacaacg attaatagac ggctggctgg tgcagcactg tcaggcaact gcattggtaa tttttaattt ttaacgtgag ttgagatcct aqoggtggtt caqcagagcg oaagaactct tgccagtggc ggcgcagcgg ctaoaccgaa gagaaaggog gcttccaggg coat tqaogt gtatcatatg ttatgcccaq catogctatt tgaotcacgg ccaaaatcaa cgqtaggcgt cactgcttao tcqagcaagc gtgacataat ttaagtgtat tatggaactg tcagaaqaaa ccaaaaaaga ttgagtcatg gaaaaagctg aggcataaca tctgctatta gttaataagq atttgtagag taaaatgaat aagoaatagc tttgtccaaa tagctagaac cooctotag cctggcgtta agcgaagagg cgcctgatgc actctcagta ccgctgacg accgtctccg cgaaagggcg atgttotagc gtcatgataa accctattt occtgataaa gtcgccctta ctggtgaaag gatctcaaca agoaotttta caactcggtc gaaaagcatc agtgataaca gcttttttgc aatqaagcca ttgcgcaaac tggatggagg tttattgctg gggccagatg atggatgaac ctgtcagaoc aaaaggatct ttttcgttcc ttttttctgc tgtttgccgg caqataccaa gtagcaccgc gataagtcgt tcgggctgaa ctgagatacc gacaggtatc ggaaacgcct caatgqgtgg ccaaqtaogc tacatgacct accatggtga gqatttccaa cgggactttc gtacggtggg tggcttatcg ggccgcgact tggacaaact aatgtgttaa atgaatggga tgccatctag agagaaaggt ctgtgtttag cactgctata gttataatca ataactatgc aatatttgat gttttaottg gcaattgttq atcacaaatt ctcatcaatg atcatgttct agcgaattaa cccaacttaa cccgcaccga ggtattttct caatctgctc cgccctgacg ggagctgcat gggtaccaga tacgaagctg taatggtttc gtttattttt tgcttcaata ttcccttttt taaaagatgc gcggtaagat aagttctgct googcataca ttacggatgg ctgcggccaa acaacat ggg taccaaacga tattaactgg cggataaagt ataaatctgg gtaagcccto gaaatagaca aagtttactc aggtgaagat actgagcgtc gcgtaatctg atoaagagot atactgtcct ctacatacct gtcttaccgg oggggggttc tacagcgtga cqgtaagcgg ggtatcttta agtatttaog cccctattga tatgggactt tgcgqttttg gtctccaocc caaaatgtcg aggtctatat aaattaatac ctactagagg acctacagag actactgatt gcagtggtqq tgatgatgaq aqaagacoc taatagaact caagaaaatt taacatactg tcaaaaattg gtatagtgcc ctttaaaaaa ttgttaactt tcacaaataa tatcttatoa gggatatcag ttcactggc tcgccttqca tcgcccttcc ccttacgcat tgatgccgca ggcttqtotg gtgtcagagg acatgatgtt gtacccoggc ttagacgtca ctaaatacat atattgaaaa tgcggcattt tgaagatcag ccttqaqagt atgtggcgcg ctattctcag catgacagta cttacttctq ggatcatgta cgaqcgtgac cgaactactt tgcaggacoa agccggtgag ccgtatcgta gatcgctgag atatataott cctttttgat agaccccgta ctgcttgcaa accaactctt tctagtgtag cgctct got a gttqgact ca gtgcacacag gctatgagaa cagggtcgga tagtcctgtc gtaaactgcc cgtcaatgac tcctacttgg gcaqtacatc cattgacgtc taacaactoc aaqoagagct gaotcaotat atctttgtqa atttaaaqct ctaattgttt aatgccttta gctactgctg aaggactttc cttgcttgct at ggaaaaat t tt t t ttt a tgtaccttta ttqactagag cctoccacac gt ttat tgca aqcatttttt tgtctggatc cttcgtagct gtcgttttac gcacatcccc caacagttgc ctgtgcggta tagttaagoc ctcccggcat ttttcaccgt ctagctacqa ctcgtgatac ggtggcactt tcaaatatgt aggaaqagta tgccttcctg ttgggtgcac tttcgccccg gt at tat ccc aatgacttgg agagaattat acaacgatcg actcgccttg accacgatgc actotagctt cttctgcgct cgtgggtotc gttatctaoa ataggtgcct tagattgatt aatctcatga gaaaaqatca acaaaaaaac tttccgaagg