EP1049779A2 - Lh-receptor gene promoter for tissue-specific gene expression - Google Patents

Lh-receptor gene promoter for tissue-specific gene expression

Info

Publication number
EP1049779A2
EP1049779A2 EP99908806A EP99908806A EP1049779A2 EP 1049779 A2 EP1049779 A2 EP 1049779A2 EP 99908806 A EP99908806 A EP 99908806A EP 99908806 A EP99908806 A EP 99908806A EP 1049779 A2 EP1049779 A2 EP 1049779A2
Authority
EP
European Patent Office
Prior art keywords
sequence
gene
nucleic acid
acid molecule
recombinant nucleic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99908806A
Other languages
German (de)
French (fr)
Inventor
Olaf G. Wilhelm
Manfred Schmitt
Sabine Wilhelm
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Heidelberg Pharma AG
Original Assignee
Wilex Biotechnology GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wilex Biotechnology GmbH filed Critical Wilex Biotechnology GmbH
Priority to EP99908806A priority Critical patent/EP1049779A2/en
Publication of EP1049779A2 publication Critical patent/EP1049779A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention generally relates to the tissue-specific expression of heterologous genes and particularly to the expression of heterologous genes in human ovarian-derived cells.
  • the luteinizing hormone receptor plays a crucial role in reproduction. Upon binding of its ligands, the luteinizing hormone (LH) and human choriongonadotropin (HCG), the receptor activates adenylate cyclase and phospholipase D which induces the synthesis and secretion of steroid hormones.
  • the luteinizing hormone receptor belongs to the family of seven transmembrane G protein coupled receptors. Short soluble variant forms of the luteinizing hormone receptor devoid of transmembrane domains have been detected in porcine and rat species.
  • Atger et al. (Mol. Cell. Endocrin. 1 1 1 ( 1 995), 1 1 3-1 23) have determined the complete organization of the human LH-R gene and the structure of 1 591 bp of its 5'-flanking region.
  • the gene spans over 70 kbp and contains 1 1 exons.
  • the first 10 exons and part of the last exon encode the extracellular domain of the receptor while the transmembrane and intracellular domains are encoded by the remaining part of the last exon.
  • the gene encodes a 701 amino acids long preprotein.
  • the transcription initiation site is located 1 085 bp upstream of the initiation codon.
  • the promoter region is different from the murine LH-R promoter, contains two putative TATA boxes at positions -34 and -47 and a CAAT box consensus sequence at position -89.
  • a consensus sequence corresponding to a cAMP responsive element is found at position -698.
  • Seven AP, consensus sequences are also found in the 5'-flanking region of the gene.
  • Huhtaniemi et al ol. Cell. Edocrinol. 88 (1992), 55-66) disclose that isolated promoter segments from the LH-receptor gene show only low functional activity as verified in transient expression studies in immature rat granulosa cells using the luciferase coding region as the reporter for promoter activity.
  • the promoter element seems to be still under tissue- specific control, since no promoter activity was detected in CHO cells.
  • Owing to the extremely low transcription activity of the LH-receptor promoter up to 3 %) as compared to the positive control of the CMV promoter, the LH-receptor promoter seems entirely unsuitable as expression system for heterologous genes, e.g. in gene therapy.
  • Dorigo and Berek (Int.J.Gynecol. Cancer 7 (1997), 1 -13) describe several strategies in gene therapy for ovarian cancer, such as gene transfer systems, immuno-gene therapy, anti-oncogene and tumor suppressor gene therapy, growth factors and cytokines, prodrug therapy and drug resistance genes.
  • gene transfer systems immuno-gene therapy, anti-oncogene and tumor suppressor gene therapy, growth factors and cytokines, prodrug therapy and drug resistance genes.
  • the object underlying the present invention was to provide novel means and methods for the diagnosis and therapy of ovarian diseases, particularly ovarian carcinoma.
  • promoter sequences of the LH-receptor gene can be used for cell-specific expression of heterologous genes. It was particularly surprising to learn that high gene expression was obtained while cell specificity was retained. In this fashion a method for the ovarian cell-specific expression of heterologous genes, e.g. reporter genes or toxic genes, is provided which allows new approaches to the diagnosis and therapy of ovarian diseases, particularly ovarian cancers.
  • a first aspect of the present invention relates to a recombinant nucleic acid molecule comprising a LH-receptor gene promoter sequence capable of selectively directing gene expression in human ovarian-derived cells.
  • LH-receptor gene promoter can act as highly tissue-specific and efficient expression systems for heterologous genes and thus are suitable for therapeutic application, e.g. in tumor therapy.
  • longer LH-receptor gene promoter fragments were incapable of directing an efficient gene expression.
  • LH-receptor gene promoter fragments comprising the nucleotide sequence from position 1 351 - 1 591 or from 2503- 2678 as shown in SEQ ID NO.1 (corresponding from position -241 to -1 or from position + 909 to 1085 according to Atger et al., 1 995) allow efficient and tissue-specific transcription of heterologous genes, e.g.
  • the LH-receptor gene promoter sequence of the present invention is a sequence which comprises tissue-specific transcription-enhancing elements but which is free from repressor elements which inhibit an efficient transcription of heterologous genes. Such repressor elements are e.g. located in the region between positions 1 1 71 and 1 350 as shown in SEQ ID NO.1 .
  • the LH-receptor gene promoter sequence of the invention may be selected from transcriptionally active and/or tissue-specific regulation elements of the nucleotide sequence as shown in SEQ ID NO.1 optionally in combination with heterologous promoter sequence elements.
  • the LH-receptor gene promoter sequence may be selected from a sequence comprising: (a) the nucleotide sequence from position 1351 -2678 as shown in SEQ
  • the recombinant nucleic acid molecule of the present invention is preferably free from further non-promoter sequences of the region of the human LH- receptor gene, in particular free from coding sequences of the LH-receptor gene.
  • the recombinant nucleic acid molecule of the present invention can be a DNA or RNA molecule.
  • the nucleic acid molecule is a vector, i.e. a nucleic acid molecule which is capable of being propagated in a suitable host c. II, e.g. by autonomous replication and/or by integration into the host cell chromosome.
  • the vector is a eukaryotic vector, more preferably a vector capable of being propagated in a mammalian, e.g. human cell.
  • Suitable vectors are plasmids or viral vectors such as retroviruses, adenoviruses, adeno-associated viruses, artificial viruses or other gene transfer vehicles (Miller and Vile, FASEB J.9 (1 995), 1 90-1 99 and references cited therein).
