WO2002053748A1 - Regulation de 3-$g(b)-hydroxysteroide deshydrogenase-isomerase - Google Patents

Regulation de 3-$g(b)-hydroxysteroide deshydrogenase-isomerase Download PDF

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WO2002053748A1
WO2002053748A1 PCT/EP2001/015257 EP0115257W WO02053748A1 WO 2002053748 A1 WO2002053748 A1 WO 2002053748A1 EP 0115257 W EP0115257 W EP 0115257W WO 02053748 A1 WO02053748 A1 WO 02053748A1
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isomerase
polypeptide
hydroxysteroid dehydrogenase
polynucleotide
seq
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PCT/EP2001/015257
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Rainer KÖHLER
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Bayer Aktiengesellschaft
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Priority to US10/451,373 priority Critical patent/US20040101877A1/en
Publication of WO2002053748A1 publication Critical patent/WO2002053748A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the regulation of human 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase.
  • pregnenolone is converted to progesterone
  • 17 alpha-hydroxy-pregnenolone is converted to 17 alpha-hydroxy- progesterone
  • dehydroisoandrosterone is converted to androstenedione, respectively beta-hydroxy-5-ene steroid dehydrogenase and 3-keto-5-ene steroid isomerase.
  • 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase catalyzes the oxidation and isomerization of 5-ene-3 beta-hydroxypregnene and 5- ene-hydroxyandrostene steroid precursors into 4-ene-ketosteroids required for the formation of steroid hormones. Because of the importance of these hormones, there is a need in the art to identify additional 3- ⁇ -hydroxysteroid dehydrogenase- isomerases, which can be regulated to provide therapeutic effects.
  • One embodiment of the invention is a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 45% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 45% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.
  • Binding between the test compound and the 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide is detected.
  • a test compound which binds to the 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the activity of the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase through interacting with the 3- ⁇ - hydroxysteroid dehydrogenase-isomerase mRNA.
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
  • a test compound is contacted with a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 45% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.
  • a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity of the polypeptide is detected.
  • a test compound which increases 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase activity of the polypeptide relative to 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
  • a test compound which decreases 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity of the polypeptide relative to 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.
  • Binding of the test compound to the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase product is detected.
  • a test compound which binds to the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.
  • the invention thus provides a human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site.
  • Human 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity.
  • Fig. 1 shows the DNA-sequence encoding a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase Polypeptide (SEQ ID NO:l).
  • Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig.1
  • FIG. 3 shows the amino acid sequence of the protein identified by trembl Accession
  • FIG. 4 shows the BLASTP - alignment of SEQ ID NO:2 against trembl
  • Fig. 5 shows the BLASTP - alignment of SEQ ID NO:2 against swissnew
  • Fig. 6 shows the HMMPFAM - alignment of SEQ ID NO:2 against pfam
  • Fig. 7 shows the HMMPFAM - alignment of SEQ ID NO:2 against pfam
  • Fig. 4 shows the BLASTP - alignment of SEQ ID NO:2 against trembl
  • Fig. 5 shows the BLASTP - alignment of SEQ ID NO:2 against swissnew
  • Fig. 6 shows the HMMPFAM
  • Fig. 8 shows the HMMPFAM - alignment of SEQ ID NO:2 against pfam
  • Fig. 9 shows the BLASTP - alignment of SEQ ID NO:2 against pdb
  • Fig. 10 shows the Psort and tmpredict analyses.
  • Fig. 11 shows the Relevant part of genscan analysis of genomic sequence.
  • Fig. 12 shows the alignment of: 428 gensca: 428 genscan analysis of AC009019.6 or
  • Fig. 13 shows the BLASTP - alignment of 428_protc (genscan predicted) against tremblnew
  • Fig. 14 shows the BLASTP - alignment of 428_protc against tremblnew
  • Fig. 15 shows the BLASTP - alignment of 428protd (genewise predicted) against tremblnew
  • Fig. 16 shows the BLASTP - alignment of 428jprotd_rel against aageneseqj AAG632211 AAG63221.
  • Fig. 17 shows the Genewise prediction of 428 using the mouse homologue.
  • the invention relates to an isolated polynucleotide being selected from the group consisting of: a) a polynucleotide encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 45% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.
  • a novel 3- ⁇ - hydroxysteroid dehydrogenase-isomerase can be used in therapeutic methods to treat cancer.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase comprises the amino acid sequence shown in SEQ ID NO:2.
  • a coding sequence for human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase is shown in SEQ ID NO:l; this sequence is localized on chromosome 16.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase is 44% identical over 193 amino acids to trembl
  • Human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase of the invention is expected to be useful for the same purposes as previously identified 3- ⁇ -hydroxysteroid dehydrogenase-isomerase enzymes.