ccgtagttag atcctgttac a ga cga tagt cccagcttgg agcgccacgc acaqgagagc ggqtttcgoc cacttggcag 240 gqtaaatgqc 300 cagtacatct 360 aatgggcgtg 420 aatgggaqtt 480 qccccattqa 540 ctctgqctaa 600 agggagaccc 660 aggaacctta 720 ctaaggtaaa 780 gtqtatttta 840 atgaggaaaa 900 actctcaaca 960 cttcagaatt 1020 ttgctattta 1080 attctgtaac 1140 ctocacacag 1200 gctttttaat 1260 atcataatca 1320 ctccccctga 1380 gcttataatq 1440 tcactgcatt 1500 cccggggggt 1560 agaacatcat 1620 aacgtcgtga 1680 ctttcgocag 1740 gcagcotgaa 1800 tttcacaccg 1860 agccccgaca 1920 ccgcttaoag 1960 catcaccgaa 2040 agctgatatc 2100 gcctattttt 2160 ttcggggaaa 2220 atccgctcat 2280 tgagtattca 2340 tttttgctca 2400 gagtgggtta 2460 aagaacgttt 2520 gtattgacgc 2580 ttgagtactc 2640 gcagtgctgc 2700 gaggaccgaa 2760 atcgttggga 2820 ctgtagcaat 2880 cccggcaaca 2940 cggcccttcc 3000 gcggtatcat 3060 cgacggggag 3120 cactgattaa 3180 taaaacttca 3240 ccaaaatccc 3300 aaggatcttc 3360 caccgctacc 3420 taactggctt 3480 gccaccactt 3540 cagtggctgo 3600 taccggataa 3660 agcgaacgac 3720 ttccogaagg 3780 gcacgaggga 3840 acctctgact 3900 WO 00/49147 WO 0049147PCT/EPOO/01368 tqagcgtcga cgcggccttt gttatcccct ccgcagccga acgcaaaccg t cccgactgg gqcaccccag ataacaattt agagctctag atgttctggg tttttgtgat ttacggttcc gattctgtgg acgaccgagc cctctccccg aaagcgggca qctttacact cacacagqaa agctctagag atatcagctt gctcgtcaqg t ggcct tt tg ataaccgtat gcagcgaqtc cgcgttggcc gtqagcgcaa ttatgcttcc acagctatga aqcttgcatq cgtagctaga qgggcggagc ctqgcctttt taccgccttt agtgagcgag gattcattaa cqcaattaat ggctcgtatg ccatqattac ccggggtacc acatcatgtt ctatggaaaa gctcacatgt gagtgagctg gaagcqqaag tgcagctggc gtgagttagc ttgtgtggaa gccaagctct agcttcgtag ctggtacccc acgccagcaa tctttcctgc ataccgctcg agcgcccaat acgacaggtt tcactcatta ttgtgagcgg ctaaaqctct ctaqaacatc ggtcga 3960 4020 4080 4140 4200 4260 4320 4380 4440 4496 <210> 17 <211> 4644 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: vector <400> 17 cgcqttgaca atagcccata cgcccaacga tagggacttt tacatcaagt ccgcctggca acgtattagt gatagcggtt tgttttggca cgcaaatggg ctagagaacc aagcttgacc cttctgtqgt tataaaattt gattccaacc cctgttttgc ttctactcct gctaagtttt caccacaaag ctttataagt gcataqagtg ttgtaaaggg gccataccac acctgaaaca gttacaaata ctagttgtgg accagcttcg gttctggtac ctggqaaaac ctggcgtaat tggcgaatgg gtaqctagaa gtgcggtatt gttaagccaq cccggcatcc ttcaccgtca acgaagctqa tacgcctatt cttttcgggg tgtatccgct gtatgagtat ttgattattg tatggagttc cccccgccca ccattgacgt gtatcatatg ttatgcccag catcgctatt tgactcacgg ccaaaat caa cggtaggcqt cactgcttac tcqagcaagc gtgacataat ttaagtgtat tatggaactq tcagaagaaa ccaaaaaaga ttgagtcatg gaaaaagctg agqcataaca tctgctatta gttaataagg atttgtagag taaaatgaat aagcaatagc tttgtccaaa tagctagaac ccccctctag cctggcgtta agcgaagagg cggggtacca catcatgttc tcacaccgca ccccgacacc gcttacagac tcaccgaaac tatcccagaa tttataggtt aaatgtgcgc catgagacaa tcaacatttc actagttatt cgcgt ta cat ttgacgtcaa caatgggtgg