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WO1998036641A1 - Systeme de transport d'implant dentaire muni d'un support de guidage a collerette amovible - Google Patents

Systeme de transport d'implant dentaire muni d'un support de guidage a collerette amovible Download PDF

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WO1998036641A1
WO1998036641A1 PCT/US1998/003049 US9803049W WO9836641A1 WO 1998036641 A1 WO1998036641 A1 WO 1998036641A1 US 9803049 W US9803049 W US 9803049W WO 9836641 A1 WO9836641 A1 WO 9836641A1
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promoter
inflammatory response
affector
mammal
function
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Santa Jeremy Ono
Vincenzo Casolaro
Michael Sheffery
Steven L. Swendeman
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Schepens Eye Research Institute Inc
Johns Hopkins University
Memorial Sloan Kettering Cancer Center
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Schepens Eye Research Institute Inc
Johns Hopkins University
Memorial Sloan Kettering Cancer Center
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/13—Decoys

Definitions

  • This invention relates to regulation of the mammalian inflammatory response and particularly to regulation of cytokine control thereof .
  • IL4 is the prototypic member of a family of cytokines able to modulate the differentiation and the biologic activities of cells of the hematopoietic lineage, including T cells (Boulay and Paul, 1992). These cytokines, including IL3 , IL5 , IL13 and granulocyte-macrophage colony-stimulating factor, are coexpressed in the T helper (Th) 2 subset of CD4 + T cells and in Fc£RI + cells (i.e., basophils and mast cells) (Paul et al . , 1993; Paul and Seder, 1994) .
  • Th T helper 2 subset of CD4 + T cells
  • Fc£RI + cells i.e., basophils and mast cells
  • Th2 and Fc£RI + cells do not produce IL-2 or interferon (IFN) - ⁇ , which are typically associated with the Thl subset of T cells and play a critical role in the development of cell-mediated responses (Paul et al . , 1993; Paul and Seder, 1994) .
  • IFN interferon
  • TL4 itself, perhaps produced by cells other than T cells, such as basophils, mast cells, or CD4 + NK1.1 + cells, is an essential signal for the preferential expression of a Th2 phenotype, while it interferes with Thl cell differentiation and function (Paul et al . , 1993; Paul and Seder, 1994; Yoshi oto and Paul, 1994) .
  • CP2 a 502 -aa nuclear protein that was originally identified as a factor binding to at least two elements within the murine ⁇ -globin gene promoter and able to activate transcription from ⁇ -globin promoter-driven templates in vi tro and in vivo (Lim et al . , 1992), is also, unexpectedly, an integral component of a transcriptional complex forming in Jurkat cells on an IL4 promoter region previously shown to interact with other factors, including NFAT-1, CBF, and HMG I (Y) . Additionally, we have shown that IL2 transcription is repressed in cells overexpressing CP2. These results suggest that CP2 is an important participant in differential cytokine gene expression in human T cells. Furthermore, we have discovered that an alternatively spliced CP2 variant lacking the DNA-binding domain functions as a dominant negative antagonist of CP2 in regulating IL4 production.
  • the invention features therapeutic compositions that include small molecule affectors of CP2 function, particularly inhibitors or activators of CP2 production or of complex formation between CP2 and a CP2 recognition element in the TL4 promoter, and methods of use of such compositions.
  • the inhibitors or activators are useful as therapeutic agents in methods of treatment of immunological disorders, to restore appropriate immunological balance.
  • the invention also includes methods of screening for such affectors of CP2 function.
  • the invention further includes methods of treatment using gene therapy, such as using CP2 cDNA to transform cells from Thl to Th2 , to decrease a harmful inflammatory response in a patient, or using CP2 dominant negative cDNA to transform cells from Th2 to Thl, in order to induce an inflammatory response.
  • gene therapy such as using CP2 cDNA to transform cells from Thl to Th2 , to decrease a harmful inflammatory response in a patient, or using CP2 dominant negative cDNA to transform cells from Th2 to Thl, in order to induce an inflammatory response.
  • Fig. 1 shows the effect of CP2 overexpression of TL2 and JL4 promoter activity in transiently transfected Jurkat cells
  • Fig. 2A is a schematic diagram of CP2 primary structure, of the structure of nine COOH-terminal truncations of CP2 and of the structure of a ⁇ Elf-1 deletion of CP2 ;
  • Fig. 2B shows the effect of COOH-terminal truncation on CP2 activity.