  • the recombinant nucleic acid molecule contains the LH-receptor gene promoter sequence operatively linked to a heterologous gene, i.e. the promoter is capable of directing the expression of the heterologous gene, i.e. a gene which is different from the human LH-receptor gene.
  • the heterologous gene may be a reporter gene such as the luciferase gene, the green fluorescent protein gene, or the ⁇ - galactosidase gene.
  • the heterologous gene may be a toxin or suicide gene such as the ricin gene, the cholera toxin gene, the E.coli cytosine deaminase gene or the Herpes simplex virus thymidine kinase gene.
  • the recombinant nucleic acid vector of the present invention is capable of being propagated in a host cell, preferably in a human host cell.
  • Such propagation may include extrachromosomal propagation, e.g. mediated by a suitable origin of replication located on said vector, or integration into the host cell genome, e.g. by using a viral vector as described above or by using a vector suitable for gene targeting.
  • a gene targeting vector comprises at least two DNA sequences homologous to a target sequence present in a target cell, e.g.
  • the LH-receptor gene promoter sequence optionally the sequence coding for a heterologous gene located between the homologous DNA sequences, and optionally a selection marker gene also located between the homologous DNA sequences.
  • the homologous DNA sequences flanking the promoter sequence preferably have a length of at least 1 50 bp each.
  • the selection marker gene may be any selection marker gene, preferably one that is suitable for eukaryotic cells, which upon selection results in a selectable phenotype, e.g. a gene resistant to antibiotics or an auxotrophy gene.
  • the LH-receptor gene promoter sequence according to the present invention is a sequence capable of selectively directing gene expression in human ovarian-derived cells.
  • the promoter sequence is capable of an at least two times, more preferably at least three times higher expression in human ovarian-derived cells than in other human cells, e.g. human fibroblast cells.
  • the promoter sequence of the present invention is capable of selectively directing gene expression in human ovarian cancer cells, e.g. with a selectivity at least two times higher than in other human cancer cells, e.g. kidney cancer cells or colon cancer cells.
  • SEQ ID NO.1 the 5'-region of the LH-receptor gene including the promoter is shown.
  • a known transcription starting point is at nucleotide 1 592.
  • the translation starts at nucleotide 2679.
  • Variants of this sequence e.g. as shown in Example 3, are also encompassed by the present invention.
  • the LH-receptor gene promoter sequence may be selected from a sequence comprising:
  • nucleotide sequence having at least 70 %, preferably at least 80 %, and more preferably at least 90 % sequence identity to the sequence of (a), and (c) a fragment of the nucleotide sequences of (a) or (b), which has a length of preferably at least 50, and more preferably at least 100 nucleotides, capable of selectively directing gene expression in human ovarian-derived cells.
  • the LH-receptor gene promoter sequence may be selected from a sequence comprising
  • (c) a fragment of the nucleotide sequences of (a) or (b) having preferably a length of at least 50 nucleotides, more preferably of at least 1 00 nucleotides, capable of selectively directing gene expression in human ovarian-derived cells.
  • promoter sequence may also include both preferred sequences as defined above.
  • the promoter of the present invention preferably comprises at least one regulatable sequence element, e.g. sequence elements that are responsive to substances selected from their own ligand LH, follicle stimulating hormone (FSH), estradiol, growth factors and gonatropin releasing hormone.
  • regulatable sequence element e.g. sequence elements that are responsive to substances selected from their own ligand LH, follicle stimulating hormone (FSH), estradiol, growth factors and gonatropin releasing hormone.
  • ovarian-derived cells comprises ovarian cells, cell lines derived from ovarian cells including ovarian cancer cells, ovarian tissues, tissues such as uterus and uterine arteries, fallopian tubes and placenta, and cells or cell lines derived therefrom.
  • the LH-receptor gene promoter sequence according to the present invention may also be a hybrid sequence comprising, on the one hand, sequence elements responsible for tissue-specific expression and, on the other hand, sequence elements from different promoter sequences, such as TATA box, CAAT box etc.
  • the present invention further relates to a method of selectively expressing genes (different from LH-receptor gene) in human ovarian-derived cells comprising the steps:
  • the nucleic acid molecule is preferably located on a vector as described above which may be extrachromosomally or chromosomally located within the cell.
  • the expression of the heterologous gene may be regulated, e.g. stimulated, by various substances as described above.
  • the method may be used for cell-specific expression of heterologous genes in human ovarian-derived cells, e.g. in vitro cultured cells or human tissue, to substitute for missing genes, to replace mutated genes, to express foreign genes which may subsequently facilitate cell-specific targeting of human ovarian-derived tissue, or to enable cell-specific killing of human ovarian-derived tissue in diseases such as human ovarian cancer.
  • a pharmaceutical composition comprising as an active agent the recombinant nucleic acid molecule as described above, optionally in combination with other pharmaceutically active agents and pharmaceutically acceptable carriers, diluents and adjuvants.
  • Administration of the pharmaceutical composition may be accomplished by known gene-therapeutic methods such as illustrated by Dorigo and Berek (Int.J.Gynecol. Cancer 7 ( 1 997), 1 -1 3) and references cited therein which describe several clinical protocols.
  • the pharmaceutical composition of the present invention is preferably for use in the diagnosis and treatment of ovarian diseases, more preferably for use in the diagnosis and treatment of ovarian cancer.
  • ovarian and non-ovarian diseases which might be important for application are tube carcinoma, endometriosis, malign trophoblast diseases, invasive complete and partial vesicular mole and chorionepithelioma (the LH receptor is also formed in the tubes and the placenta).
  • Fig. 1 shows a comparison of the expression of different LH-R promoter luciferase constructs in human fibroblast cells and human ovarian cancer cells;
  • Fig. 2 shows a comparison of the expression of LH-R promoter
  • Fig. 3 shows a comparison of the expression of different LH-R promoter HSVtk constructs in the cell line HT-1 080
  • Fig. 4 shows a comparison of the expression of different LH-R promoter HSVtk constructs in the cell line NIH:OVCAR-3