ccaagtacgc tacatgacct accatggtga ggatttccaa cgggactttc gtacggtggg tqgcttatcg ggccgcgact tgqacaaact aatgtgttaa atgaatggga tgccatctag agagaaaggt ctqtgtttaq cactgctata gttataatca ataactatgc aatatttgat gttttacttg gcaattgttg atcacaaatt ctcatcaatg atcatgttct agcgaattaa cccaacttaa cccgcaccga gcttcgtagc tggtaccccg tatggtgcac cgccaacacc aagctgtgac gcgcgagacg catgatgttc aatgtcatga ggaaccccta taaccctgat cgtgtcgccc aatagtaatc aacttacggt taatqacgta agtatttacg cccctattga tatgggactt tgcggttttg gtctccaccc c 'aaaatgtcg aggtctatat aaattaatac ctactagagg acctacagag actactqatt gcagtggtgg tgatgatqag aqaagacccc taatagaact caagaaaatt taacatactg tcaaaaattg gtatagtgcc ctttaaaaaa ttgttaactt tcacaaataa tatcttatca ggqatatcag ttcactggcc t cgccttqca tcqcccttcc tagaacatca cctgatgcgg tctcagtaca cgctqacgcg cgtctccggg aaaqggctac tagctacgaa taataatggt tttgtttatt aaatgcttca ttattccctt aattacgggg aaatggcccg tgttcccata gtaaactqcc cgtcaatgac tcctacttgg gcagtacatc cattgacgtc taacaactcc aagcagagct gactcactat atctttgtga atttaaagct ctaattgttt aatgccttta gctactgctg aaggactttc cttgcttgct atggaaaaat ttttttctta tgtaccttta.

ttgactagag cctcccacac qtttattgca agcatttttt tgtctggatc cttcgtagct gtcgttttac gcacatcccc caacaqttgc tgttctggga tattttctcc atctgctctg ccctgacqggg agctgcatgt cagaacatga gctggtaccc ttcttagacg tttctaaata.

ataatattga ttttgcggca tcattagttc cctggctgac 120 gtaacgccaa 180 cacttggcag 240 qgtaaatggc 300 cagtacatct 360 aatgggcqtg 420 aatgggagtt 480 gccccattga 54.0 ctctggctaa 600 agggagaccc 660 aggaacctta 720 ctaaggtaaa 780 gtgtatttta 840 atgaqgaaaa 900 actctcaaca 960 cttcagaatt 1020 ttgctattta 1080 attctgtaac 1140 ctccacacag 1200 gctttttaat 1260 atcataatca 1320 ctccccctga 1380 gcttataatg 1440 tcactgcatt 1500 cccgggggqt 1560 aqaacatcat 1620 aacgtcgtqa 1680 ctttcqccag 1740 gcagcctqaa 1800 tatcagcttc 1860 ttacgcatct 1920 atgccgcata 1980 cttgtctgct 2040 gtcagaggtt 2100 tgttctagct 2160 cgcctcgtga 2220 tcaggtggca 2280 cattcaaata 2340 aaaaggaaga 2400 ttttgccttc 2460 WO 00/49147 WO 0049147PCT/EPOO/01368 ctgtttttqc cacgagtggq ccgaaqaacg cccgtattga tggttgagta tatgcagtgc tcgqaggacc ttqatcgttq tgcctgtagc cttcccggca gctcggccct ctcgcggtat acacgacggg cctcactgat atttaaaact tgaccaaaat tcaaagqatc aaccaccgct aggtaactgg tagqccacca taccagt.ggc agttaccgga tggagcgaac cgcttcccga agcgcacgag gccacctctg aaaacgccag tgttctttcc ctgataccgc agaacatgat ctggtacccc ttcattaatg caattaatgt ctcgtatqtt atgattacqc ggggtaccag atcatgttct tcacccagaa ttacatcgaa ttttccaatg cgccgqcaa ctcaccagtc tgccataacc gaaggagcta ggaaccggag aatqqcaaca acaattaata tccgqctggc cattgcagca gagtcaggca taagcattgg t cat ttt taa cccttaacgt ttcttgagat accagcggtg cttcagcaga cttcaagaac tgctgccagt taaggcgcaq gacctacacc agggagaaag ggagcttcca acttgagcgt caacgcggcc tgcgttatcc tcgccgcagc gttctagcta agcggaagag caqctggcac gagttagctc gtgtggaatt caagctctct cttcgtaqct ggtaccccgg acgctggtga ctqgatctca atgagcactt