  • Human 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase is believed to be useful in therapeutic methods to treat disorders such as cancer.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase also can be used to screen for human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activators and inhibitors.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 510, 520, 530, 535, or 538 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 1
  • a 3- ⁇ -hydroxy- steroid dehydrogenase-isomerase polypeptide of the invention therefore can be a portion of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase protein, a full-length 3- ⁇ - hydroxysteroid dehydrogenase-isomerase protein, or a fusion protein comprising all or a portion of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase protein.
  • 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polypeptide variants have amino acid sequences which are at least about 45, 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO:2 or a fragment thereof. Percent identity between a putative 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucme with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence.
  • Fusion proteins are useful for generating antibodies against 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide amino acid sequences and for use in. various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide.
  • Protein affinity chromatography or library-based assays for protein-protein interactions such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 510, 520, 530, 535, or 538 contiguous amino acids of SEQ ID NO:2 or of a biologically active - in ⁇
  • the first polypeptide segment also can comprise full-length 3- ⁇ -hydroxysteroid dehydrogenase-isomerase protein.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include ⁇ -galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transf erase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags,
  • VSV-G tags VSV-G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • a fusion protein also can be engineered to contain a cleavage site located between the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide-encoding sequence and the heterologous protein sequence, so that the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:l in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the
  • DNA construct in a host cell as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-
  • Species homologs of human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide can be obtained using 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide, and expressing the cDNAs as is known in the art.
  • a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide.
  • a coding sequence for human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase is shown in SEQ ID NO: 1
  • nucleotide sequences encoding human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO:l or its complement also are 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • Complementary DNA (cDNA) molecules, species homologs, and variants of 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotides that encode biologically active 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides also are 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotides.
  • Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID
  • amino acids NO:l or its complement also are 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.
  • Variants and homologs of the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polynucleotides described above also are 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polynucleotides.
  • homologous 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotides under stringent conditions, as is known in the art.
  • homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain
  • Species homologs of the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973).
  • Variants of human 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polynucleotides or 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotides of other species can therefore be identified by hybridizing a putative homologous 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or the complement thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polynucleotides or their complements following stringent hybridization and/or wash conditions also are 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • T m of a hybrid between a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotide having a nucleotide sequence shown in SEQ ID NO:l or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A.
  • Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% form- amide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C.
  • Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.
  • a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises 3- ⁇ - hydroxysteroid dehydrogenase-isomerase nucleotide sequences.
  • Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase cDNA molecules can be made with standard molecular biology techniques, using 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase mRNA as a template. Human 3- ⁇ -hydroxysteroid dehydrogenase- isomerase cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • synthetic chemistry techniques can be used to synthesize 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotides.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof.
  • PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988).
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 °C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991).
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ fiowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides can be obtained, for example, by purification from human cells, by expression of 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotides, or by direct chemical synthesis.
  • Human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptides can be purified from any cell that expresses the enzyme, including host cells that have been transfected with 3- ⁇ -hydroxysteroid dehydrogenase-isomerase expression constructs.
  • a purified 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide is separated from other compounds that normally associate with the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art.
  • Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • a preparation of purified 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
  • the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower
  • control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions ⁇ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected depending upon the use intended for the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide. For example, when a large quantity of a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • a sequence encoding the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pIN vectors Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • Plant and Insect Expression Systems the expression of sequences encoding 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results Probl.
  • constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g. , Hobbs or Murray, in MCGRAW HILL YEARBOOK OF
  • An insect system also can be used to express a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polypeptide.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in
  • Sequences encoding 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S.
  • a number of viral-based expression systems can be used to express 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptides in mammalian host cells.
  • sequences encoding 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides.
  • Such signals include the ATG initiation codon and adjacent sequences.
  • sequences encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including the
  • ATG initiation codon should be provided.
  • the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl. Cell Differ. 20, 125-162, 1994).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO,
  • HeLa, MDCK, HEK293, and WI38 are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.
  • herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosylrransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tic or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci.
  • npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Mu ⁇ ay, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988).
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system
  • marker gene expression suggests that the 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotide.
  • host cells which contain a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polynucleotide and which express a 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide to detect transformants that contain a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide.
  • a variety of protocols for detecting and measuring the expression of a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can be used, or a competitive binding assay can be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptides can be designed to contain signal sequences which direct secretion of soluble 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp. Purifi 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide from the fusion protein.
  • IMAC immobilized metal ion affinity chromatography
  • Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND
  • composition of a synthetic 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.