  • Example 1 shows a comparison of the expression of different LH-R promoter HSVtk constructs in the cell line NIH:OVCAR-3
  • Total genomic DNA was isolated from human ovarian cancer cell line NIH:OVCAR-3 (Hamilton et al., Cancer Res.43 ( 1 983), 3379-5389) .
  • the 5'- flanking region of the LH-R gene promoter was isolated by PCR using primers based on the published gene sequence (Atger et al. (1 995), supra) .
  • the promoter fragments were cloned into pGL2-basic vector (Promega Corporation, USA). Transient transfection experiments to analyze promoter activity were performed as follows:
  • 6/3-LHR is a vector containing the 241 bp upstream of the transcription initiation site.
  • Vector 6/9-LHR contains the 721 bp upstream of the transcription initiation site.
  • the vectors were transfected into human fibroblast HT-1080 cells (Rasheed et al. Cancer 33 ( 1 974), 1027-1033) and human ovarian cancer NIH:OVCAR-3 cells.
  • a vector containing /?-galactosidase as reporter gene was co-transfected in order to normalize transfection efficacy.
  • luciferase activity was detected according to the manufacturer's protocol (Promega Corp., Madison, Wisconsin, USA) .
  • the 6/3-LHR vector was compared with the pGL2- control vector (Promega Corporation, USA) containing the SV40 promoter in order to determine promoter activity in different cell lines.
  • the activity of the SV40 promoter was set as 100 %. After 48 hours luciferase activity in cell extracts was assayed.
  • LHCGR-P luteinizing hormone-choriogonadotropin receptor promoter
  • HSVtk herpes simplex virus thymidine kinase
  • the 240 bp fragment was identified on an agarose gel and ligated into the multiple cloning site of the luciferase vector pGL2 Basic (Promega) which was digested with the restriction enzymes Kpnl and Bglll. The correct sequence was verified by sequencing.
  • LHCGR-P fragment 421 (consisting of 421 bp and corresponding to position -1 to -420 of the LHCGR-P sequence according to Atger et al., 1 995) was isolated from genomic DNA of lymphocytes by polymerase chain reaction (PCR) (5'primer: 20 nt containing a Kpnl restriction site at the 5'priming end and the 3'primer: 21 nt containing a Bglll restriction site at the 3'priming site).
  • PCR polymerase chain reaction
  • the 421 bp fragment was identified on an agarose gel and ligated into the linearized TA cloning vector pCR2.1 from Invitrogen (USA/Netherlands) . The correct sequence was verified by sequencing. After digestion with the restriction enzymes Kpnl and Bglll, the 421 bp fragment was isolated from the TA-cloning vector and ligated into the luciferase vector pGL2 Basic (Promega) which was digested with the restriction enzymes Kpnl and Bglll.
  • the LHCGR-P fragment 1 76 (consisting of 1 76 bp and corresponding to position + 909 to + 1085 of the LHCGR-P sequence according to Atger et al., 1 995) was isolated from genomic DNA of ovarian cancer cells NIH:OVCAR-3 by polymerase chain reaction (PCR) (5'primer: 1 8 nt containing a Kpnl restriction site at the 5'priming site and the 3'primer: 1 8 nt containing a Bglll restriction site at the 3'priming site).
  • the 1 76 bp fragment was identified on an agarose gel and ligated into the linearized TA cloning vector pGlow-TOPO from Invitrogen (USA/Netherlands).
  • the 1 76 bp fragment was isolated from the TA-cloning vector and ligated into the luciferase vector pGL2 Basic (Promega) which was digested with the restriction enzymes Kpnl and Bglll.
  • the HSVtk DNA sequence (accession # J02224) corresponding to position ATG 51 5-1 646 of the HSVtk gene was amplified by PCR from pBR322 plasmid (5'primer: atggcttcgtacccctgc containing a Bglll restriction site at the 5'priming site and the 3'primer: tcagttagcctccccat containing a Hindlll restriction site at the 3'priming site) .
  • LHCGR-P fragments 1 76, 241 , 421 operatively linked to HSVtk in the pCDNA3.1 + vector (Invitrogen).
  • pCDNA3.1 - vector (Invitrogen) which contains the CMV promoter and HSVtk gene.
  • the pCDNA3.1 - vector was restricted with BamHI and Hindlll and the amplified HSVtk sequence (51 5-1 646) was cloned with Bglll and Hindlll restriction sites into this plasmid such that the HSVtk gene is under the control of the CMV promoter.
  • the pcDNA3.1 + vector alone without LHCGR-P/HSVtk insert As a negative control we used the pcDNA3.1 + vector alone without LHCGR-P/HSVtk insert.
  • the herpes simplex virus thymidine kinase suicide gene expressed by cells is capable of converting the normally nontoxic substance ganciclovir into a toxic metabolite which leads to cell death. Since the expression of the LHCG-R gene mainly occurs in gonadal tissue, we studied the expression of the HSVtk gene under control of cell-specific promoter fragments ( 1 76, 241 , 421 ) in ovarian cancer cells (OVCAR-3) and control cells (HT-1080) . Cells were then treated with ganciclovir and cell viability was measured in the MTT assay (Alley et al, Cancer Res., 48:589-601 , 1 988) which measures viable cell dehydrogenase activity.
  • the cell lines OVCAR-3 and HT-1 080 were seeded and cultured in 24-well plates at a density of 1 .6 x 10 5 and 8 x 10 4 respectively in 500 ⁇ of DMEM ( 10 % fetal calf serum) overnight. One hour before transfection another change of DMEM ( 10 % FCS) was made. Immediately before transfection the culture medium was removed from the wells and suspensions of vectors containing LHCGR-P fragments 241 , 421 , 1 76 operatively linked to the HSV tk gene at a concentration of 1 //g/ml- DNA and 0.5 //g/ml lipofectin reagent (Gibco) in DMEM without FCS were added to the cells.
  • DMEM 10 % fetal calf serum
  • Fig. 3 shows the ganciclovir activity of the control cell line HT-1080 in the MTT viability assay as described above.
  • the data are representative of three experiments done in quadruplicate. The standard deviation (SD) is shown in bars. It can be gathered from Fig. 3 that the growth of the control cell line is not significantly inhibited by the HSVtk gene under the control of the LH-R promoter fragments.
  • the negative control (pcDNA3.1 ) and the positive control (HSVtk) yield the expected results.
  • Fig. 4 shows the measurement of ganciclovir sensitivity of NIH:OVCAR-3 cells in the MTT viability assay.
  • the data are representative of three experiments done in quadruplicate.
  • the standard deviation (SD) is shown in bars.
  • nucleotide sequences from LH-R promoter regions as published by Atger et al., 1 995 (supra) were compared to own analyzed sequences of genomic DNA from ovarian cancer cell (NIH:OVCAR-3) lymphocytes and ovarian cancer tissue.
  • lymphocyte DNA we found a T in position 91 6, a G in position 1098, an A in position 1 120, a T in position 1 31 5, an A in position 141 1 , a C in position 1 560, a G in position 1 672, a G in position 1 700 and a G in position 1 879.
  • the sequence obtained from the ovarian cancer cell line NIH:OVCAR-3 was identical to the published sequence.