gagcaactcg acagaaaagc atgagtgata accgcttttt ctgaatgaag acgttgcgca gactggatgg tggtttattg ctggqccag actatggatg taactgtcag tttaaaagqa gagttttcgt cctttttttc gtttgtttgc gcgcagatac tctgtagcac ggcgataagt cggtcgggct gaactgagat gcgqacaggt gggggaaacg cgatttttgt t t tt tacggt cctgattctg cgaacgaccg cgaagctgat cgcccaatac gacaggtttc actcattagg gtgagcggat agagctctag agaacatcat tcga aagtaaaaga acaqcggtaa ttaaagttct gtcgccgcat atcttacgga acactgcggc tgcacaacat ccataccaaa aactattaac aggcggataa ctgataaatc atggtaaqcc aacqaaataq accaagttta tctaggtqaa tccactqagc tgcgcgtaat cggatcaaga caaatactgt cgcctacata cgtgtcttac gaacgggggg acctacagcg atccggtaag cctggtatct gatgctcgtc tcctggcctt tggataaccg agcgcagcga atcccagaac gcaaaccgcc ccgactggaa caccccaggc aacaatttca agctctagag gttctgggat tgctgaagat gatccttgag gctatgtggc acactattct tggcatgaca caacttactt qggggatcat cgacgagcgt tggcgaacta agttgcaqga tggagccgqt Ctcccgtatc acagatcgct ctcatatata gatccttttt gtcagacccc ctgctgcttg qctaccaact ccttctagtg cctcqctctq cgqgttggac ttcgtgcaca tgagctatga cggcaqggtc ttatagtcct aggggggcqg ttgctggcct tattaccgcc gtcagtgagc atgatgttct tctccccgcg agcgggcagt tttacacttt cacaggaaac ctctagagag atcagcttcg cagttqggtg 2520 agttttcgcc 2580 qcggtattat 2640 cagaatgact 2700 gtaagagaat 2760 ctgacaacga 2820 gtaactcgcc 2880 gacaccacga 2940 cttactctag 3000 ccacttctqc 3060 gagcgtqggt 3120 gtagttatct 3180 gagataggtg 3240 ctttagattg 3300 gataatctca 3360 gtagaaaaga 3420 caaacaaaaa 3480 ctttttccga 3540 tagccgtagt 3600 ctaatcctat 3660 tcaagacgat 3720 cagcccagct 3780 gaaagcgcca 3840 ggaacaggaq 3900 gtcgggtttc 3960 agcctatgga 4020 tttgctcaca 4080 tttgagtgaq 4140 gaggggtacc 4200 agctacgaag 4-2-60 cgttggccga 4320 gagcgcaacq 4380 atgcttccgg 4440 aqctatgacc 4500 cttgcatgcc 4560 tagctagaac 4620 4644 '<210> 18 <211> 933 <212> PRT <213> Homo sapiens <400> 18 Met Thr Giu Leu Ly; 1 Ala Lys Gly Pro Ala Pro His Val Ala Gly Gly Pro Pro Ser Ala Gly Pro Phe Pro Glu Val Gly Ser 25 Pro Leu Leu Cys Pro Gly Ser Gin 40 Thr Ser Asp Thr Leu Arg Pro Ala Pro Giu Val Arg Pro Cys Ser Ala Ile Pro Ile Ser Leu 55 Gin Gly Gln Asp Pro Ser Asp 70 Ser Asp Val Giu Gly Ala Tyr Asp Gly Leu Leu Phe Pro Giu Lys Thr Gin Asp Gin Gin Ser Ser Arg Ala Giu Ala Thr Arg 90 Gly Ala WO 00/49147 PCT/EP00/01368 17 Gly Gly Ser Ser Ser Ser Pro Pro Glu Lys Asp Ser Gly Leu Leu Asp 100 105 110 Ser Val Leu Asp Thr Leu Leu Ala Pro Ser Gly Pro Gly Gin Ser Gin 115 120 125 Pro Ser Pro Pro Ala Cys Glu Val Thr Ser Ser Trp Cys Leu Phe Gly 130 135 140 Pro Glu Leu Pro Glu Asp Pro Pro Ala Ala Pro Ala Thr Gin Arg Val 145 150 155 160 Leu Ser Pro Leu Met Ser Arg Ser Gly Cys Lys Val Gly Asp Ser Ser 165 170 175 Gly Thr Ala Ala Ala His Lys Val Leu Pro Arg Gly Leu Ser Pro Ala 180 185 190 Arg Gin Leu Leu Leu Pro Ala Ser Glu Ser Pro His Trp Ser Gly Ala 195 200 205 Pro Val Lys Pro Ser Pro Gin Ala Ala Ala Val Glu Val Glu Glu Glu 210 215 220 Asp Gly Ser