  • Mean + SEM fold induction of ⁇ l through ⁇ 9 cloned into a pRc/CMV mammalian expression vector and cotransfected in Jurkat cells with a p/L4.311 reporter plasmid from three independent experiments is indicated relative to samples cotransfected with a noncoding Prc/CMV vector (designated by the dashed horizontal line) .
  • Full-length CP2 expression vector is designated by FL;
  • Fig. 2C shows the effect of Elf-1 factor deletion on CP2 activity under the same conditions as Fig. 2B;
  • Fig. 3A is a schematic representation of the IL4 promoter and of PCR-generated deletional mutants
  • Fig. 3B shows the effect of the IL4 deletional mutants of Fig. 3A on constitutive and induced CAT expression.
  • Jurkat cells were transiently transfected with 1 ⁇ g each of the pXL4.741 reporter plasmid or its deletional mutants (pIL4.311 through pTL4.65) and 2 ⁇ g of a Prc/CMV-CP2 expression vector or its corresponding noncoding control (indicated by the dashed line) . Mean + SEM fold induction in three independent transfections are shown;
  • Fig. 4A shows the nucleotide sequence of the IL4 promoter regions required for full (225-176) and partial (175-176) transactivation in vivo . Boxed sequences ⁇ Box II, ISRE, P3 , CCAAT d , and P2) have been characterized as competent recognition sites for the indicated transcription factors. Also shown are oligonucleotide probes 225-176, 195-146, and 175-146, spanning the IL4 promoter regions included between the indicated bp; Fig. 4B is a representation of binding of enriched, bacterially expressed CP2 (rCP2) to a 5' end-labeled ⁇ -globin canonical site and to the T 4 oligonucleotides shown in Fig. 4A;
  • rCP2 enriched, bacterially expressed CP2
  • Fig. 4C shows (OP) 2 Cu + footprinting of a 195-146 oligonucleotide, 5' end-labeled on the coding strand;
  • Fig. 4D shows alignment of the IL4 promoter region protected by RCP2 with a series of CPRE identified in some cellular and viral promoters .
  • the two CNRG boxes are indicated in bold letters. Shown are the distal and proximal CPRE from the mouse ⁇ -globin promoter, high-affinity sites from the rat ⁇ -fibrinogen promoter and the HIV LTR, and a low-affinity site from the MHC class II Ea promoter.
  • Cyp 2d- 9 steroid 16 ⁇ -hydroxylase gene recently found to bind the CP2-related protein LBP-la (Sueyoshi et al . , 1995) . Numbers indicate the positions relative to the transcription initiation sites;
  • Fig. 5A shows binding of Jurkat nuclear proteins to a 5 ' end-labeled ⁇ -globin or a 195-146 oligonucleotide
  • Fig. 5B shows the effect of RCP2 on the binding of Jurkat nuclear proteins to a 195-146 probe.
  • Jurkat nuclear extracts (5 ⁇ g) were incubated with the probe with (lane 2) or without RCP2 (lane 3) .
  • the white arrow to the left of the gel indicates the complex formed with RCP2 alone (lane 1) , while the black arrow indicates the appearance of an additional complex (shown in lane 2) of intermediate mobility between the NFAT-1/CBF complexes;
  • Fig. 6A shows detection of endogenous CP2 in complexes forming on full-length PCR-generated IL4 promoter fragments via Dnase I footprinting of an TL4.265 deletional fragment, 5' end- labeled oh the noncoding strand.
  • the positions of protected nucleotides are determined by alignment with a Maxam-Gilbert G+A ladder of the same fragment .
  • Numbers to the left of the gel refer to the nucleotides around which elements characterized so far in the human or mouse promoters (schematized to the far left) are centered. Shown is the pattern of Dnase I cleavage of free DNA (F) and DNA incubated with 20 ⁇ g Jurkat nuclear extracts
  • FIG. 6B shows EMSA of PCR-generated, PCR-labeled IL4 promoter deletional fragments with Jurkat nuclear extracts. Following exposure to the probes, extracts (2.5 ⁇ g) were incubated with equivalent amounts (1 ⁇ g protein) of rabbit preimmune serum (PS) or the indicated antibodies. The arrow to the left indicates a CP2 immunoreactive complex selectively forming on an IL4 .225 probe; and
  • Fig. 6C shows the results of an experiment in which prior to incubation with an JL4.225 probe, 1 ⁇ g Jurkat nuclear extracts (JKT; lanes 1-6) or 20 ng RCP2 (lanes 7-12) were exposed to a 50- fold molar excess of a panel of oligonucleotides spanning the indicated regions of the human IL4 promoter.
