EP0769023A1 - Recepteur epsilon d'acide retinoique - Google Patents

Recepteur epsilon d'acide retinoique

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Publication number
EP0769023A1
EP0769023A1 EP94924450A EP94924450A EP0769023A1 EP 0769023 A1 EP0769023 A1 EP 0769023A1 EP 94924450 A EP94924450 A EP 94924450A EP 94924450 A EP94924450 A EP 94924450A EP 0769023 A1 EP0769023 A1 EP 0769023A1
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EP
European Patent Office
Prior art keywords
polypeptide
rare
polynucleotide
dna
sequence
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.)
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EP94924450A
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German (de)
English (en)
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EP0769023A4 (fr
Inventor
Liang Queen Mary Hospital CAO
Jian Ni
Robert D. Fleischmann
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Priority claimed from PCT/US1994/007266 external-priority patent/WO1996000242A1/fr
Publication of EP0769023A1 publication Critical patent/EP0769023A1/fr
Publication of EP0769023A4 publication Critical patent/EP0769023A4/fr
Withdrawn legal-status Critical Current

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Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is Retinoic Acid Receptor Epsilon (RARe) . The invention also relates to inhibiting the action of such polypeptides.
  • RARe Retinoic Acid Receptor Epsilon
  • Retinoids (Vitamin A derivatives) are crucial for normal growth, vision, maintenance of numerous tissues, reproduction and overall survival (Wolbach, S.B., J. Exp. Med.. 42:753-777 (1925)) .
  • offspring of Vitamin A deficient (VAD) dams exhibit a number of developmental defects, indicating that retinoids are also important during embryogenesis (Wilson, J.G., et al., Am. J. Anat.. 92:189-217 (1953)).
  • the effects of Vitamin A deficiency in fetuses and young and adult animals can be prevented and/or reversed by retinoic acid (RA) administration (Wilson, J.G., et al., Am. .
  • Retinoids are also crucial for normal growth, vision, maintenance of numerous tissues, reproduction and overall survival (Wolbach, S.B. and Howe, P.R., J. Exp. Med. , 42:753- 777 (1925)) .
  • the retinoic acid receptor genes belong to the superfamily of genes known as the steroid hormone receptor family. All genes in this family can be divided into discrete regions or domains that are sometimes referred to as regions A/B, C, D, E, and F.
  • the C region encodes the DNA- binding domain
  • the E region encodes the ligand-binding domain
  • the F region encodes the carboxy-terminus domain.
  • the D region is believed to function as a "hinge”.
  • the function of the A/B (or N-terminus) region is not entirely clear but it may be involved with enhancement and repression of receptor transcription activity. (Hollenberg et al., Cell, 55:899-906 (1988)).
  • RAR RA receptor
  • RARc ⁇ , ⁇ and and their isoforms are activated by both all-trans and non-cis RA.
  • RAR ⁇ has also been cloned (Mech, Dev. , 40:99-112 (1993)).
  • the DNA-binding region and the ligand-binding region domains of the three RAR types are highly similar, whereas the C-terminal region and the middle region exhibit no or little similarity.
  • amino acid sequences of the three RAR types are also notably different in their B regions, and their main isoforms ( ⁇ l and ⁇ _2, ⁇ l to ⁇ 4, 1 and ⁇ 2 and ⁇ l and ⁇ 2) further differ in their N- terminal A regions (Leid, M. et al., Trends Biochem. Sci., 17:427-433 (1992) ) .
  • RAR7 mutant mice exhibit growth deficiency, early lethality, and male sterility due to squamous metaplasia of the seminal vesicles and prostate (Lohnes, D. et al., Cell, 73:643-658 (1993)).
  • RAR7 transcripts are found in precartilaginous condensations, with subsequent restriction to cartilage and differentiating squamous keratinizing epithelia, regardless of their embryonic origin (Dolle, P. et al., Nature, 342:702- 705 (1989) ) . These observations suggest a role for RAR ⁇ in morphogenesis and chondrogenesis (Dolle et al., Development, 100:1133-1151 (1990)) .
  • the DNA sequence and polypeptide encoded by such DNA sequence of the present invention belongs to the retinoic acid receptor family and is most homologous to retinoic acid receptor gamma. Both modulator and the DNA binding domains share the highest homology with the corresponding parts of the ecdysone receptor of Drosophila. The ecdysone receptor plays an important role during development.
  • a novel mature polypeptide which is RARe, as well as fragments, analogs and derivatives thereof.
  • the polypeptide of the present invention is of human origin.
  • polynucleotides (DNA or RNA) which encode such polypeptides.
  • Figure 1 depicts the polynucleotide sequence and the corresponding deduced amino acid sequence of the putative mature RARe polypeptide.
  • the standard one letter abbreviation for amino acids is used.
  • Figure 2 illustrates the high homology between RARe and RAR7 at the DNA binding region and ligand binding region.
  • Figure 3 depicts the results of a Northern blot analysis of the RARe in human adult tissues.
  • Figure 4 shows the results of electrophoresing RARe on a gel after in vi tro transcription/translation.
  • Figure 5 illustrates the baculovirus transfer plasmid pA2 used for expression of RARe .
  • nucleic acid which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75754 on March 18, 1994.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained from testes, spleen and thymus.
  • the polynucleotide of this invention was discovered in a cDNA library derived from human adult lung. It is structurally related to the retinoic acid receptor family. It contains an open reading frame encoding a protein of approximately 433 amino acid residues. The protein exhibits the highest degree of homology to retinoic acid receptor gamma with 48% identity and 58% similarity over a 70 amino acid stretch of the modulator region.