Glu Ser Glu Glu Ser Ala Gly Pro Leu Leu Lys Gly Lys 225 230 235 240 Pro Arg Ala Leu Gly Gly Ala Ala Ala Gly Gly Gly Ala Ala Ala Val 245 250 255 Pro Pro Gly Ala Ala Ala Gly Gly Val Ala Leu Val Pro Lys Glu Asp 260 265 270 Ser Arg Phe Ser Ala Pro Arg Val Ala Leu Val Glu Gin Asp Ala Pro 275 280 285 Met Ala Pro Gly Arg Ser Pro Leu Ala Thr Thr Val Met Asp Phe Ile 290 295 300 His Val Pro Ile Leu Pro Leu Asn His Ala Leu Leu Ala Ala Arg Thr 305 310 315 320 Arg Gin Leu Leu Glu Asp Glu Ser Tyr Asp Gly Gly Ala Gly Ala Ala 325 330 335 Ser Ala Phe Ala Pro Pro Arg Ser Ser Pro Cys Ala Ser Ser Thr Pro 340 345 350 Val Ala Val Gly Asp Phe Pro Asp Cys Ala Tyr Pro Pro Asp Ala Glu 355 360 365 Pro Lys Asp Asp Ala Tyr Pro Leu Tyr Ser Asp Phe Gin Pro Pro Ala 370 375 380 Leu Lys Ile Lys Glu Glu Glu Glu Gly Ala Glu Ala Ser Ala Arg Ser 385 390 395 400 Pro Arg Ser Tyr Leu Val Ala Gly Ala Asn Pro Ala Ala Phe Pro Asp 405 410 415 WO 00/49147 PCT/EP00/01368 18 Phe Pro Leu Gly Pro Pro Pro Pro Leu Pro Pro Arg Ala Thr Pro Ser 420 425 430 Arg Pro Gly Glu Ala Ala Val Thr Ala Ala Pro Ala Ser Ala Ser Val 435 440 445 Ser Ser Ala Ser Ser Ser Gly Ser Thr Leu Glu Cys Ile Leu Tyr Lys 450 455 460 Ala Glu Gly Ala Pro Pro Gin Gin Gly Pro Phe Ala Pro Pro Pro Cys 465 470 475 480 Lys Ala Pro Gly Ala Ser Gly Cys Leu Leu Pro Arg Asp Gly Leu Pro 485 490 495 Ser Thr Ser Ala Ser Ala Ala Ala Ala Gly Ala Ala Pro Ala Leu Tyr 500 505 510 Pro Ala Leu Gly Leu Asn Gly Leu Pro Gin Leu Gly Tyr Gin Ala Ala 515 520 525 Val Leu Lys Glu Gly Leu Pro Gin Val Tyr Pro Pro Tyr Leu Asn Tyr 530 535 540 Leu Arg Pro Asp Ser Glu Ala Ser Gin Ser Pro Gin Tyr Ser Phe Glu 545 550 555 560 Ser Leu Pro Gin Lys Ile Cys Leu Ile Cys Gly Asp Glu Ala Ser Gly 565 570 575 Cys His Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys 580 585 590 Arg Ala Met Glu Gly Gin His Asn Tyr Leu Cys Ala Gly Arg Asn Asp 595 600 605 Cys Ile Val Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Leu 610 615 620 Arg Lys Cys Cys Gin Ala Gly Met Val Leu Gly Gly Arg Lys Phe Lys 625 630 635 640 Lys Phe Asn Lys Val Arg Val Val Arg Ala Leu Asp Ala Val Ala Leu 645 650 655 Pro Gin Pro Leu Gly Val Pro Asn Glu Ser Gin Ala Leu Ser Gin Arg 660 665 670 Phe Thr Phe Ser Pro Gly Gin Asp Ile Gin Leu Ile Pro Pro Leu Ile 675 680 685 Asn Leu Leu Met Ser Ile Glu Pro Asp Val Ile Tyr Ala Gly His Asp 690 695 700 Asn Thr Lys Pro Asp Thr Ser Ser Ser Leu Leu Thr Ser Leu Asn Gin 705 710 715 720 Leu Gly Glu Arg Gin Leu Leu Ser Val Val Lys Trp Ser Lys Ser Leu 725 730 735 WO 00/49147 WO 0049147PCT/EPOO/01368 Pro Gly Phe Tyr Ser Trp 755 Asn Leu His Ile Asp 745 Asp Gin Ile Thr Leu Ile Gin 750 Arg Ser Tyr Met Ser Leu Met Val1 760 Phe Gly Leu Gly Trp 765 Lys His 770 Vai Ser Giy Gin Leu Tyr Phe Aia Pro 780 A-sp Leu Ile Leu Asn 785 Giu Gin Arg Met Lys 790 Giu Ser Ser Phe Tyr 795 Ser Leu Cys Leu Thr 800 Met Trp Gin Ile Gin Giu Phe Val