  • the arrows to the left indicate complexes whose formation is selectively diminished by competition with a 195-146 oligonucleotide.
  • CP2 is a critical and relatively specific transactivator of the IL4 gene in human T cells.
  • Our conclusions are supported by the following lines of evidence: 1) IL4 promoter activity is markedly enhanced, while IL2 promoter activity is repressed, in Jurkat cells overexpressing CP2 ; 2) overexpression of a CP2 dominant negative, specifically inhibiting CP2 binding and function, represses IL4 promoter activity; 3) transactivation by CP2 maps to an IL4 promoter region protected by rCP2 in footprinting experiments; and 4) native CP2 is an integral component of a transcriptional complex forming exclusively on IL4 promoter fragments including the region required for optimal CP2 binding and function.
  • ⁇ Elf-1 CP2 While repression by the ⁇ Elf-1 dominant negative suggests that endogenous CP2 critically accounts for TL4 constitutive transcription in Jurkat cells, transactivation by CP2 was less marked in Jurkat cells stimulated with A23187 and/or PMA. On the other hand, ⁇ Elf-1 CP2 overexpression also repressed T 4 transcription in stimulated cells. As previously shown in HeLa, K562 and MEL cells (Lim et al . , 1993), CP2 is constitutively expressed in the nuclei of Jurkat cells. Stimulation of these cells did not apparently affect CP2 expression and/or binding, with the only exception being a slight but noticeable downregulation by PMA. Therefore, it appears that CP2 accounts for the formation of a constitutive TL4- specific transcriptional complex, as shown in Figure 6B, while inducible factors, possibly interacting with CP2 , might mediate IL4 activation in stimulated cells .
  • a low-affinity CP2 recognition element is located between bp -177 and -158 of the human TL4 promoter.
  • This region includes a sequence ( "174 CTGATTTCACAGG “162 ) diverging by one bp from the published CP2 consensus, which has been defined by assessing the effect of in- frame clustered mutations within canonical, high-affinity CP2 sites, such as the proximal c-globin site or the ⁇ -fibrinogen site, on RCP2 binding in EMSA (Lim et al . , 1993) .
  • the IL4 CPRE is surrounded by binding elements for the factors NFAT-1, CBF, IRF-2, STAT6 , and HMG I (Y) (Szabo et al . , 1993; Li-Weber et al . , 1994; Klein-Hessling et al . , 1996; Lederer et al . , 1996) , suggesting that CP2 may interact with any of these factors.
  • the 3' half of the CPRE partially overlaps the P2 sequence, which in a previous study of the mouse TL4 promoter has been shown to interact with NFAT-1 or a related protein (Szabo et al . , 1993).
  • NFAT-1 can bind to a 175-146 oligonucleotide in EMSA.
  • a CBF-binding Y/CCAAT box the most distal of three similar functional elements identified in the IL4 promoter — lies immediately upstream of the 5' end of the T 4 CPRE ( Figure 4A) (Li-Weber et al . , 1994) .
  • CP2 and CBF/CP1 also bind to adjacent elements in the oi-globin promoter, suggestive of possible interactions between the two factors (Kim et al . , 1990) .
  • CP2 might not cooperate with CBF and/or NFAT-1 in this particular system.
  • immunoreactive CP2 was undetectable in EMSA with nuclear extracts from Jurkat cells, expressing fair amounts of the protein, and an oligonucleotide probe (195-146) including the P2 and P3 NFAT-binding sequences and the distal CCAAT.
  • a major complex was formed that contained CBF, as previously demonstrated in EMSA using a similar oligonucleotide (Li-Weber et al . , 1994).
  • the first preferentially binds CP2 as a homodimer
  • second-class CPRE including the ⁇ -globin SSE and another site in the ⁇ -globin .promoter
  • CP2 as an obligate heterodimer, where stage- and/or lineage-specific cofactors possibly mediate CP2 binding and function (Jane et al . , 1995)
  • stage- and/or lineage-specific cofactors possibly mediate CP2 binding and function (Jane et al . , 1995) .
  • the IL4 CPRE exhibits an imperfect half site, we cannot conclude that this site belongs to this second class of CP2 elements.
