WO1993023548A2 - Procede de detection d'arn messagers codant la proteine de liaison fk506 a specificite tissulaire et ses utilisations - Google Patents
Procede de detection d'arn messagers codant la proteine de liaison fk506 a specificite tissulaire et ses utilisations Download PDFInfo
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- WO1993023548A2 WO1993023548A2 PCT/US1993/004916 US9304916W WO9323548A2 WO 1993023548 A2 WO1993023548 A2 WO 1993023548A2 US 9304916 W US9304916 W US 9304916W WO 9323548 A2 WO9323548 A2 WO 9323548A2
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- C07—ORGANIC CHEMISTRY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- FK506 and cyclosporin A are potent imirtunosuppressants that block the early steps of T lymphocyte activation. These imirtunosuppressants interfere with the Ca 2+ -dependent signaling pathways mediated by the T cell receptor.
- imirtunosuppressants interfere with the Ca 2+ -dependent signaling pathways mediated by the T cell receptor.
- serine/threonine phosphatase (Klee, C.B. and Krinks, M.H. Biochemistry 17:120-126 (1978)), is a common target of these immunosuppressants when they are bound to specific intracellular receptors in vitro and in T lymphocytes. (Liu, J. et al., Cell 66:807-815 (1991); Friedman and Weissman, 1991). These receptors, termed immunophilins, apparently play key roles in
- FKBPs FK506-binding proteins
- FKBP12 an abundant cytosolic immunophilin of M. 11,800. More recently, FKBP13 (Jin, Y.J. et al., Proc. Natl. Acad. Sci. USA 88:6677-6681 (1991)) and FKBP25 (Galat, A., et al.. Biochemistry 31:2427-2434 (1991) have been characterized as immunophilins that bind both FK506 and rapamycin. All of these FKBPs share a core consensus sequence with FKBP12 but only the FKBP12-FK506 complex inhibits calcineurin. This finding implies that the different FKBPs are likely to have diverse effects on signal transduction pathways even though their
- immunosuppressant binding properties reflect aspects of structural similarity.
- RNA messenger RNA
- SEQ ID NO:1 sequence of a messenger RNA (mRNA) encoding an FK506 binding protein in human T lymphocytes and to the sequencing of its corresponding cDNA (SEQ ID NO:1). Sequencing of the cDNA reveals that the mRNA, also referred to as the RNA transcript, is identical in sequence in two regions (i.e., the 5' untranslated region (5'UTR) and the open reading frame region (ORF) ) to two known FKBP12-encoding RNA sequences and clearly distinct from the two known sequences in the 3' untranslated region
- 5'UTR 5' untranslated region
- ORF open reading frame region
- tissue distribution of the mRNA of the present invention which is expressed predominantly in heart, placental and pancreatic tissue, differs from the tissue distribution of the other two mRNAs.
- the present invention also relates to
- isolated DNA which is indicative of tissue-specificity FK506 binding protein mRNAs, and to DNA or RNA probes for use in the detection of nucleic acid sequences (mRNA and DNA) encoding FK506 binding protein mRNA present in specific tissue.
- This invention further relates to a method of detecting the expression of tissue-specific FKBP12 mRNA as a means of monitoring tissue rejection in transplant patients.
- indication of tissue rejection has been diagnosed by a combination of clinical,
- liver allograft rejection is based on clinical manifestation of fever without a source of infection, bile output or increased ascites; the biochemical indices of total bilirubin, SGOT, SGPT, alkaline phosphatase and other enzyme levels; and the
- RNA from tissue of a human is combined with a DNA or RNA probe which is complementary to all or a portion of the RNA sequence of the 3'UTR of a mRNA encoding FKBP12, the cDNA is shown in SEQ ID NO: 2. Detection of hybridization is an indication of the presence of a mRNA encoding FKBP12 in specific tissue. The extent to which FKBP12 is
- the present method is particularly advantageous because it makes it possible to detect tissue rejection due to an ineffective dosage of FK506 at an earlier stage than is possible with other available methods and, thus, to modify the drug dosage to increase the chances of tissue survival.