Lys 810 Leu Gin Val Ser Gin Giu 815 Giu Phe Leu Giu Giy Leu 835 Cys 820 Met Lys Vai Leu Leu 825 Leu Leu Asn Thr Ile Pro Leu 830 Ser Ser Tyr Arq Ser Gin Thr Phe Giu Giu Met Ile Arg 850 Giu Leu Ile Lys Aia 855 Ile Gly Leu Arg Gin 860 Lys Giy Val Vai 7 eu Asp Asn Leu 880 Ser 865 Ser Ser Gin Arg Tyr Gin Leu Thr Lys Leu 875 Cys Leu Asn His Asp Leu Val Gin Ser Arg Ala 900 Lys 885 Gin Leu His Leu Thr Phe Ile 895 Ser Giu Val 910 Lys Pro Leu Leu Ser Val Glu Phe 905 Pro Glu Met Met Ile Ala Ala 915 Gin Leu Pro Lys Ile 920 Leu Aia Gly Met Vali 925 Leu Phe 930 His Lys Lys <210> 19 <211> 2970 <212> DNA <213> Homo sapiens <400> 19 ctgaccagcg cacattcaac tcccttttcc tgagctgaag ggtcgqatcc ggacaccttg gccctgccag cgt ggagggc tcccccagaa aggtcccggg gtttggcccc cccgctcatg taaagtgctg ccctcactgg ggaggaggat ggctctgggt ccgccctccc acccactttc ctcctccctg gcaaagggtc ccactgctgt cctgaagttt ggacaggacc gcatattcca aaggacagcg cagagccaac gaacttcccg agccggtccg ccccggggcc tccggggccc ggctctgagt ggcgcggcgg ccgcccccga tcctccctct gagacggggg cccgggctcc gtcgcccagc cggccatacc cctccgacga gagctgaagc gactgctgga ccagccctcc aagatccacc ggtgcaaggt tgtcaccagc cagtgaagcc ccgagqagtc ctggaggagg cccaggaggt gcccctatat agqagaaaag ccacgtqgcq cgcagqtccg tatctccctg aaagacgcag tacaaggggt cagtgtcttg cgcctgcgag gqctgccccc tggagacagc ccggcagctg gtctccgcag tgcgggtccg agccgcggct ggagatccct tcccqaaacc gggagtccaq ggcggcccgc ttcccgggga gacgggctac gaccagcagt gctggaggca qacactctgt gtcaccagct gccacccagc t ccgggacgg ctgctcccgg gccgctgcqq cttctgaaqg gtcccgccgg ccggtccagc ccctcctcct tcqtcatgac cctcccccga gccagacctc tcttccctcq cgctgtcgga gcagttctag tggcgccctc cttggtgcct gggtgttgtc cagctgccca cctctgagag tggaggttga gcaaacctcg gggcggcagc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 WO 00/49147 WO 0049147PCT/EPOO/01368 agqaggcgtc ggtggagcag tttcatccac gctgctggaa gcgqagttca gtacccgccc gcccgctcta ttcctacctt gcccccgctg acccgccagt gtacaaaqcg gCCqggcgcg cgccqccgcc gctcggctac caactacctg acctcagaag tacctgtggg atgtgctgga tcgccttaga caataaagtc tccaaatqaa gttgattcca acatgacaac cgagaggcaa acatattgat tctaggatgg aatactaaat gcagatccca agtattgtta ggaqatgagg agttgtgtcg tcttgtcaaa tgttgaattt agggatggtg gccctggtcc gacgcgccga gtgcctatcc gacgaaagtt ccctgtgcct gacgccgagc aagataaagg gtggccggtg ccgccgcgag gcctcagtct gagggcgcgc agcggctqcc ggggcggccc caggccgccg aggccggatt atttgtttaa agctgtaagg agaaatgact aaqtqctgtc aqagttgtga agccaagccc ccactgatca acaaaacctg ct tct t tcag gaccagataa agatcctaca gaacagcgga caggagtttg cttcttaata tcaagctaca agctcacagc caacttcatc ccagaaatga aaaccccttc ccaaggaaga tgqcgcccgg tgcctctcaa acgacggcgq cgtccacccc ccaaggacga aggaggagga ccaaccccgc cgaccccatc cgtctgcgtc cgccccagca tgctcccgcg ccgcgctcta tgctcaagga cagaagccag tctgtgggga tcttctttaa gcatcgttga aggctggcat qagcact gga taagccagag acctgttaat acacctccag tagtcaagtg ctctcattca aacatgtcag tgaaagaatc tcaagcttca caattccttt