Abstract

The invention relates to a recombinant nucleic acid molecule comprising a LH-receptor gene promoter sequence capable of selectively directing gene expression in human ovarian-derived cells.

Description

LH-receptor gene promoter for tissue-specific gene expression
Description
The invention generally relates to the tissue-specific expression of heterologous genes and particularly to the expression of heterologous genes in human ovarian-derived cells.
The luteinizing hormone receptor (LH-R) plays a crucial role in reproduction. Upon binding of its ligands, the luteinizing hormone (LH) and human choriongonadotropin (HCG), the receptor activates adenylate cyclase and phospholipase D which induces the synthesis and secretion of steroid hormones. The luteinizing hormone receptor belongs to the family of seven transmembrane G protein coupled receptors. Short soluble variant forms of the luteinizing hormone receptor devoid of transmembrane domains have been detected in porcine and rat species. These variants are generated by alternative splicing (for an overview see: Segaloff and Ascoli, Endocrine Reviews 14 ( 1 993), 324-347; McFarland et al., Science 245 ( 1 989), 494- 499; Misrahi et al. in: The Ovary (Adashi and Leung, eds.), pp. 57-92 ( 1 993), Raven Press, New York; Loosfelt et al., Science 245 ( 1 989), 525- 528).
Atger et al. (Mol. Cell. Endocrin. 1 1 1 ( 1 995), 1 1 3-1 23) have determined the complete organization of the human LH-R gene and the structure of 1 591 bp of its 5'-flanking region. The gene spans over 70 kbp and contains 1 1 exons. The first 10 exons and part of the last exon encode the extracellular domain of the receptor while the transmembrane and intracellular domains are encoded by the remaining part of the last exon. The gene encodes a 701 amino acids long preprotein. The transcription initiation site is located 1 085 bp upstream of the initiation codon. The promoter region is different from the murine LH-R promoter, contains two putative TATA boxes at positions -34 and -47 and a CAAT box consensus sequence at position -89. A consensus sequence corresponding to a cAMP responsive element is found at position -698. Seven AP, consensus sequences are also found in the 5'-flanking region of the gene.
Huhtaniemi et al ( ol. Cell. Edocrinol. 88 (1992), 55-66) disclose that isolated promoter segments from the LH-receptor gene show only low functional activity as verified in transient expression studies in immature rat granulosa cells using the luciferase coding region as the reporter for promoter activity. The promoter element seems to be still under tissue- specific control, since no promoter activity was detected in CHO cells. Owing to the extremely low transcription activity of the LH-receptor promoter (up to 3 %) as compared to the positive control of the CMV promoter, the LH-receptor promoter seems entirely unsuitable as expression system for heterologous genes, e.g. in gene therapy.
Human ovarian cancer spreads from the ovaries into the entire abdominal cavity. Distant metastases, for example in the lung, bones or liver are rarely found. Ovarian cancer is treated, on the one hand, by radical elimination of all tumors within the intraperitoneal cavity (removal of the uterus, removal of the ovaries, tubes, the greater omentum if necessary, parts of the intestine, the lymph nodes within the range of large vessels etc.) and, on the other hand, by postoperative chemotherapy (cisplatin, carboplatin and/ortaxol). Literature: John T.Soper. Epithelial Ovarian Cancer. In: Clinical Obstetrics and Gynecology (eds. Roy M.Pitkin, James R.Scott) Vol.37, No.2, 375-493, 1994).
Dorigo and Berek (Int.J.Gynecol. Cancer 7 (1997), 1 -13) describe several strategies in gene therapy for ovarian cancer, such as gene transfer systems, immuno-gene therapy, anti-oncogene and tumor suppressor gene therapy, growth factors and cytokines, prodrug therapy and drug resistance genes. However, none of these methods has found clinical approval up to now.
The object underlying the present invention was to provide novel means and methods for the diagnosis and therapy of ovarian diseases, particularly ovarian carcinoma. Surprisingly, it was found that promoter sequences of the LH-receptor gene can be used for cell-specific expression of heterologous genes. It was particularly surprising to learn that high gene expression was obtained while cell specificity was retained. In this fashion a method for the ovarian cell-specific expression of heterologous genes, e.g. reporter genes or toxic genes, is provided which allows new approaches to the diagnosis and therapy of ovarian diseases, particularly ovarian cancers.
A first aspect of the present invention relates to a recombinant nucleic acid molecule comprising a LH-receptor gene promoter sequence capable of selectively directing gene expression in human ovarian-derived cells.
Surprisingly, it was found that short fragments from the LH-receptor gene promoter can act as highly tissue-specific and efficient expression systems for heterologous genes and thus are suitable for therapeutic application, e.g. in tumor therapy. In contrast thereto, longer LH-receptor gene promoter fragments were incapable of directing an efficient gene expression. More particularly, it was found that LH-receptor gene promoter fragments comprising the nucleotide sequence from position 1 351 - 1 591 or from 2503- 2678 as shown in SEQ ID NO.1 (corresponding from position -241 to -1 or from position + 909 to 1085 according to Atger et al., 1 995) allow efficient and tissue-specific transcription of heterologous genes, e.g. the luciferase gene or the herpes simplex virus thymidine kinase gene. In contrast thereto, however, long fragments, e.g. 421 or 721 bp fragments comprising the nucleotide sequence from position 1 1 71 -1 591 or from 870-1 591 as shown in SEQ ID NO.1 (corresponding from -1 to -420 or from -1 to -720 according to Atger et al., 1 995), did not exhibit any significant transcriptional activity. Thus, the LH-receptor gene promoter sequence of the present invention is a sequence which comprises tissue-specific transcription-enhancing elements but which is free from repressor elements which inhibit an efficient transcription of heterologous genes. Such repressor elements are e.g. located in the region between positions 1 1 71 and 1 350 as shown in SEQ ID NO.1 .
Thus, the LH-receptor gene promoter sequence of the invention may be selected from transcriptionally active and/or tissue-specific regulation elements of the nucleotide sequence as shown in SEQ ID NO.1 optionally in combination with heterologous promoter sequence elements.
More particularly, the LH-receptor gene promoter sequence may be selected from a sequence comprising: (a) the nucleotide sequence from position 1351 -2678 as shown in SEQ
ID NO.1 , (b) a nucleotide sequence having at least 70 %, preferably at least