  • the DNA binding region and ligand binding region are also highly homologous to retinoid acid receptor gamma ( Figure 5) .
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure l or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa- histidine tag supplied by a pQE-9 (Qiagen) vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • HA hemagglutinin
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984) ) .
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides .
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA.
  • the present invention further relates to a RARe polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
  • fragment when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the RARe genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence (s) (promoter) to direct mRNA synthesis.
  • s expression control sequence
  • promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or _ the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • bacterial cells such as E. coli. Streptomvces, Salmonella t phimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila and Sf_9
  • animal cells such as CHO, COS or Bowes melanoma
  • plant cells etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs are described above.
  • SUBSTITUTE SHEET (RULE 25) comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen) , pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia), pRS and pGEM.
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R/ P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986) ) .
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers .
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al . , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989) , the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , ctf-factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplas ic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the RARe polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.15-5 mM) of calcium ion present during purification. (Price et al., J. Biol. Chem. , 244:917 (1969)). Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedure ' s, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • the RARe hormone receptor may be used for the identification of its novel hormones, DNA binding sites, gene targets and tissue specificity.
  • the RARe polypeptide may also be employed as a therapeutic target for tissue regeneration, reproduction, stimulation of the immune and hematopoietic system, and for the treatment of male sterility. This is particularly important since RAR mutant mice exhibited the previously stated abnormalities. Accordingly, the RARe receptor may be used to treat these disorders.
  • the RARe polypeptide may also be used for screening of putative hormone molecules, such as steroid agonists that can modulate the hormone response mediated through this receptor.
  • putative hormone molecules such as steroid agonists that can modulate the hormone response mediated through this receptor.
  • the gene encoding for RARe binding sites is cloned in the pRS eukaryotic expression vector (Giguere et al., Cell, 46:645 (1986)) producing pRShRARe .
  • the plasmid is then introduced into monkey kidney CV-1 cells via calcium- phosphate transfection together with a reporter plasmid DELTA MTV-TRE sub p-CAT.
  • TRE means thyroid receptor response element and TRE sub p is a TRE that has been engineered to maximize the palindorminicity of this element.
  • pRSerbA sup-1 encodes no protein, stands as a negative control
  • the transfected cells are incubated in the presence or absence of 100 nM retinoic acid, or other potential ligands, for 36 hours, and the induced CAT activities were analyzed by chromatography. The results will indicate which ligands bind to RARe .
  • This invention further provides a method of screening drugs to identify those which enhance (agonists) interaction of ligands to the RARe.
  • a reporter plasmid and pRShRARe are introduced into monkey cells as discussed above. The transfected cells are then incubated with labeled retinoic acid in the presence of drug. The ability of drug to enhance this interaction could then be measured.
  • the reporter gene can be a CAT or luciferase gene whose activities with respect to the interaction of ligand and receptor are capable or being measured (Promega Technical Bulletin No. 126, July, 1993) .
  • Transfection systems are useful as living cell cultures for competitive binding assays between known or candidate drugs and ligands which bind to the receptor and which are labeled by radioactive, spectroscopic or other reagents.
  • a transfection system constitutes a "drug discovery system" useful for the identification of natural or synthetic compounds with potential for drug development that can be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the human RARe.
  • the transfection system is also useful for determining the affinity and efficacy of known drugs at the human RARe sites.
  • the agonists and drugs which are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA polynucleotide
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • the RARe polypeptides of the present invention are also useful for identifying other molecules which have similar biological activity.
  • An example of a screen for this is isolating the coding region of the RARe gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • RARe Availability of the RARe allows its use in diagnostic assays to determine levels of retinoic acid present in various tissues and body fluids. The determination of these levels allows the diagnosis and treatment of diseases related to low levels of retinoic acid.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in si tu hybridization (FISH) of a cDNA clones to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