- the present invention also relates to a method of determining the effects of FK506 therapy on an individual. Clinical management of
- RNA which is present in specific tissue of a human can be hybridized to a DNA or RNA probe which is complementary to all or a portion of the RNA sequence of the 3'UTR of an FKBP12 mRNA, the cDNA is shown in SEQ ID NO: 2. Detection of hybridization is indicative of mRNA present which encodes FKBP12, and in what amount. The amount of FKBP12 mRNA present is a reasonable indication of the amount of FKBP12 being produced in the target tissue. If insufficient FKBP12 is produced to be effective in immunosuppressive therapy, another line of therapy may be initiated before rejection or toxicity occurs.
- the present invention also relates to a method of modifying production of FKBP12 in vivo.
- a synthetic oligonucleotide which
- FKBP12 mRNA can be administered to an individual.
- the hybridization of the nucleotide sequence could result in modification of the production of FKBP12.
- the nucleotide sequence in a preferred embodiment, the nucleotide sequence
- administered will be a DNA sequence, such that when it hybridizes to the 3'UTR of an FKBP12 mRNA translation will be modified.
- Figure 1 shows the FKBP12A nucleic acid sequence. (SEQ ID NO: 1).
- Figure 2 shows the FKBP12A 3' untranslated region (3'UTR) sequence. (SEQ ID NO: 2).
- FIG. 3 shows the comparison of the FKBPs, FKBP12A, FKBP12B and FKBP12C, including their
- Figure 4 shows the electrophoretic gel pattern of human genomic DNA digested with one of five different restriction endonucleases and then used for Southern analysis.
- Figure 5 is a schematic representation of the human FKBP12 gene which spans approximately 17-30 kb and contains five exons that encode three different FKBP12 mRNAs.
- Figure 6 is a schematic representation of three FKBP12 mRNAs (12A, 12B, 12C) encoded by the
- FKBP12 gene (SEQ ID NOS:14-25).
- Figure 7A shows results of Northern analysis which indicates that poly(A)+RNA from E + T cells enriched from human PBLs contains FKBP12A (0.9 kb), FKBP12B (1.7 kb), and FKBP12C (0.7kb) mRNA transcripts.
- Figure 7B shows PCR analysis results of first-strand cDNA transcribed from cytoplasmic RNA and demonstrates that the FKBP12A and FKBP12B mRNAs are produced in an enriched, PHA-stimulated population of T lymphocytes.
- Figure 8A shows poly(A)+RNA isolated from enriched populations of human E + T cells that were stimulated for 0, 4, 16, or 48 hr with 10 ⁇ g ml -1 PHA.
- Figure 8B shows that a 72 hour exposure of the membrane used in 8A reveals that PHA stimulation also increases the abundance of the 0.9 kb FKBP12A mRNA transcripts.
- Figure 9 depicts the electrophoretic gel pattern demonstrating that the 1.7 kb FKBP12 mRNA transcript is present in a variety of human tissues.
- Figure 10 is a schematic representation showing the synthetic oligonucleotides and restriction enzyme cleavage sites used to generate probes specific to the different FKBP12 cDNAs and mRNA and the FKBP12 gene (SEQ ID NOS:8-11 and 26-33). Detailed Description of the Invention
- mRNA messenger RNA
- FK506 binding protein in human T lymphocytes
- sequencing of its corresponding cDNA revealed that the mRNA, or RNA transcript, is identical in sequence in two regions (i.e., the 5' untranslated region (5'UTR) and the open reading frame region (ORF)) to two known FKBP12-encoding RNA
- tissue distribution of the mRNA of the present invention which is expressed predominantly in heart, placental and pancreatic tissue, differs from the tissue distribution of the other two mRNAs.
- the present invention more particularly relates to the cDNA clone containing an open reading frame that encodes a specific FK506 binding protein hereinafter referred to as FKBP12A (SEQ ID NO: 1), as shown in Figure 1.
- oligonucleotide probes specific to the FKBP12 open reading frame (ORF) sequence were used to screen T cell cDNA libraries for other full-length FKBP12 cDNAs.