  • the JL4 element apparently binds rCP2 homodimers, the resulting complex having identical mobility to that forming on an ⁇ f-globin first- class site.
  • IL4 transcriptional regulators have been identified by assessing the effect of their overexpression or ectopic expression in cells transiently transfected with IL4 promoter-driven reporter constructs.
  • NFAT-1, c-Maf, C/EBP/3 and NF- ⁇ Bl activate transcription from a panel of human or mouse IL4 promoter constructs (Casolaro et al . , 1995; Davydov et al . , 1995; Ho et al . , 1996; Luo et al . , 1996), while other factors, such as RelA and HMG I (Y) , exert a distinct inhibition of IL4 promoter activity (Casolaro et al .
  • IL4 promoter activation we analyzed the effect of CP2 overexpression on chloramphenicol acetyltransferase (CAT) gene expression driven by a region of the human JL4 gene extending from bp -311 through +55 relative to the transcription initiation site. This region includes all the IL4 promoter elements characterized to date in human or mouse T cells, and confers on heterologous reporter genes proper lineage- and activation-specific expression (Bruhn et al., 1993; Todd et al . , 1993). The human T cell line Jurkat has been widely used as a model for the study of human IL4 transcription.
  • CAT chloramphenicol acetyltransferase
  • IL4 transcription IL4 transcription
  • Jurkat cells are in a preactivated state, characterized by constitutively elevated IL4 promoter activity and mRNA accumulation (Li-Weber et al . , 1992) . This may reflect dysregulation of the calcineurin-NFAT pathway in a leukemic cell line, although constitutive NFAT-1 nuclear translocation and IL4 activation ' have also been observed in nontransformed Th2 clones (Lederer et al . , 1994) . Referring to Fig.
  • the CAT reporter plasmids IL2-15 ⁇ CX ( IL2) , bearing bp -319 to +52 of human IL2, or pJL4.311 (JL4) , bearing bp -311 to +55 of human IL4 , were transfected (1 ⁇ g each) in Jurkat cells along with 2 ⁇ g of Prc/CMV-CP2 expression plasmid ( ⁇ ) or a control pRc/CMV noncoding vector (D) as described in Materials and Methods.
  • CP2 overexpression also resulted in significant, although less noticeable, increase of T 4 promoter activity in cells stimulated with PMA (10 ng/ml) and Ca 2+ -ionophore (A23187; 1 ⁇ M) or Ca 2+ ionophore alone, which was sufficient to induce maximal IL4 transactivation.
  • PMA 10 ng/ml
  • Ca 2+ -ionophore A23187; 1 ⁇ M
  • Ca 2+ ionophore alone
  • CP2 appears to represent a novel family of dimeric transcription factors binding direct DNA repeats. CP2 dimerizes in solution, and heterodimers of CP2 with other LBP-1 family members and nonrelated proteins have been described (Uv et al . ,
  • CP2 dimerization is not essential for DNA binding, as would be suggested by the dyad symmetry of the CP2 consensus recognition sequence, but it does affect the stability of CP2 nucleoprotein complexes (Uv et al . , 1994; Zhong et al . , 1994).
  • the major CP2 dimerization domain has been located within a COOH-terminal region (aa 426 to 502) sharing significant homology to the Drosophila factor Elf-1 (Uv et al . , 1994).
  • Fig. 2A shows a schematic diagram of CP2 primary structure and of the structure of COOH-terminal truncations ⁇ l through ⁇ 9.
  • the DNA- binding and dimerization domains, sharing homology to the Drosophila regulatory protein Elf-1, are indicated; a black box labelled "Elf-1" indicates the region encoded by exon 6 of the CP2 gene (aa 189-239) , which is the most closely related to Elf-1 and is essential for DNA binding.
  • a domain (SPXX; ' aa 250 to 405) , rich in serine/threonine (17.5%) and proline (11%) residues, that contains several SPXX motifs.
  • This domain is followed by a stretch of 10 glutamines or a sequence of alternating glutamine and proline residues (Q/P domain; ⁇ ) in human or mouse CP2 , respectively (Lim et al . , 1992) .
  • the COOH-terminal dimerization domain is included in a region having a net moderately negative charge (net acidic) .
  • a ⁇ Elf-1 deletion (lacking aa 189-239) .