  • Verma et al. Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .
  • Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • the present invention further provides a method for diagnosing a predisposition to a disorder associated with the underexpression of the RARe polypeptide, which comprises obtaining DNA of subjects suffering from the disorder, performing a restriction digest of the DNA with a panel of
  • SUBSTITUTE SHEET (RULE 25) restriction enzymes, electrophoretically separating the resulting DNA fragments on a sizing gel, contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding an RARe polypeptide and labelled with a detectable marker, detecting labelled bands which have hybridized to the DNA encoding an RARe polypeptide with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder, preparing DNA obtained for diagnosis by the above steps and comparing the unique band pattern specific to the DNA of subjects suffering from the disorder and the DNA obtained from the diagnosis step to diagnose predisposition to the disorder.
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • SUBSTITUTE SHEET (RULE 25) analytical purposes, typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37"C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980) .
  • Oligonucleotide ⁇ refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146) . Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • Example 1 Expression of RARe by in vi tro transcription and translation
  • the in vi tro transcription and translation of RARe was carried out using the TN ⁇ Coupled Reticulocyte Lysate System (Promega, 2800 Woods Hollow Road, Madison, WI 53771-5399) .
  • the cDNA encoding for RARe ATCC # 75754, was cloned directionally EcoRI to Xhol with the EcoRI site defining the 5' end of the gene and the Xhol site defining the 3' end of the gene.
  • the gene was inserted in the T3 direction.
  • T3 defines a bacteriophage RNA polymerase which recognizes a specific promoter, and transcribes the DNA into a mRNA.
  • a rabbit reticulocyte lysate is supplemented with T3 RNA polymerase and directs the expression of proteins with a T3 promoter utilizing the T3 RNA polymerase to transcribe the message, and the reticulocyte lysate to translate the nascent RNA.
  • I ⁇ g of circular (or linear) plasmid containing the RARe DNA was added directly to TNTTM lysate and incubated in a 50 ⁇ l reaction volume at 30°C for 1 hour with T3 RNA polymerase and [ 35 S] -Methionine. After incubation, the translation product was separated by 10% SDS-PAGE gel electrophoresis.
  • Sf9 insect cells are maintained in culture using Grace's Insect medium (Gibco) supplemented with 10% FBS at 27°C.
  • the baculovirus transfer plasmid pA2 was constructed by Gentz et al (unpublished results) ( Figure 5) .
  • the entire human RARe cDNA including the BamHl site at 5' end and Asp 718 site at 3' end were amplified using PCR.
  • the PCR oligonucleotide primers used for the amplification of the human RARe are:
  • the amplified fragment containing human RARe cDNA, and plasmid pA2 were digested by BamHI and Asp 718 and purified by Geneclean kit.
  • the amplified fragment containing human RARe cDNA was inserted into vector pA2 to generate the recombinant transfer vector pA2RARe .
  • the accuracy of the construct was verified using restriction endonuclease, PCR and DNA sequencing.
  • Transfer of the human RARe cDNA from and pA2RAR to an AcNPV genome can be achieved by co-transfection using Lipofectin (Gibco) .
  • AcNPV is a wild-type virus abailable from Pharmagene (San Diego, CA) .
  • Techniques for manipulation of baculovirus and insect cell culture such as cultivation, co-transfection, virus infection, isolation, screening and purification of putative recombinant plaques and virus titer determination was described by O'Reilly D.R. et al [Baculovirus expression vectors: A Laboratory Manual (1992) , W. H. Freeman and Company] .
  • the plaques will be screened for recombinant baculovirus by using the blue-white selection.
  • the visually screened plaques are to be analyzed and confirmed by PCR screening.
  • Sf9 cells (2X10 6 cells/ml) can be infected with recombinant RAR virus (10 8 plaque-forming units/ml) for 2 hr at room temperature. After transfection, the viral inoculum is replaced with fresh medium. The supernatant and cells are harvested after 60 - 72 hr. The extra cellular and intracellular fractions are then analyzed for the expression of RARe by SDS-PAGE.
  • the receptor can be partially purified by sequential anion-exchange, gel-filtration and DNA affinity chromatography.
  • Example 3 Expression pattern of RARe in human tissue Northern blot analysis was carried out to examine the levels of expression of RARe in human tissues. Total cellular RNA samples were isolated with RNAzolTM B system
  • RNA isolated from each human tissue specified was separated on 1% agarose gel and blotted onto a nylon filter.
  • the labeling reaction was done according to the Stratagene Prime-It kit with 50ng DNA fragment.
  • the labeled DNA was purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303) .
  • the filter was then hybridized with radioactive labeled full length RARe gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65 ° C. After wash twice at room temperature and twice at 60°C with 0.5 x SSC, 0.1% SDS, the filter was then exposed at -70°C overnight with an intensifying screen.
  • the message RNA for RARe is abundant in testes, placenta, spleen, thymus and lung.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • Gin Leu Gly Met lie Glu Lys Leu Val Pro Ala Gin Gin Gin Gin Cys