- ORF open reading frame
- polyadenylated cDNA has a 5'UTR and ORF sequence identical to the cDNAs reported by Maki, N. et al.. Proc. Natl. Acad. Sci. USA 87:5440-5443 (1990) and Standaert, R.F. et al., Nature 346:671-674 (1990), and a 3'UTR distinct from those of the previously known FKBP12 cDNA clones ( Figures 2 SEQ ID NO: 12, and 3 SEQ ID NOS: 1, 3 and 4). The newly cloned cDNA was
- FKBP12A SEQ. ID NO: 1
- FKBP12B SEQ ID NO: 3
- FKBP12C SEQ ID NO: 4
- FKBP12A is 738 base pairs in length and is predicted to encode a protein of 108 amino acid residues (SEQ ID NO: 5). This protein sequence is identical to the deduced amino acid
- FKBP12 sequence of FKBP12 based on two published FKBP12 cDNA sequences. (Standaert, R.F., et al. Nature 346:671-674 (1990); Maki, N. et al., Proc. Natl. Acad. Sci. USA 87:5440-5443 (1990).
- Radiolabeled DNA fragments specific to the three FKBP12 cDNAs were used as hybridization probes against human genomic DNA cut to completion with one of five
- transcript abundance is FKBP12B>FKBP12C>FKBP12A.
- FKBP12A 3'UTR probe also hybridized to a 4.7 kb
- transcript population ( Figure 7A, Lane 1) and, to a lesser degree, to two larger transcript populations (approximately 5.7 kd and 7.8 kb, visible in Figure 7A, Lane 2). These could represent cross-hybridizing mRNAs unrelated to FKBP12 production or precursor populations of mature FKBP12 transcripts.
- cytoplasmic RNA of T lymphocytes stimulated with PHA contains FKBP12A and FKBP12B transcripts.
- the amplified FKBP12A sequences were consistently less abundant than the amplified FKBP12B sequences (Lane 2 and 3 in Figure 7B),
- FKBP12C is a subset of the FKBP12B sequence, internal primers could not distinguish the two
- transcripts The first two time points examined after stimulation with 10 ⁇ g ml PHA, 4 hour and 16 hour, showed a progressive increase in these mRNA levels.
- FKBP12 transcripts predominate in poly(A)+RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas.
- the radiolabeled probe was the DNA oligomer described in Figure 8. Positions of RNA molecular weight markers are shown in kb.
- RNAs contained uncharacterized, faintly hybridizing RNAs around 4.2 kb.
- the three FKBP12 transcripts are produced from a single gene that contains five exons ( Figures 5 and 6 (SEQ ID NOS: 14-25)) and is located on chromosome 20 (DiLella, A.G. and Craig R.J., Biochemistry 30:8512-8517 (1991)).
- the four intron-exon splice junctions contain the conserved elements characteristic of eukaryotic genes ( Figure 5) and FKBP12 is encoded by the first four exons.
- the fifth exon provides a 3'UTR sequence exclusive to the FKBP12B and 12C transcripts.
- the four protein-coding exons correspond to the FKBP12 sequence as follows: Exon 1 encodes amino acids 1-11, Exon 2 encodes amino acids 13-27, Exon 3 encodes amino acids 29-65, and Exon 4 encodes amino acids 66-107 (arrows in Figure 3 designate exon junctions). Amino acids 12 and 28 are encoded by codons that are split by exon boundaries. There is a correlation between the exon-encoded domains of FKBP12 and the structural motifs of the protein (Moore, J.M. et al., Nature
- the unique 3 'UTRs also suggest that stability, translatability, cellular location, and tissue specificity may affect FKBP12 production.
- transcript diversity generated by these elements is reasonably viewed as a means to control the final production level of FKBP12 in cells.
- 3'UTRs are known to regulate spatial expression of proteins by controlling proper transcript localization within cells (Lipshitz, H.D. Current
- FKBP12A, FKBP12B and FKBP12C transcripts are regulated in a tissue-specific fashion, because the distribution of FKBP12B transcripts appears greater in heart, brain, placenta, and lung than in skeletal muscle, kidney, liver, and pancreas ( Figure 9). This variance could affect the immunophilin:calcineurin ratio postulated to be important for FK506- and CsA-specificity.
