EP1049779A2 - Lh-rezeptorgenpromoter für gewebenspezifischen genexpression - Google Patents

Lh-rezeptorgenpromoter für gewebenspezifischen genexpression

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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Withdrawn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
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English (en)
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/de
Publication of EP1049779A2 publication Critical patent/EP1049779A2/de
Withdrawn legal-status Critical Current

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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.

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EP99908806A 1998-01-20 1999-01-20 Lh-rezeptorgenpromoter für gewebenspezifischen genexpression Withdrawn EP1049779A2 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP99908806A EP1049779A2 (de) 1998-01-20 1999-01-20 Lh-rezeptorgenpromoter für gewebenspezifischen genexpression

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 (de) 1998-01-20 1999-01-20 Lh-rezeptorgenpromoter für gewebenspezifischen genexpression

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EP1049779A2 true EP1049779A2 (de) 2000-11-08

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EP (1) EP1049779A2 (de)
AU (1) AU2828899A (de)
CA (1) CA2318660A1 (de)
WO (1) WO1999036530A2 (de)

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WO1990013643A2 (en) * 1989-05-05 1990-11-15 Genentech, Inc. Glycoprotein hormone receptor molecules

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See references of WO9936530A3 *

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

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