80 %, and more preferably at least 90 % sequence identity to the sequence of (a), and (c) a fragment of the nucleotide sequences of (a) or (b) which has a length of preferably at least 50, and more preferably at least 100 nucleotides, capable of selectively directing gene expression in human ovarian-derived cells
The recombinant nucleic acid molecule of the present invention is preferably free from further non-promoter sequences of the region of the human LH- receptor gene, in particular free from coding sequences of the LH-receptor gene.
The recombinant nucleic acid molecule of the present invention can be a DNA or RNA molecule. Preferably, the nucleic acid molecule is a vector, i.e. a nucleic acid molecule which is capable of being propagated in a suitable host c. II, e.g. by autonomous replication and/or by integration into the host cell chromosome. Preferably, the vector is a eukaryotic vector, more preferably a vector capable of being propagated in a mammalian, e.g. human cell. Examples for suitable vectors are plasmids or viral vectors such as retroviruses, adenoviruses, adeno-associated viruses, artificial viruses or other gene transfer vehicles (Miller and Vile, FASEB J.9 (1 995), 1 90-1 99 and references cited therein).
In a preferred embodiment of the present invention the recombinant nucleic acid molecule contains the LH-receptor gene promoter sequence operatively linked to a heterologous gene, i.e. the promoter is capable of directing the expression of the heterologous gene, i.e. a gene which is different from the human LH-receptor gene. The heterologous gene may be a reporter gene such as the luciferase gene, the green fluorescent protein gene, or the β- galactosidase gene. On the other hand, the heterologous gene may be a toxin or suicide gene such as the ricin gene, the cholera toxin gene, the E.coli cytosine deaminase gene or the Herpes simplex virus thymidine kinase gene.
The recombinant nucleic acid vector of the present invention is capable of being propagated in a host cell, preferably in a human host cell. Such propagation may include extrachromosomal propagation, e.g. mediated by a suitable origin of replication located on said vector, or integration into the host cell genome, e.g. by using a viral vector as described above or by using a vector suitable for gene targeting. Such a gene targeting vector comprises at least two DNA sequences homologous to a target sequence present in a target cell, e.g. a human cell, the LH-receptor gene promoter sequence, optionally the sequence coding for a heterologous gene located between the homologous DNA sequences, and optionally a selection marker gene also located between the homologous DNA sequences. The homologous DNA sequences flanking the promoter sequence preferably have a length of at least 1 50 bp each. For more details reference is made to the gene targeting methods described in WO 90/1 1 354 and WO 91 /09955.
The selection marker gene may be any selection marker gene, preferably one that is suitable for eukaryotic cells, which upon selection results in a selectable phenotype, e.g. a gene resistant to antibiotics or an auxotrophy gene.
The LH-receptor gene promoter sequence according to the present invention is a sequence capable of selectively directing gene expression in human ovarian-derived cells. Preferably, the promoter sequence is capable of an at least two times, more preferably at least three times higher expression in human ovarian-derived cells than in other human cells, e.g. human fibroblast cells. More preferably, the promoter sequence of the present invention is capable of selectively directing gene expression in human ovarian cancer cells, e.g. with a selectivity at least two times higher than in other human cancer cells, e.g. kidney cancer cells or colon cancer cells.
In SEQ ID NO.1 the 5'-region of the LH-receptor gene including the promoter is shown. A known transcription starting point is at nucleotide 1 592. The translation starts at nucleotide 2679. Variants of this sequence, e.g. as shown in Example 3, are also encompassed by the present invention.
According to one preferred embodiment, the LH-receptor gene promoter sequence may be selected from a sequence comprising:
(a) the nucleotide sequence from position 1 351 to 1 591 as shown in SEQ ID NO.1 ,
(b) a nucleotide sequence having at least 70 %, preferably at least 80 %, and more preferably at least 90 % sequence identity to the sequence of (a), and (c) a fragment of the nucleotide sequences of (a) or (b), which has a length of preferably at least 50, and more preferably at least 100 nucleotides, capable of selectively directing gene expression in human ovarian-derived cells.
Surprisingly, it was found that a sequence immediately located upstream the known translation starting point is also capable of effectively directing expression of heterologous genes. Thus, according to another preferred embodiment, the LH-receptor gene promoter sequence may be selected from a sequence comprising
(a) the nucleotide sequence from position 2503 to position 2678 as shown in SEQ ID NO.1 ( 1 76 bp upstream to 1 bp downstream of the translation initiation site of the LH-receptor gene),
(b) a nucleotide sequence having at least 70 %, preferably at least 80 %, and more preferably at least 90 % sequence identity to the sequence of (a), and
(c) a fragment of the nucleotide sequences of (a) or (b) having preferably a length of at least 50 nucleotides, more preferably of at least 1 00 nucleotides, capable of selectively directing gene expression in human ovarian-derived cells.
Further, the promoter sequence may also include both preferred sequences as defined above.
The promoter of the present invention preferably comprises at least one regulatable sequence element, e.g. sequence elements that are responsive to substances selected from their own ligand LH, follicle stimulating hormone (FSH), estradiol, growth factors and gonatropin releasing hormone.
The term "ovarian-derived cells" according to the present invention comprises ovarian cells, cell lines derived from ovarian cells including ovarian cancer cells, ovarian tissues, tissues such as uterus and uterine arteries, fallopian tubes and placenta, and cells or cell lines derived therefrom.
The LH-receptor gene promoter sequence according to the present invention may also be a hybrid sequence comprising, on the one hand, sequence elements responsible for tissue-specific expression and, on the other hand, sequence elements from different promoter sequences, such as TATA box, CAAT box etc.
The present invention further relates to a method of selectively expressing genes (different from LH-receptor gene) in human ovarian-derived cells comprising the steps:
(a) providing a recombinant nucleic acid molecule as described above,