- FKBP12A is expressed in heart, placental and pancreatic tissues; FKBP12B, also found in these tissues, is additionally found in brain, lung, skeletal muscle, kidney and liver, in various
- the 3'UTR provides a useful marker sequence to determine tissue-specificity of FKBP12 mRNAs.
- the present invention also relates to probes which hybridize to all or a portion of the 3'UTR of FKBPs, and the use of these probes to detect the presence of tissue-specific FKBP12 mRNAs.
- one type of DNA probe is comprised of a DNA sequence complementary to the 3'UTR of FKBP12A mRNA.
- hybridization of the FKBP12A DNA probe to RNA present in a tissue sample would be useful to detect FKBP12A mRNA present.
- an RNA probe could be used in a similar manner.
- the present invention further relates to a method of monitoring rejection of transplanted tissue, or determining the effects of FK506 therapy to an individual.
- Production of FKBP12 is essential to form the inhibitory complex of FK506/FKBP12.
- FK506/FKBP12 complex acts as a potent
- FK506 therapy modifications of FK506 therapy may be made or another line of therapy may be initiated, before tissue rejection or systemic toxicity occurs.
- the present invention also relates to a method of modifying production of FKBP12 in vivo.
- a nucleotide fragment comprising either DNA or RNA complementary to DNA or RNA encoding FKBP12 is administered to an individual and hybridizes to all or a portion of the DNA or RNA encoding an FKBP. It is reasonable to predict that hybridization could result in modified production of the FKBP.
- the nucleotide sequence administered can be a DNA sequence complementary to the 3'UTR of FKBP12A mRNA. When the sequence hybridizes to the 3'UTR, translation of the mRNA would be inhibited. Thus, production of FKBP12 in specific tissues such as heart, is decreased.
- a decreased production of heart FKBP12A could result in less competition for FK506 by heart tissue relative to, for example, kidney tissue. If a kidney had been the transplanted organ, the immunosuppressant activity could be concomitantly increased in that specific tissue. Thus, in vivo modification of tissue-specific FKBP12 could increase effective immunosuppressant therapy for targeted tissues.
- the present invention further relates to in vivo and in vitro methods of evaluating the
- Measurement of an agent as an inhibitor may be accomplished by first, obtaining a baseline value of FKBP12 mRNA in cells a specific mammalian organ (i.e., the level of FKBP12 mRNA in the cells prior to or in the absence of treatment with an agent to be evaluated for its effectiveness as an immunosuppressant) using nucleic acid probes which hybridize to all or a portion of the 3'UTR of FKBP12 mRNAs, as described above.
- the baseline value is compared with the FKBP12 mRNA level in the same type of cells (e.g., a second sample or cells) after treatment with or in the presence of the agent being evaluated.
- Lower levels of FKBP12 mRNA after treatment with or in the presence of the agent i.e., a post-treatment level of FKBP12 mRNA is an indication that the agent
- the agent to be evaluated is administered to a mammal under conditions appropriate for the immunosuppressive agent to act upon production of FKBP12 mRNA.
- cells may be isolated from mammalian tissue of interest, cultured in vitro, and a baseline value indicating the presence of FKBP12 mRNA in the cultured cells obtained as previously described. The cells are then treated with an agent to be
- FKBP12 mRNA production is an indication of its ability to inhibit FKBP12 mRNA production.
- the presence or absence of FKBP12 mRNA is determined, with the difference between the two values providing a reasonable basis of evaluating the effectiveness of the agent in immunosuppressive therapy.
- Candidate FKBP12 cDNA clones were isolated from two human T lymphocyte cDNA libraries.
- a ⁇ gt10 cDNA library (Clontech Laboratories, Inc., Palo Alto, CA) of peripheral blood T cells stimulated with
- phytohaemagglutinin (PHA) for 48 hr was screened under nonstringent conditions with two synthetic oligomers derived from the open reading frame (ORF) sequence of two FKBP12 cDNAs (Maki, N. et al.. Proc.Natl. Acad. Sci. USA 87:5440-5443 (1990); Standaert, R.F. et al.. Nature 346:671-674 (1990).