Abstract

L'invention concerne un polypeptide récepteur epsilon d'acide rétinoïque (RARε) ainsi qu'un ADN (ARN) codant ces polypeptides. L'invention concerne également un procédé de production de ce polypeptide par génie génétique et son utilisation à des fins thérapeutiques, par exemple, dans la régénération et la stimulation des tissus du système immun et hématopoïétique. L'invention concerne également des procédés d'identification de ligands stimulant le RARε.
EP94924450A 1994-06-24 1994-06-24 Recepteur epsilon d'acide retinoique Withdrawn EP0769023A4 (fr)

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PCT/US1994/007266 WO1996000242A1 (fr) 1994-06-24 1994-06-24 Recepteur epsilon d'acide retinoique

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989012687A1 (fr) * 1988-06-16 1989-12-28 La Jolla Cancer Research Foundation Recepteur d'acide retinoique
WO1991012258A1 (fr) * 1990-02-09 1991-08-22 The Salk Institute For Biological Studies Compositions de recepteurs de retinoides et leurs procedes d'obtention
WO1993006215A1 (fr) * 1991-09-17 1993-04-01 The Salk Institute For Biological Studies Recepteurs de la superfamille des recepteurs d'hormones thyroidiennes/steroides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989012687A1 (fr) * 1988-06-16 1989-12-28 La Jolla Cancer Research Foundation Recepteur d'acide retinoique
WO1991012258A1 (fr) * 1990-02-09 1991-08-22 The Salk Institute For Biological Studies Compositions de recepteurs de retinoides et leurs procedes d'obtention
WO1993006215A1 (fr) * 1991-09-17 1993-04-01 The Salk Institute For Biological Studies Recepteurs de la superfamille des recepteurs d'hormones thyroidiennes/steroides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9600242A1 *

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EP0769023A4 (fr) 1999-05-26
AU7471294A (en) 1996-01-19

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