(b) introducing said recombinant nucleic acid molecule in human ovarian- derived cells, whereby a LH-promoter gene sequence operatively linked tc a heterologous gene in said cells is obtained, and
(c) expressing said heterologous gene.
The nucleic acid molecule is preferably located on a vector as described above which may be extrachromosomally or chromosomally located within the cell. The expression of the heterologous gene may be regulated, e.g. stimulated, by various substances as described above.
The method may be used for cell-specific expression of heterologous genes in human ovarian-derived cells, e.g. in vitro cultured cells or human tissue, to substitute for missing genes, to replace mutated genes, to express foreign genes which may subsequently facilitate cell-specific targeting of human ovarian-derived tissue, or to enable cell-specific killing of human ovarian-derived tissue in diseases such as human ovarian cancer.
Further, according to the present invention a pharmaceutical composition is provided comprising as an active agent the recombinant nucleic acid molecule as described above, optionally in combination with other pharmaceutically active agents and pharmaceutically acceptable carriers, diluents and adjuvants. Administration of the pharmaceutical composition may be accomplished by known gene-therapeutic methods such as illustrated by Dorigo and Berek (Int.J.Gynecol. Cancer 7 ( 1 997), 1 -1 3) and references cited therein which describe several clinical protocols.
The pharmaceutical composition of the present invention is preferably for use in the diagnosis and treatment of ovarian diseases, more preferably for use in the diagnosis and treatment of ovarian cancer. Further concrete examples of ovarian and non-ovarian diseases which might be important for application are tube carcinoma, endometriosis, malign trophoblast diseases, invasive complete and partial vesicular mole and chorionepithelioma (the LH receptor is also formed in the tubes and the placenta).
The present invention shall be further elucidated by the following Figures and Examples:
Fig. 1 shows a comparison of the expression of different LH-R promoter luciferase constructs in human fibroblast cells and human ovarian cancer cells;
Fig. 2 shows a comparison of the expression of LH-R promoter and
SV40 promoter luciferase constructs in different tumor cells.
Fig. 3 shows a comparison of the expression of different LH-R promoter HSVtk constructs in the cell line HT-1 080
Fig. 4 shows a comparison of the expression of different LH-R promoter HSVtk constructs in the cell line NIH:OVCAR-3 Example 1
Luciferase expression using different LH-R gene promoter fragments
Total genomic DNA was isolated from human ovarian cancer cell line NIH:OVCAR-3 (Hamilton et al., Cancer Res.43 ( 1 983), 3379-5389) . The 5'- flanking region of the LH-R gene promoter was isolated by PCR using primers based on the published gene sequence (Atger et al. (1 995), supra) . The promoter fragments were cloned into pGL2-basic vector (Promega Corporation, USA). Transient transfection experiments to analyze promoter activity were performed as follows:
pGL2-basic vector containing different fragments of the LH-R promoter region were constructed. 6/3-LHR is a vector containing the 241 bp upstream of the transcription initiation site. Vector 6/9-LHR contains the 721 bp upstream of the transcription initiation site.
The vectors were transfected into human fibroblast HT-1080 cells (Rasheed et al. Cancer 33 ( 1 974), 1027-1033) and human ovarian cancer NIH:OVCAR-3 cells. A vector containing /?-galactosidase as reporter gene was co-transfected in order to normalize transfection efficacy.
Expression was determined using luciferase as reporter gene. The luciferase activity was detected according to the manufacturer's protocol (Promega Corp., Madison, Wisconsin, USA) .
The results of this experiment are illustrated in Figure 1 . By using the vector 6/9-LHR an expression of luciferase could neither be detected in human fibroblast cells nor in human ovarian cancer cells. This means that the 721 bp sequence upstream of the transcription initiation site does not provide a significant gene expression. When using the 6/3-LHR vector containing a 241 bp fragment of the LH-R promoter region, an expression in human ovarian cancer cells more than 5 times higher than in human fibroblast cells was found. This proves that the LH-R promoter is capable of inducing tissue-specific expression in human ovarian-derived cells.
In a further experiment the 6/3-LHR vector was compared with the pGL2- control vector (Promega Corporation, USA) containing the SV40 promoter in order to determine promoter activity in different cell lines. The activity of the SV40 promoter was set as 100 %. After 48 hours luciferase activity in cell extracts was assayed.
The results of this experiment are shown in Figure 2. While in liver cancer HepG2 cells (Aden et al., Nature 283 (1 979), 61 5-61 6) and colon cancer cells SW 707 (DKFZ, Tumorbank Heidelberg, In Vitro 1 8 (1 982), 1 65-172) the expression by the SV40 promoter is comparable with that by the LH-R promoter, the expression by the LH-R promoter was found to be more than 4 times higher in the human ovarian cancer NIH:0VCAR-3 cells. Both the high degree of expression and the specificity are utterly surprising.
Unless otherwise indicated, all molecular-biological work has been carried out using the standard techniques as set out in Sambrook et al., Molecular
Cloning, A Laboratory Manual (2nd ed.), Cold Spring Harbour Press ( 1 989) .
Example 2
HSV thγmidine kinase expression using different LH-R promoter fragments
2.1 Methods
2.1 .1 Preparation of different luteinizing hormone-choriogonadotropin receptor promoter (LHCGR-P) gene fragments controlling herpes simplex virus thymidine kinase (HSVtk) expression LHCGR-P-fragment 241 (consisting of 241 bp and corresponding to position -1 to -240 of the LHCGR-P sequence according to Atger et al., 1 995) was isolated from genomic DNA of ovarian cancer cells NIH:OVCAR-3 (ATCC) by polymerase chain reaction (PCR) (5'primer: 22 nt containing a Kpnl restriction site at the 5'priming end and the 3'primer: 21 nt containing a Bglll restriction site at the 3'priming site).
The 240 bp fragment was identified on an agarose gel and ligated into the multiple cloning site of the luciferase vector pGL2 Basic (Promega) which was digested with the restriction enzymes Kpnl and Bglll. The correct sequence was verified by sequencing.
LHCGR-P fragment 421 (consisting of 421 bp and corresponding to position -1 to -420 of the LHCGR-P sequence according to Atger et al., 1 995) was isolated from genomic DNA of lymphocytes by polymerase chain reaction (PCR) (5'primer: 20 nt containing a Kpnl restriction site at the 5'priming end and the 3'primer: 21 nt containing a Bglll restriction site at the 3'priming site).