- the oligomers, (SEQ ID NOS: 6 and 7) were synthesized on an Applied Biosystems 380A DNA synthesizer and were radiolabeled with ⁇ - 32 P-ATP.
- FKBP12A A full-length FKBP12 cDNA obtained from this screen (FKBP12A) was used to screen a second stimulated T lymphocyte cDNA library for additional FKBP12 cDNAs.
- the genomic clone ⁇ VX18 was obtained by screening an EMBL-3 SP6/T7 library (Clontech) of human placental genomic DNA with 32 P-labeled FKBP12A cDNA under
- Genomic clones cVX5 and cVX8 were obtained from a pWE15 cosmid library (Strategene Cloning Systems, La Jolla, CA) of human placental genomic DNA using hybridization probes under stringent conditions.
- cVX5 was isolated with a 4.5 kb fragment of ⁇ VX18 that extended from an Sfi I site just outside the SP6 promoter to the closest internal Kpn I site.
- cVX8 was isolated with a 764 bp fragment specific to the FKBP12B cDNA (generated by PCR using VX10230 and VX10232 as primers as shown in Figure 10) and was analyzed further after demonstrating that it also hybridized to a 319 bp Ban II/EcoR I fragment specific to the FKBP12 cDNA.
- the 738 bp cDNA insert of FKBP12A was excised by EcoR I (all restriction enzymes obtained from New England Biolabs, Beverly, MA) digestion and cloned into pUC19 for sequence analysis.
- the FKBP12A cDNA sequence (SEQ ID NO: 1) has been deposited in GenBank with accession number X52220.
- the intron and exon sequences of the human FKBP12 gene have been deposited in GenBank with accession numbers
- Human genomic DNA (Clontech Laboratories, Inc. Palo Alto, CA) was digested with the restriction endonucleases BamH I, BsaB I, EcoR I, Hind III, or Pst I and fractionated (10 ⁇ g per lane) in 1% agarose.
- the DNA was transferred to GeneScreen (New England Nuclear Research Products, Boston, MA) by capillary transfer using standard conditions and was crosslinked to the membrane with ultraviolet light (Stratalinker,
- the DNA probes were radiolabeled using ⁇ - 32 P-dCTP and a
- a lambda library of human placental genomic/DNA was screened at high stringency.
- Four clones were selected and purified, and DNA from each clone was digested with restriction er.; .nucleases and examined by Southern analysis. All clones hybridized to probes from the ORF, but they exhibited variable hybridization to probes from the 3' termini of the different FKBP12 cDNAs.
- One clone hybridized specifically to probes derived from the 3'UTRs of FKBP12B and FKBP12C, and sequence analysis indicated this clone was a processed pseudogene.
- the three remaining clones hybridized specifically to probes from the 3'UTR of FKBP12A and were extremely similar by restriction enzyme analysis.
- FKBP12B and FKBP12C cDNA sequences but only the first 165 bases of Exon 4 (the 126 bp of ORF, the TGA stop codon, and the next 36 bp) were found in these cDNA sequences ( Figure 6). Additional library screens were performed to isolate genomic clones containing the 5' exons of the FKBP12 mRNAs and the 3' exons of the 12B and 12C mRNAs.
- cosmid library with inserts averaging 38 kb in size was screened.
- Two independent screening approaches were used: (1) cosmid selection with a fragment from the 5' end of ⁇ VX18 and (2) cosmid selection with a DNA fragment from the 3'UTR of FKBP12B followed by secondary selection with a DNA fragment from the 3'UTR of FKBP12B followed by secondary
- Clone cVX5 was isolated by the first approach and contained the 5' end of the FKBP12 gene; clone cVX8 was isolated by the second approach and contained the entire FKBP12 gene ( Figure 5).