  • CP2 dominant negative has been described as lacking the domain encoded by exon 6 (aa 189 to 239) , which is the one most closely related to Elf-1 (Uv et al . , 1994; Zhong et al . , 1994).
  • This protein named in different studies LBP-ld, aCP2 , or LSF-ID, is spontaneously generated in HeLa and other cells by alternative splicing or secondary splicing of CP2 transcripts and exhibits no DNA binding activity (Shirra et al .
  • FIG. 3A shows a schematic representation of the JL4 promoter and of PCR-generated deletional mutants bearing 5' deletions of the human JL4 promoter to the indicated nucleotide upstream of the transcription initiation site. These fragments were inserted into pCAT-Basic to generate the reporter plasmids pTL4.741, pJL4.311, pI 4.265, pJL4.225, pTL4.175, pIL4.145, pIL4.95 and pTL4.65.
  • Open and closed rectangles indicate the relative positions of positive and negative regulatory cis elements, respectively, identified to date in the human or mouse IL4 promoter. Deletions were specifically designed to analyze the relative impact of J 4 regulatory elements characterized to date on CP2 -mediated activity. Positive regulatory elements include: the P0-P4 sequences, scattered throughout the promoter and shown to bind NFAT-1 and other factors, such as NF- ⁇ B (PI), CBF (PI), HMG I (Y)
  • Negative regulatory elements include: NRE-I and NRE-II, contiguously located at bp -307, which bind as yet unidentified transcriptional repressor(s) (Li -Weber et al . , 1992); an IRF-2- binding element (ISRE), located at bp -195 (Li-Weber et al . , 1994) ; and an A/T-rich region starting at bp -209 and termed Box II, presumably mediating repression by HMG I (Y) (Klein-Hessling et al. , 1996) .
  • CPRE CP2 -recognition element
  • EXAMPLE IV Definition of a CPRE Within the JL4 Promoter
  • the TL4 promoter region included between bp -225 and -146 contains binding sites for numerous factors, including the P2 and P3 sequences (NFAT-1 and STAT6) , a CCAAT box partially overlapping the P3 sequence (CBF) , an ISRE (IRF-2) and the HMG I (Y) -binding A/T-rich Box II, as shown in Fig. 4A (Szabo et al . , 1993; Li-Weber et al . , 1994; Klein-Hessling et al . , 1996; Lederer et al . , 1996) .
  • enriched, bacterially expressed CP2 (rCP2) , used at a concentration sufficient for saturation of an c-globin consensus oligonucleotide (100 ng in 15 ⁇ l) , did not bind to an oligonucleotide (175-146) including the TL4 promoter region whose removal resulted in loss of CP2 -mediated transactivation in vivo (Fig. 4B, lane 2) . Additionally, rCP2 did not bind to oligonucleotide 225-176, spanning the TL4 promoter region necessary for maximal transactivation (Fig. 4B, lane 4) .
  • a hypersensitive site at bp -151 and a footprint extending from bp -158 to -177 are indicated to the right as an asterisk and a dashed vertical line, respectively.
  • rCP2 protected a DNA sequence extending from bp -177 to -158.
  • An imperfect CPRE (- 174 CTGATTTCACAGG ⁇ 162 ) is recognizable within this sequence.
  • rCP2 only partially protected an adjacent CCAAT box (- 180 ATTGG ⁇ 176 ) , confirming that, in contrast to initial observations (Chodosh et al . , 1988), ' CP2 is not a CCAAT-binding protein.
  • Fig. 4D shows the alignment of the T 4 promoter region protected by rCP2 with a series of CPRE identified in some cellular and viral promoters.
  • Fig. 5A binding of Jurkat nuclear proteins (5 ⁇ g) to a 5' end- labeled ⁇ -globin (lanes 1-5) or 195-146 oligonucleotides (lanes 6-10) was examined. Following incubation with the DNA probes, extracts were incubated with rabbit preimmune serum (PS; lanes 1 and 6) or the indicated antibodies (1 ⁇ g each) . Complexes containing immunoreactive CP2 (lane 2) , NFAT-1 and/or CBF-A are indicated.
  • NFAT-1 reported to interact with at least four sites within the proximal 225 bp of the TL4 promoter (Szabo et al . , 1993) , is not apparently a component of this complex. NFAT- 1-immunoreactive complexes were instead detected on the JL4.175 and TL4.95 fragments, suggesting that the exposure of different regions of the IL4 promoter leads to the formation of alternative transcriptional complexes of different composition and perhaps function.