  • an antibody which specifically binds to a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide from solution.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • a 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • mineral gels e.g., aluminum hydroxide
  • surface active substances e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies that specifically bind to a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256,
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985).
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues.
  • rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described in GB2188638B.
  • Antibodies that specifically bind to a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides.
  • Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11).
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J Biol. Chem. 269, 199-206.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al,
  • Antibodies which specifically bind to 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).
  • chimeric antibodies can be constructed as disclosed in
  • Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev..90, 543-583, 1990.
  • Modifications of 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a 3- ⁇ -hydroxysteroid dehydro- genase-isomerase polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent 3- ⁇ -hydroxysteroid dehydrogenase-isomerase nucleotides, can provide sufficient targeting specificity for 3- ⁇ - hydroxysteroid dehydrogenase-isomerase mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • intemucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curt: Opin.
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).
  • Specific ribozyme cleavage sites within a 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate 3- ⁇ -hydroxysteroid dehydrogenase-isomerase RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease 3- ⁇ -hydroxysteroid dehydrogenase-isomerase expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • genes whose products interact with human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase may represent genes that are differentially expressed in disorders including, but not limited to, cancer. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human 3- ⁇ - hydroxysteroid dehydrogenase-isomerase gene or gene product may itself be tested for differential expression.
  • RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al., ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.
  • Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subrractive hybridization (Hedrick et al,
  • the differential expression information may itself suggest relevant methods for the treatment of disorders involving the human 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase.
  • treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human 3- ⁇ - hydroxysteroid dehydrogenase-isomerase.
  • the differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase gene or gene product are up-regulated or down-regulated.
  • the invention provides assays for screening test compounds that bind to or modulate the activity of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide or a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polynucleotide.
  • a test compound preferably binds to a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide or polynucleotide. More preferably, a test compound decreases or increases 3- ⁇ - hydroxysteroid dehydrogenase-isomerase activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • Test compounds can be screened for the ability to bind to 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptides or polynucleotides or to affect 3- ⁇ -hydroxy- steroid dehydrogenase-isomerase activity or 3- ⁇ -hydroxysteroid dehydrogenase- isomerase gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in peti ⁇ dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995).
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel.
  • beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polypeptide, such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the 3 - ⁇ -hydroxy steroid dehydro- genase-isomerase polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound that is bound to the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide.
  • a microphysio- meter e.g., CytosensorTM
  • a microphysio- meter is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS).
  • Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide (McConnell et al, Science 257, 1906-1912, 1992).
  • BIA Bimolecular Interaction Analysis
  • a 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Barrel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey” or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey” proteins are able to interact in vivo to form an protein-dependent complex, the
  • DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the reporter gene.
  • a reporter gene e.g., LacZ
  • DNA sequence encoding the protein that interacts with the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide is a sequence encoding the protein that interacts with the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide.
  • either the 3 - ⁇ -hydroxy steroid dehydrogenase- isomerase polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide is a fusion protein comprising a domain that allows the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide to be bound to a solid support.
  • gluta- thione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the test compound or the test compound and the non-adsorbed 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide are then combined with the test compound or the test compound and the non-adsorbed 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide or polynucleotide
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to the 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Any cell which comprises a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide or polynucleotide can be used in a cell-based assay system.
  • a 3- ⁇ -hydroxy- steroid dehydrogenase-isomerase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide or polynucleotide is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease the 3- ⁇ - hydroxysteroid dehydrogenase-isomerase activity of a human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide.
  • 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity can be measured, for example, as described in Cherradi, et al, J. Steroid. Biochem. Mol. Biol. 41(3-8), 831-36 (1997).
  • Enzyme assays can be carried out after contacting either a purified 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound that decreases a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase activity of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity.
  • a test compound which increases a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity of a human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human 3- ⁇ -hydroxysteroid dehydrogenase- isomerase activity.
  • test compounds that increase or decrease 3- ⁇ -hydroxysteroid dehydrogenase-isomerase gene expression are identified.
  • a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison.
  • test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radio- immunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide.
  • screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell that expresses a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide can be used in a cell-based assay system.
  • the 3 - ⁇ -hydroxy steroid de- hydrogenase-isomerase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the invention can comprise, for example, a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide, 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide, or mimetics, activators, or inhibitors of a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide activity.
  • compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • agent such as stabilizing compound
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be penneated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the conesponding free base forms.
  • the prefened preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency, and method of administration.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase can be regulated to treat cancer.
  • Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these abenant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.
  • Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins.
  • proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities.
  • Agonists and/or antagonists of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.