- Subcloning and sequencing part of cVX5 revealed two exons (Exons 1 and 2) separated by an 80 bp intron. These exons encoded the 5'UTR and first 85 bp of ORF in each FKBP12 cDNA. Specifically, Exon 1 contained the upstream sequence and first 37 bp of ORF while Exon 2 encoded the next 48 bp of ORF ( Figure 6).
- the synthetic oligomers VX10027 and VX10247 (SEQ ID NOS: 8 and 9 respectively) served as the 5' and 3' primers, respectively. Each primer was used at a final concentration of 10 ⁇ M in conjunction with 50 ng of each cell line DNA, and the total reaction volume was 50 ⁇ l.
- the amplified product was of the expected size (395 bp) and hybridized to synthetic DNA oligomers derived from the gene sequence and internal to the PCR primers.
- PBLs peripheral blood lymphocytes
- lymphocytes were isolated and separated by density centrifugation over Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) according to the manufacturer's instructions. The population was enriched for T lymphocytes by rosetting with sheep red blood cells for 1 hr at 4°C followed by another
- E + PBLs Erythrocyte receptor positive PBLS
- RPMI 1640 Gibco BRL, Grand Island, NY
- Poly(A)+RNA for Northern analysis was fractionated in 1% agarose containing formaldehyde before being transferred and crosslinked to nylon membrane as described above.
- Northern blot was purchased from Clontech. DNA probes were radiolabeled with ⁇ - 32 P-dCTP as described above while RNA probes were transcribed from fragments cloned into pBluescript ® II (Stratagene Cloning Systems La
- Marker sizes were determined by the migration profile of lambda DNA digested with EcoR I and Hind III and are indicated in kb.
- RNA was prepared for polymerase chain
- cytoplasmic RNA was primed with oligo-dT 12-18
- DNA oligomers used to amplify specific portions of the different FKBP12 mRNAs were as follows: VX10172 (SEQ ID NO:28)-VX10247 (SEQ ID NO: 9) for FKBP12A mRNA, VX10027 (SEQ ID NO: 8) -VX10173 (SEQ ID NO:29) for
- FKBP12B mRNA, and VX10172 (SEQ ID NO:28)-VX10028 (SEQ ID NO:26) for the ORF common to all FKBP mRNAs see Figure 10).
- a Perkin Elmer Cetus GenAmp 9600 was used for all PCR reactions that involved cytoplasmic RNA, and the DNA products were fractionated on 2% NuSieve 3:1 (FMC BioProducts, Rockland, ME) agarose gels.
- Lane 1 VX10172 (SEQ ID NO: 28)-VX10028 (SEQ ID NO:26): specific to the ORF of all FKBP12 mRNAs, 299 bp fragment expected and obtained;
- Lane 2 VX10172 (SEQ ID NO:28) -VX10247 (SEQ ID N0:9): specific to FKBP12A mRNA, 683 bp fragment expected and obtained;
- Lane 3 VX10027 (SEQ ID NO: 28) -VX10173 (SEQ ID NO:29): specific to FKBP12B mRNA, 607 bp fragment expected and obtained;
- Lane 4 VX10201 (SEQ ID NO: 30)-VX10801 (SEQ ID NO:33): specific to FKBP12C mRNA, 608 bp fragment expected but not obtained;
- Lane 5 control primers SEQ ID NOS: 12 and 13, respectively to produce a 335 bp fragment specific for ß2-microglobulin.
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US6852496B1 (en) | 1997-08-12 | 2005-02-08 | Oregon Health And Science University | Methods of screening for agents that promote nerve cell growth |
US7338976B1 (en) | 1998-08-14 | 2008-03-04 | Gpi Nil Holdings, Inc. | Heterocyclic esters or amides for vision and memory disorders |
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- 1993-05-20 WO PCT/US1993/004916 patent/WO1993023548A2/fr active Application Filing
- 1993-05-20 AU AU43888/93A patent/AU4388893A/en not_active Abandoned
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WO1992019745A1 (fr) * | 1991-05-08 | 1992-11-12 | Vertex Pharmaceuticals Incorporated | Rfkbp: une nouvelle isomerase de prolyle et proteine de liaison de rapamycine/fk506 |
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