ttagagagct gtttctatca tgtactgctt tgtctgaagt tctttcataa ttcccgcttc gcgct ccccg tcacgcctta ggccggggct ggtcgctgta cgcgtaccct aggcgcggag agccttcccg cagacccqggg ctcctcgggg gggcccgttc ggacggcctg ccctgcactc gggcctgccq ccagagccca tgaagcatca gagggcaatg taaaatccgc ggtccttgga tgctqttqct attcactttt gagcat tgaa ttctttgctq gtctaaatca gtattcttgg tgggcagatg atcattctat agttagccaa ggaagggcta catcaaggca acttacaaaa gaatacattt tattgctgca tcagcgccca ctgqccacca ttggcagccc qccagcgcct ggcgacttcc ctctatagcg gcct ccgcgc gatttcccgt gaagcggcgg tcgaccctgg gcgccgccgc Ccctccacct ggcct caacg caggtctacc caatacagct ggctgtcatt qaagggqagc agaaaaaact ggtcgaaaat ctcccacaac tcaccaggtc ccagatgtga acaagtctta ttgccaggtt atgagcttaa ctgtattttg tcattatgcc gaagagttcc cgaagtcaaa attggtttga cttcttgata atccagtccc caattaccca gggtcgccct 1020 cggtgatgga 1080 gcactcggca 1140 ttgccccgcc 1200 ccgactgcgc 1260 acttccagcc 1320 gctccccgcg 1380 tggqgccacc 1440 tgacggccgc 1500 agtgcatcct 1560 cctgcaaggc 1620 ccgcctctgc 1680 ggctcccgca 1740 cgccctatct 1800 tcgagtcatt 1860 atggtgtcct 1920 acaactactt 1980 gcccagcatg 2040 ttaaaaagtt 2100 cattgggcgt 2160 aagacataca 2220 tctatqcagg 2280 atcaactagg 2340 ttcgaaactt 2400 tggtgtttgg 2460 cacctgatct 2520 ttaccatgtg 2580 tctgtatgaa 2640 cccagtttga 2700 ggcaaaaagg 2-7-60 acttgcatga 2820 gggcactgag 2880 agatattggc 2940 2970

Claims (2)

1. 28-02-'05 09:04 FROM- T-683 P007/022 F-605 MOtflnMA,,a..4.Ufl~ *n~aOAFMZ44WV+i-.imddums dftcc.ZL ooooo 9 9 9 9 099* oo@o 0 9. 9 9 9.* 99 9 99 9. 9* .9* 9 9 *9 9 9 9 99 9* 9 9 9 9 9. 9 9 *9 9 9 9. 9 99 9 99 9 9 o o: *oo oo 47 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. Use of 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 use of claim 3, wherein the blood clotting factor is a human blood clotting factor. 5. The use of claim 4, wherein the human blood clotting factor is selected from the group consisting of factor VIII, factor IX, and von Willebrand Factor (vWF). 6. The use of anyone of claims 1 to 5, wherein the nucleic acid construct comprises 1 to 20 HRE(s). 7. The use of claim 6, wherein the nucleic acid construct comprises 3 to 10 HREs. 8. The use of anyone of claims 1 to 7, wherein the at least one HRE is a steroid responsive element. 9. The use of claim 8, wherein the steroid responsive element a progesterone responsive element (PRE). COMS ID No: SBMI-01139823 Received by IP Australia: Time 09:12 Date 2005-02-28 28-02-'05 09:05 FROM- T-683 P008/022 F-605 P. ttFsir0awt44-MW AmmnUVAS0Wx"frnPw&=ntc2WSS 48 The use of claim 4, wherein the 1RE is a PRE and the blood clotting factor is factor Ix. 11. The use of claim 10, wherein the factor IX has a nucleotide sequence corresponding to nucleotides 689 to 2071 of SEQ ID NO: 1. 12. The use of claim 6, wherein the HRE is a PRE and the blood clotting factor is factor VIM. .13 The use of anyone of claims 8 to 12, wherein the PRE has a double stranded DNA sequence comprising SEQ ID NOs: 3 and 4. 