  • rCP2 bound only to IL4 promoter fragments extending upstream of bp -175.
  • Fig. 6C both rCP2 binding and the formation of a CP2-immunoreactive native complex on an TL4.225 probe were specifically inhibited by competition with a 50-fold molar excess of unlabelled 195-146 oligonucleotide, but not by oligonucleotides spanning other regions of the human IL4 promoter, consistent with our footprinting analysis.
  • JL4 promoter fragments used in this study were initially generated by PCR using human genomic DNA as a template and cloned into the HindiII and Xbal sites of a pBluescript vector (Stratagene Cloning Systems, La Jolla, CA) .
  • an Xbal -tailed oligonucleotide corresponding to bp +36 to +55 of human IL-4 and one of four Hindi11 -tailed oligonucleotides, corresponding to bp -741 to -722, -311 to -292, -265 to -246, -225 to -206, -175 to -156, -145 to -126, -95 to -76, or -65 to -46, were used to introduce the appropriate restriction sites at the invariant 3' and at the 5' ends, respectively.
  • each fragment was inserted into the Hindi11 and Xbal sites of the pCAT-Basic vector (Promega Corporation, Madison, WI) to construct the corresponding reporter plasmids pIL4.741, pIL4.311, pIL4.265, pIL4.225, pIL4.175, pIL4.145, pIL4.95 and pTL4.65.
  • the CP2 expression plasmid (in pRc/CMV; Invitrogen Corporation, San Diego, CA) has been described in previous studies (Lim et al . , 1993). COOH- terminal truncations of the full-length polypeptide and the Elf-1 deletion were prepared by PCR as described (Zhong et al .
  • the JL2.15 ⁇ CX reporter plasmid bearing bp -319 to +52 of human TL2 (Shaw et al . , 1988), has been kindly donated by Dr. G. R. Crabtree (Stanford University, Stanford, CA) .
  • FBS heat-inactivated fetal bovine serum
  • Cells (3 x 10 6 ) were transfected with 1 ⁇ g CAT reporter plasmid and 2 ⁇ g expression plasmids by 48-h culture in RPMI 1640 containing 5.2 mg/ml Lip ⁇ fectamine (Life Technologies, Gaithersburg, MD) , according to the manufacturer's specifications (Casolaro et al . , 1995) . Equal amounts of the corresponding noncoding vectors were added to control samples to yield a constant amount (3 ⁇ g) of DNA in each transfection. Where indicated, cells were stimulated 16-18 h before harvest.
  • CAT concentrations have been expressed as pg/ ⁇ g total protein.
  • Nuclear Extracts and Recombinant Proteins have been prepared by a modification of a described protocol (Li et al . , 1991). Cells (5 x 10 7 ) were allowed to swell in 10 mM HEPES, pH 7.9, 30 mMKCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM phenyl methyl sulfonyl fluoride (PMSF) , 0.5 ⁇ g/ml leupeptin, and 1 ⁇ g/ml aprotinin, then lysed by the addition of 0.075% NP40.
  • DTT dithiothreitol
  • PMSF phenyl methyl sulfonyl fluoride
  • Nuclei were separated from the cytosolic extract by centrifugation for 4 min at 3,000 rpm in microfuge, resuspended in 20 mM HEPES, pH 7.9, 420 mMKCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, 0.5 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin, and 20% glycerol , and left on ice for 40 min. Nuclear debris and membranes were pelleted for 10 min at 14,000 rpm in microfuge. The supernatant was removed, aliquoted, quick- frozen in liquid nitrogen, and stored at -80°C.
  • Double-stranded oligonucleotides were 5 ' -end-labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) using ⁇ - 32 P-ATP (Amersham Corporation, Arlington Heights, IL) and purified by electrophoresis on 4% polyacrylamide gels.
  • T4 polynucleotide kinase New England Biolabs, Beverly, MA
  • ⁇ - 32 P-ATP Amersham Corporation, Arlington Heights, IL
  • larger regions of the human IL4 promoter corresponding to the deletional fragments inserted into CAT vectors, were generated by PCR of a pIL4.741 template using the Xbal and HindiII primers described in an earlier section. These fragments were labeled by inclusion of either 5' end-labeled primers or cv- 32 P-dCTP in the PCR reaction.