  • This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase polypeptide binding molecule
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • a reagent which affects 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity can be administered to a human cell, either in vitro or in vivo, to reduce 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase activity.
  • the reagent preferably binds to an expression product of a human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase gene. If the expression product is a protein, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours.
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells.
  • a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More prefened liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
  • Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151).
  • polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993);
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases 3- ⁇ -hydroxysteroid dehydrogenase- isomerase activity relative to the 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in
  • LD 50 the dose lethal to 50% of the population
  • LD 0 the dose lethal to 50% of the population
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the EDs 0 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage fonn employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, elecfroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g ofDNA.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase gene or the activity of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase gene or the activity of a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase- specific mRNA, quantitative RT-PCR, immunologic detection of a 3- ⁇ -hydroxy- steroid dehydrogenase-isomerase polypeptide, or measurement of 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding 3- ⁇ -hydroxysteroid dehydrogenase-isomerase in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags. Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis.
  • DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85, 4397-4401, 1985).
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
  • Altered levels of a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
  • the polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase activity is measured in the following assay: Monitered by using 20 ⁇ M [ 3 H]DHEA as substrate and 0.2 mM NAD + in buffer C (50 mM potassium phosphate pH 7.4 containing 0.1 mM EDTA). Incubation time is 5 min at 37 °C.
  • the steroid mixture is separated by silica gel thin-layer chromatography in chloroform-acetone (90:10, v/v) using authentic DHEA, 5-ene-androstenedione and 4-ene-androstenedione as internal standards.
  • the reaction products are scrapped off the gel and counted for the radioactivity content.
  • the 3 ⁇ -OH dehydrogenase activity is measured spectrophotometrically by following the increase of absorbance at 340 nm using 20 ⁇ M DHEA, 50 ⁇ M NAD + and the cell extract.
  • the isomerase activity is measured spectrophotometrically (241 nm) in buffer C using 50 ⁇ M 5-ene- androstenedione as substrate with (50 ⁇ M) NAD + and the cell extract. It is shown that the polypeptide of SEQ ID NO: 2 has a 3- ⁇ -hydroxysteroid dehydrogenase- isomerase activity.
  • the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptides in yeast.
  • the 3- ⁇ -hydroxysteroid dehydrogenase- isomerase-encoding DNA sequence is derived from SEQ ID NO:l.
  • the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.
  • the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human 3- ⁇ -hydroxy- steroid dehydrogenase-isomerase polypeptide is obtained.
  • test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptides comprise the amino acid sequence shown in SEQ ID NO:2.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells.
  • Binding of a test compound to a 3- ⁇ -hydroxysteroid dehydrogenase-isomerase polypeptide is detected by fluorescence measurements of the contents of the wells.
  • a test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase polypeptide.
  • test compound is administered to a culture of human cells transfected with a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase expression construct and incubated at
  • RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18,
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase-specific probe at 65 °C in Express-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:l.
  • a test compound that decreases the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase gene expression.
  • a test compound is administered to a culture of human cells transfected with a 3- ⁇ - hydroxysteroid dehydrogenase-isomerase expression construct and incubated at 37 °C for 10 to 45 minutes.
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • 3- ⁇ -hydroxysteroid dehydrogenase-isomerase activity is measured using the method of Chenadi, et al, J. Steroid. Biochem. Mol. Biol. 41(3-8), 831-36 (1997).
  • a test compound which decreases the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase activity of the 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase relative to the 3- ⁇ - hydroxysteroid dehydrogenase-isomerase activity in the absence of the test compound is identified as an inhibitor of 3- ⁇ -hydroxysteroid dehydrogenase- isomerase activity.
  • RT-PCR RT-PCR
  • expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes.
  • Expression in the following cancer cell lines also is determined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205
  • colon A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon), MDA-
  • MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignant and normal tissue from the same patient also are tested.
  • Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis” firstly described in Higuchi et al, BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology
  • the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al., Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996).
  • the amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction.
  • the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used.
  • RNA extraction and cDNA preparation Total RNA from the tissues listed above are used for expression quantification. RNAs labeled "from autopsy” were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.
  • RNA Fifty ⁇ g of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/ ⁇ l RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/ ⁇ l RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM MgCl 2 ; 50 mM NaCl; and 1 mM DTT.
  • RNA is extracted once with 1 volume of phenolxhloro- formtisoamyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol.
  • RNA from the autoptic tissues Fifty ⁇ g of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectrophoto- metric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200ng/ ⁇ L. Reverse transcription is canied out with 2.5 ⁇ M of random hexamer primers.
  • TaqMan quantitative analysis Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.