14. The use of anyone of claims 1 to 13, 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. use of claim 14, wherein the construct further comprises a tissue specific :promoter. 16. The use of claim 15, wherein the tissue specific promoter is an c-antitrypsin promoter. 17. The use according to anyone of claims 1 to 16, wherein the hormone- hormone receptor complex is a steroid-steroid receptor complex. 18. The use of claim 17, wherein the molar ratio of TRE within the nucleic acid construct to hormone receptor is from 1:1 to 1:10 and/or the molar ratio of hormone to hormone receptor is at least 1,000:1. COMS ID No: SBMI-01139823 Received by IP Australia: Time 09:12 Date 2005-02-28 28-02-'05 09:05 FROM- T-683 P009/022 F-605 49 19. The use of claim 18, wherein the molar ratio of HRE within the nucleic acid construct to hormone receptor is from 1:2 to 1:5 and/or the molar ratio of hormone to hormone receptor is at least 10,000:1. The use of any one of claims 17 to 19, wherein the receptor is a progesterone receptor and the steroid is progesterone or a progesterone derivative. 21. The use of claim 20, 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 corresponding to nucleotides 557 to 933 SEQ ID NO: 18. o 22. A pharmaceutical composition comprising a nucleic acid construct comprising at least one HRE and a transgene as defined in claims 1 to 16 and/or (ii) a vector comprising said nucleic acid construct, said at least one HRE being bound to a hormone-hormone receptor complex. o* 23. The pharmaceutical composition of claim 22, wherein the hormone- hormone receptor complex is as defined in claims 17 to 21. 24. The pharmaceutical composition of claim 22, wherein the transgene is a gene encoding Sa blood clotting factor. The pharmaceutical composition of claim 24 wherein the blood clotting factor is factor IX. 26. The pharmaceutical composition of claim 24 wherein the blood clotting factor is factor VIII. 27. The pharmaceutical composition of anyone of claims 24 to 26, which is suitable for gene transfer. COMS ID No: SBMI-01139823 Received by IP Australia: Time 09:12 Date 2005-02-28 28-02-'05 14:10 FROM- T63P0/1 -1 T-693 P005/010 F-624 P ~OPM 28. The pharmaceutical composition of anyone of claims 24 to 26, which is suitable for treating hemophilia.
2. 29. A use according to anyone of claims 1 to 21 substantially as described hereinibefore with reference to the examples and/or figures. A pharmaceutical composition according to anyone of claims 22 to 28 substantially as described hereinbefore wvith reference to the examples and/or figures. DATED tiS 2 80' day of FEBRUARY, 2005 Octoagene GmnbH by DAVIES COLLISON CAVE Patent attorneys for the applicant COMS ID No: SBMI-01140374 Received by IP Australia: Time 14:15 Date 2005-02-28