The 421 bp fragment was identified on an agarose gel and ligated into the linearized TA cloning vector pCR2.1 from Invitrogen (USA/Netherlands) . The correct sequence was verified by sequencing. After digestion with the restriction enzymes Kpnl and Bglll, the 421 bp fragment was isolated from the TA-cloning vector and ligated into the luciferase vector pGL2 Basic (Promega) which was digested with the restriction enzymes Kpnl and Bglll.
The LHCGR-P fragment 1 76 (consisting of 1 76 bp and corresponding to position + 909 to + 1085 of the LHCGR-P sequence according to Atger et al., 1 995) was isolated from genomic DNA of ovarian cancer cells NIH:OVCAR-3 by polymerase chain reaction (PCR) (5'primer: 1 8 nt containing a Kpnl restriction site at the 5'priming site and the 3'primer: 1 8 nt containing a Bglll restriction site at the 3'priming site). The 1 76 bp fragment was identified on an agarose gel and ligated into the linearized TA cloning vector pGlow-TOPO from Invitrogen (USA/Netherlands). After digestion with the restriction enzymes Kpnl and Bglll, the 1 76 bp fragment was isolated from the TA-cloning vector and ligated into the luciferase vector pGL2 Basic (Promega) which was digested with the restriction enzymes Kpnl and Bglll.
The HSVtk DNA sequence (accession # J02224) corresponding to position ATG 51 5-1 646 of the HSVtk gene was amplified by PCR from pBR322 plasmid (5'primer: atggcttcgtacccctgc containing a Bglll restriction site at the 5'priming site and the 3'primer: tcagttagcctcccccat containing a Hindlll restriction site at the 3'priming site) .
The three LHCGR-P fragments in pGL2 Basic vector obtained by digestion with Smal and Bglll were cloned together with HSVtk containing Bglll and Hindlll restriction sites into the pcDNA3.1 + vector (Invitrogen) which was digested with Nrul and Hindlll.
For preparation of LHCGR-P fragments 1 76, 241 , 421 operatively linked to HSVtk in the pCDNA3.1 + vector (Invitrogen), we eliminated the CMV promoter of pCDNA3.1 + vector with restriction enzymes Nrul at the 5'priming site and Hindlll at the 3'priming site and ligated LHRCGR-P fragments 1 76, 241 and 421 linked to the HSVtk into pCDNA3.1 + vector.
As a positive control we used the pCDNA3.1 - vector (Invitrogen) which contains the CMV promoter and HSVtk gene. For that the pCDNA3.1 - vector was restricted with BamHI and Hindlll and the amplified HSVtk sequence (51 5-1 646) was cloned with Bglll and Hindlll restriction sites into this plasmid such that the HSVtk gene is under the control of the CMV promoter. As a negative control we used the pcDNA3.1 + vector alone without LHCGR-P/HSVtk insert.
2.1.2 In vitro ganciclovir (GCV) sensitivity of ovarian cancer cells transfected with LHCGR-P/HSVtk constructs
The herpes simplex virus thymidine kinase suicide gene expressed by cells is capable of converting the normally nontoxic substance ganciclovir into a toxic metabolite which leads to cell death. Since the expression of the LHCG-R gene mainly occurs in gonadal tissue, we studied the expression of the HSVtk gene under control of cell-specific promoter fragments ( 1 76, 241 , 421 ) in ovarian cancer cells (OVCAR-3) and control cells (HT-1080) . Cells were then treated with ganciclovir and cell viability was measured in the MTT assay (Alley et al, Cancer Res., 48:589-601 , 1 988) which measures viable cell dehydrogenase activity.
The cell lines OVCAR-3 and HT-1 080 were seeded and cultured in 24-well plates at a density of 1 .6 x 105 and 8 x 104 respectively in 500 μ\ of DMEM ( 10 % fetal calf serum) overnight. One hour before transfection another change of DMEM ( 10 % FCS) was made. Immediately before transfection the culture medium was removed from the wells and suspensions of vectors containing LHCGR-P fragments 241 , 421 , 1 76 operatively linked to the HSV tk gene at a concentration of 1 //g/ml- DNA and 0.5 //g/ml lipofectin reagent (Gibco) in DMEM without FCS were added to the cells. After incubation for another 24 hours the medium was washed twice with PBS and replaced with fresh DMEM (10 % FCS) containing 400 M ganciclovir (Cymevene, Roche) or no ganciclovir. The cells were then cultured at 37 °C in a 5 % C02 humidified atmosphere for another 3 days, and the medium containing GCV was exchanged every day. Cell growth was then assessed by means of the MTT assay. To each well of the 24-well plate 50 μ\ of MTT solution (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma in PBS (5 mg/ml) was added to 500 μl culture supernatant and properly mixed with the pipet. Afterwards the culture was incubated for 2 hours at 37°C. The reaction was stopped by the addition of 500 /I isopropanol/HCI (0.04 N) to each well. The solution in each well was mixed properly and measured photometrically at 570 nm and 620 nm. The experiments were carried out twice in quadruplicate and calculated as % inhibition of cell growth transfected with the LHCGR-P constructs in comparison to the vector without LHCGR-P/HSVtk insert but treated with ganciclovir.
2.2 Results
Fig. 3 shows the ganciclovir activity of the control cell line HT-1080 in the MTT viability assay as described above. The data are representative of three experiments done in quadruplicate. The standard deviation (SD) is shown in bars. It can be gathered from Fig. 3 that the growth of the control cell line is not significantly inhibited by the HSVtk gene under the control of the LH-R promoter fragments. The negative control (pcDNA3.1 ) and the positive control (HSVtk) yield the expected results.
Fig. 4 shows the measurement of ganciclovir sensitivity of NIH:OVCAR-3 cells in the MTT viability assay. The data are representative of three experiments done in quadruplicate. The standard deviation (SD) is shown in bars.
While the 421 bp fragment shows no inhibitory effect at all, the two other fragments (241 and 1 76 bp) show significant inhibition. This means that the examined LH-R promoter fragments effect significant and tissue-specific expression of the HSVtk gene. The degree of expression lies in a range of from 30-50 % of the construct HSVtk used as control (which contains the strong CMV promoter). Example 3
Sequence comparison
The nucleotide sequences from LH-R promoter regions as published by Atger et al., 1 995 (supra) were compared to own analyzed sequences of genomic DNA from ovarian cancer cell (NIH:OVCAR-3) lymphocytes and ovarian cancer tissue.
In lymphocyte DNA we found a T in position 91 6, a G in position 1098, an A in position 1 120, a T in position 1 31 5, an A in position 141 1 , a C in position 1 560, a G in position 1 672, a G in position 1 700 and a G in position 1 879.
In ovarian cancer tissue DNA we found a C in position 1 592 and a G in position 1 879 (all sequences refer to the numbering in SEQ ID NO.1 ).
The sequence obtained from the ovarian cancer cell line NIH:OVCAR-3 was identical to the published sequence.