  • FAM 6-carboxy-fluorescein
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents
  • the assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 20X stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 ⁇ l.
  • This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus.
  • Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c).
  • test compound p.o., i.p., i.v., i.m., or s.c
  • Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the honnone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m.
  • a biologic stimulus i.e., LHRH may be injected i.m.
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • specific readout assay protocol these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇
  • Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for a predetermined duration (i.e., 1 week).
  • animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded.
  • Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent.
  • Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol.
  • Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • a marker of cell number i.e., MTT or LDH
  • the cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • Hydron pellets with or without growth factors or cells are implanted into a micropocket surgically created in the rodent cornea.
  • Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet).
  • Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Rank Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p
  • ⁇ 0.05 as compared to the growth factor or cells only group.
  • Matrigel containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p ⁇ 0.05 as compared to the vehicle control group.
  • Tumor cells or fragments are implanted subcutaneously on Day 0.
  • Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden.
  • Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day.
  • Anti- tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p ⁇ 0.05.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan- Meier curves from the times for individual tumors to attain the evaluation size. Significance is p ⁇ 0.05.
  • Tumor cells are injected intraperitoneally or intracranially on Day 0.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting on Day 1. Observations of morbidity and/or mortality are recorded twice daily. Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long- term survivors is indicated separately. Survival times are used to generate Kaplan- Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
  • Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Tumor cells or fragments, of mammary adenocarcinoma origin are implanted directly into a surgically exposed and reflected mammary fat pad in rodents.
  • the fat pad is placed back in its original position and the surgical site is closed.
  • Honnones may also be administered to the rodents to support the growth of the tumors.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.
  • Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • Tumor measurements may be recorded after dosing has stopped to momtor tumor growth delay.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells or fragments, of prostatic adenocarcinoma origin are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents.
  • the prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles.
  • the successfully inoculated prostate is replaced in the abdomen and the incisions throught e abdomen and skin are closed.
  • Hormones may also be administered to the rodents to support the growth of the tumors.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c.
  • Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea.
  • the trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
  • the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termina- tion of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizing the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t- test to compare tumor sizes in the treated and control groups at the end of treatment.
  • Significance is p ⁇ 0.05 as compared to the control group.
  • This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor.
  • Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells are inoculated s.c. and the tumors allowed to grow to a predetennined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined.
  • Survival data is used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment for both of these endpoints.
  • Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
  • the mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance at p ⁇ 0.05 compared to the vehicle control group in the experiment for both endpoints.
  • the cell line used for testing is the human colon cancer cell line HCT116.
  • Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37 °C in a 95% air/5%CO 2 atmosphere.
  • Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems
  • oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration. Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 ⁇ M once per day for seven days.
  • test oligonucleotide for seven days results in significantly reduced expression of human 3 - ⁇ -hydroxy steroid dehydrogenase-isomerase as determined by Western blotting. This effect is not observed with the control oligonucleotide.
  • the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide. The number of cells in cultures treated with the test oligonucleotide is not more than 30% of control, indicating that the inhibition of human 3- ⁇ -hydroxysteroid dehydrogenase-isomerase has an anti-proliferative effect on cancer cells.

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Abstract

L'invention concerne des réactifs régulant la 3-ß-hydroxystéroïde déshydrogénase-isomérase humaine et des réactifs se liant à des produits géniques de 3-ß-hydroxystéroïde déshydrogénase-isomérase humaine, qui peuvent jouer un rôle dans la prévention, l'amélioration ou l'atténuation de dysfonctionnements ou de maladies comprenant notamment, mais pas exclusivement, le cancer.
PCT/EP2001/015257 2001-01-02 2001-12-21 Regulation de 3-$g(b)-hydroxysteroide deshydrogenase-isomerase WO2002053748A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001075067A2 (fr) * 2000-03-31 2001-10-11 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001075067A2 (fr) * 2000-03-31 2001-10-11 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 1 July 2000 (2000-07-01), XP002196416, Database accession no. AC009164 *
DATABASE EMBL [online] 13 February 2002 (2002-02-13), XP002196419, Database accession no. ABG01955 *
DATABASE EMBL [online] 13 February 2002 (2002-02-13), XP002196420, Database accession no. AAS66142 *
DATABASE EMBL [online] 4 March 2000 (2000-03-04), XP002196417, Database accession no. B52363 *
DATABASE EMBL [online] 5 April 2000 (2000-04-05), XP002196418, Database accession no. AC021336 *
LACHANCE Y. ET AL.: "Characterization of human 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase gene and its expression in mammalian cells", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 265, no. 33, 25 November 1990 (1990-11-25), pages 20469 - 20475, XP002944255, ISSN: 0021-9258 *

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