  • EMSA EMSA. Probes (10,000-30,000 c.p.m., corresponding to 5-20 fmol) were incubated (20-30 min, 25°C) with 1-5 ⁇ g nuclear extracts or 10-100 ng rCP2 in 15 ⁇ l of 12 mMHepes, pH 7.9, 50 mMKCl, 0.5 m MgCl 2 , 0.12 mM EDTA, 0.12 mM EGTA, 4 mM DTT, 0.1% NP40, 12% glycerol, 0.1 mg/ml bovine serum albumin (BSA) and 30 ⁇ g/ml (10 ⁇ g/ml in the case of rCP2) poly(dl-dC) (Pharmacia Biotech Inc., Piscataway, NJ) .
  • BSA bovine serum albumin
  • extracts were incubated 10 min at 25 °C with a 50- fold molar excess of competitor unlabelled oligonucleotides.
  • the binding reactions were incubated 30 min at 4°C with rabbit antisera specific for the transcription factors CP2 , NFAT-1 (Upstate Biotechnology, Lake Placid, NY) , CBF-A (Accurate Chemical and Scientific Corp. , Westbury, NY) , HMG I (Y) (kindly provided by Dr. D. Thanos, Columbia University, New York, NY) .
  • This treatment led, under the experimental conditions described, to specific ablation of the immunoreactive complexes, with almost no detectable "supershift” .
  • Free probes and DNA-protein complexes were resolved by electrophoresis on 4% native polyacrylamide gels in 45 mM Tris, pH 8.2, 45 mM boric acid, 1 mM EDTA and 1% glycerol and visualized by autoradiography of fixed and dried gels.
  • MgCl 2 was added to 5 mM, then samples were digested with DNase I
  • Free DNA and DNA-protein complexes visualized by autoradiography, were eluted (18 h at 37°C) from the gel matrix in 0.5 M ammonium acetate, pH 7.5 , 1 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS) . Equivalent amounts of DNA from each sample and of a Maxam-Gilbert G+A ladder of the same probes were resolved onto an 8% acrylamide/7 M urea gel.
  • CP2 has now been shown to regulate both TL4 and IL2 expression
  • interruption or enhancement of CP2 activity and thus regulation of Thl/Th2 cell balance can be used for therapeutic * control of the immune response and immunologic disease in a variety of conditions. These include, but are not restricted to: allergic rhinitis, allergic conjunctivitis, asthma, dermatitis, urticaria, multiple sclerosis, type I diabetes mellitus, arthritis and parasitic infection.
  • CP2 or dominant negative CP2 may also be useful in the management of immunodeficiency disorders or malignancies by amplifying T helper cell responses to viral antigen.
  • T helper cell response to autoantigen or foreign antigen plays an important part in disease onset and/or severity.
  • Ono and coworkers first showed that interferon-gamma mRNA is found in the pancreas at very early stages of insulitis (Ono et al . , 1988). Rabinovitch and coworkers confirmed this finding, and showed that expression of this and other Thl cytokines correlates with beta-cell destruction in BB rats (Rabinovitch et al . , 1996). Additional evidence for a role for Thl cells in IDDM came from the laboratory of Adorini (Trembleau et al .
  • autoimmune diseases also appear to depend upon the nature of the T helper cell response to autoantigen.
  • Multiple sclerosis and its experimental model (experimental allergic encephalomyelitis (EAE) ) also appears to be a Thl mediated disease.
  • EAE experimental allergic encephalomyelitis
  • Most T cell lines which are capable of inducing disease in animal models are Thl cells, and TL4 treatment results in induction of autoantigen-specific Th2 cells, diminished ' demyelination and significant amelioration of clinical disease (Cua et al . , 1995) .
  • allergic diseases show a strong correlation with Th2 responses.
  • Nasal biopsies from adult patients with seasonal allergic rhinitis or conjunctivitis exhibit elevated levels of mRNA for the Th2 cytokines, IL2 , IL4 , and IL5 , and little or no Thl cytokine gene expression (Karlsson et al . , 1995) .
  • Very similar findings are found in allergic asthmatics. Analysis of late phase allergic asthmatics typically show recruitment of eosinophils to the airways, IL4 mRNA in BAL fluids, and Th2 cytokines by ELISA (Bell et al . , 1996). In summary, an elevated Thl response appears to be critical for autoimmune diseases, while an elevated Th2 response appears to be critical for allergic diseases.