Claims

Claims
1 . A recombinant nucleic acid molecule comprising a LH-receptor gene promoter sequence capable of selectively directing gene expression in human ovarian-derived cells.
2. The recombinant nucleic acid molecule of claim 1 which comprises tissue-specific transcription elements and which is free from repressor elements.
3. The recombinant nucleic acid molecule of claim 1 or 2 which is a vector.
4. The recombinant nucleic acid molecule of claim 3 wherein said vector is a eukaryotic vector.
5. The recombinant nucleic acid molecule of claim 3 or 4 wherein said vector is a plasmid or a viral vector.
6. The recombinant nucleic acid molecule of any one of claims 1 to 5 wherein the LH-receptor gene promoter sequence is operatively linked to a heterologous gene.
7. The recombinant nucleic acid molecule of claim 6 wherein the heterologous gene is a reporter gene or a toxin gene.
8. The recombinant nucleic acid molecule of any one of claims 1 to 7 suitable for gene targeting, comprising: (a) at least two DNA sequences homologous to a target sequence present in human cells, (b) the LH-receptor gene promoter sequence located between the homologous DNA sequences of (a), and
(c) optionally a selection marker gene located between the homologous DNA sequences of (a).
9. The recombinant nucleic acid molecule of any one of claims 1 to 8 wherein said LH-receptor gene promoter sequence is selected from a sequence comprising:
(a) the nucleotide sequence from position 1 351 to 1 591 as shown in SEQ ID NO.1 ,
(b) a nucleotide sequence having at least 70 % sequence identitity to the sequence of (a), and
(c) a fragment of the nucleotide sequences of (a) or (b) capable of selectively directing gene expression in human ovarian-derived cells.
10. The recombinant nucleic acid molecule of any one of claims 1 to 8 wherein said LH-receptor gene promoter sequence comprises
(a) the nucleotide sequence from position 2503 to position 2678 as shown in SEQ ID NO.1 ,
(b) a nucleotide sequence having at least 70 % sequence identity to the sequence of (a), and
(c) a fragment of the nucleotide sequences of (a) or (b) capable of selectively directing gene expression in human ovarian-derived cells.
1 1 . The recombinant nucleic acid molecule of any one of claims 1 to 10 wherein said promoter sequence comprises at least one regulatable sequence element.
1 2. The recombinant nucleic acid molecule of claim 1 1 wherein said regulatable sequence elements are responsive to substances selected from LH, FSH, estradiol, growth factors and gonatropin releasing hormone.
1 3. The recombinant nucleic acid molecule of any one of claims 1 to 1 2 wherein said promoter sequence is a hybrid sequence comprising sequence elements from different promoter sequences.
14. A method of selectively expressing genes in human ovarian-derived cells, comprising the steps: (a) providing a recombinant nucleic acid molecule of any one of claims 1 to 1 3,
(b) introducing said recombinant nucleic acid molecule in human ovarian-derived cells, whereby a LH-promoter gene sequence operatively linked to a heterologous gene in said cells is obtained, and
(c) expressing said heterologous gene.
1 5. The method of claim 14 wherein said recombinant nucleic acid molecule is integrated in the chromosome of the cells.
1 6. A pharmaceutical composition comprising as an active agent the recombinant nucleic acid molecule of any one of claims 1 to 1 3.
1 7. The pharmaceutical composition of claim 1 6 for use in the diagnosis and treatment of ovarian diseases.
1 8. The pharmaceutical composition of claim 1 7 for use in the diagnosis and treatment of ovarian cancer.
EP99908806A 1998-01-20 1999-01-20 Lh-receptor gene promoter for tissue-specific gene expression Withdrawn EP1049779A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP99908806A EP1049779A2 (en) 1998-01-20 1999-01-20 Lh-receptor gene promoter for tissue-specific gene expression

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP98100919 1998-01-20
EP98100919 1998-01-20
PCT/EP1999/000361 WO1999036530A2 (en) 1998-01-20 1999-01-20 Lh-receptor gene promoter for tissue-specific gene expression
EP99908806A EP1049779A2 (en) 1998-01-20 1999-01-20 Lh-receptor gene promoter for tissue-specific gene expression

Publications (1)

Publication Number Publication Date
EP1049779A2 true EP1049779A2 (en) 2000-11-08

Family

ID=8231287

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99908806A Withdrawn EP1049779A2 (en) 1998-01-20 1999-01-20 Lh-receptor gene promoter for tissue-specific gene expression

Country Status (4)

Country Link
EP (1) EP1049779A2 (en)
AU (1) AU2828899A (en)
CA (1) CA2318660A1 (en)
WO (1) WO1999036530A2 (en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1199361A3 (en) * 1989-05-05 2004-03-10 Genentech Inc. Glycoprotein hormone receptor molecules. Their uses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9936530A3 *

Also Published As

Publication number Publication date
WO1999036530A2 (en) 1999-07-22
WO1999036530A3 (en) 1999-10-21
AU2828899A (en) 1999-08-02
CA2318660A1 (en) 1999-07-22

Similar Documents

Publication Publication Date Title
Lessnick et al. Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene
Adler et al. Steroid receptor-mediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain
Plowman et al. The amphiregulin gene encodes a novel epidermal growth factor-related protein with tumor-inhibitory activity
Werner et al. Structural and functional analysis of the insulin-like growth factor I receptor gene promoter.
Kaling et al. Synergism of closely adjacent estrogen-responsive elements increases their regulatory potential
Santoso et al. Adenovirus-based p53 gene therapy in ovarian cancer
EP1019499B1 (en) Methods and compositions for inducing tumor-specific cytotoxicity
US5916763A (en) Promoter for VEGF receptor
US20040203153A1 (en) Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made
Jakobovits et al. The human transforming growth factor alpha promoter directs transcription initiation from a single site in the absence of a TATA sequence
Kistler et al. Identification of a functional cyclic adenosine 3′, 5′-monophosphate response element in the 5′-flanking region of the gene for transition protein 1 (TP1), a basic chromosomal protein of mammalian spermatids
US6653132B1 (en) IRES sequences with high translational efficiency and expression vectors containing the sequence
CA2215387A1 (en) Modulators of ob gene and screening methods therefor
US7381813B2 (en) Nucleic acids encoding estrogen receptor ligand binding domain variants
JP2001520875A (en) Gene therapy to stimulate angiogenesis
US5925548A (en) Modified receptors that continuously signal
Frendo et al. An isoform of the human calcitonin receptor is expressed in TT cells and in medullary carcinoma of the thyroid
Coulson et al. Tumour-specific arginine vasopressin promoter activation in small-cell lung cancer
JP2002523106A (en) Inducible expression system
GORDON et al. Human Cart-1: structural organization, chromosomal localization, and functional analysis of a cartilage-specific homeodomain cDNA
WO1999036530A2 (en) Lh-receptor gene promoter for tissue-specific gene expression
JP2002537807A (en) Regulatory construct containing intron 3 of prostate specific membrane antigen gene
Howard Vectors for introducing genes into cells of higher eukaryotes
EP1305409B1 (en) Renal regulatory elements and methods of use thereof
WO1999050411A2 (en) Tumour-specific expression control region and the use thereof

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000801

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB IE IT LI NL SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

17Q First examination report despatched

Effective date: 20031205

18D Application deemed to be withdrawn

Effective date: 20030801