  • Nishimura et al . were able to apply IL12 antitumor gene therapy to B lymphoma cells (Nishimura et al . , 1996) . Tumor cells transfected with both the B7-1 and JL12 genes almost completely lost their
  • Retroviral mediated transfer of the TL2 gene into tumor infiltrating lymphocytes was shown to have minimal side effects when re-infused into the chest cavity of patients in a Phase I clinical trial, and some of the patients showed resolution of pleural effusions and decrease in tumor burden.
  • the methods for transfer of CP2 based genes into lymphocytes and their reintroduction into patients are in the public domain.
  • these methods have been shown to be safe and can be efficacious.
  • IL10 gene vectors were successfully transferred into murine cardiac allografts (Qin et al . , 1996) .
  • adenovirus mediated gene therapy is also a viable alternative mood. Replication-deficient recombinant adenovirus has been routinely used to transfer a variety of genes into most types of human tissue (Wilson et al . , 1995; Goebel et al . , 1996). CP2 expression vectors can also be administered locally, e.g., near the ocular surface, so that transfection can take place in vivo .
  • the search for small molecules that may interfere with endogenous CP2 , and therefore endogenous cytokine gene transcription can be carried out using existing or newly prepared small molecule libraries, which can be tested in high throughput screens.
  • One such screen is an electrophoretic gel mobility shift assay in which recombinant CP2 bound to the CP2 recognition element (CPRE) described herein is resolved from unbound CPRE on nondenaturing acrylamide gels. The complex runs more slowly through the gel than the free probe (radiolabeled CPRE) .
  • the screen for compounds that inhibit CP2 interaction with the recognition element would involve the addition of physiologic concentrations of each compound to separate binding reactions. An effective competitor would abolish CP2/CPRE complex formation, which is easily seen on the mobility shift experiment.
  • peptides derived from the CP2 DNA-binding domain or other small molecules can also be tested for inhibitory capacity.
  • Such peptides can be chosen randomly as overlapping peptides (LeSêt et al . , 1995; Digard et al . , 1995), or after rational drug design, by methods well known to those of skill in the art.
  • peptides corresponding to the DNA binding domain of CP2 (SEQ ID NO: 2) or to the Elf-1 domain (SEQ ID NO: 3) within the DNA binding domain, or to fragments thereof of 10 amino acid residues or greater, are likely to be effective competitors to CP2/CPRE complex formation.
  • Oligonucleotides that might interfere with CP2/CPRE interaction include those that are homologous to the CPRE region itself as shown in Fig. 4D.
  • Such oligonucleotides include 5 ' GTCTGATTTCA-CAGGAA3 ' ( SEQ ID NO 4 ) 5 ' AACAAGTTTTA-CTGGGT3 ' ( SEQ ID NO 5 ) 5 ' GCAAGCACAAACCAGCC3 ' ( SEQ ID NO 6 ) 5 ' GACCAGTTCCAGCCACTC3 ' ( SEQ ID NO 7 ) 5 ' TACTGGGTCTCTCTGGTT3 ' (SEQ ID NO 8); 5 ' TTCTGCCTCAGTCTGCGA3 ' (SEQ ID NO 9) ; 5 ' TCCTCCCTATTCCGGGCC3 ' (SEQ ID NO 10) ; or generically: 5 ' CNRGNNNNNNCNRG3 ' (SEQ ID NO: 11).
  • oligonucleotides can effectively permeate cell and nuclear membranes; therefore, the compounds described above, and modifications and derivatives thereof, can be expected to interfere effectively with CP2/CPRE interaction in vivo.
  • modifications to improve nuclease resistance and cellular uptake such as incorporating phosphorothiote or phosphonodithioate residues into the phosphodiester backbone, are disclosed in U.S. Pat. Nos. 5,378,825 and 5,194,599, hereby incorporated by reference herein.
  • Other common modifications to enable a compound to be used as a therapeutic agent include alkylation and glycosylation.
  • An oligonucleotide used in the therapeutic composition of the invention is preferably 20-100 residues in length.
  • antisense oligonucleotides and derivatives thereof complementary to the sequence: 5'cctggggcaa ggaaggagcc aggatggcct gggctctgaa gctgcctctg3 ' (SEQ ID NO: 12) would be expected to interfere with CP2 production by inhibiting translation of the CP2 mRNA by ribosomes (Alama et al. , 1997) .
  • the therapeutic methods described herein may be tested for effectiveness in the various animal models that have now been developed for specific human diseases.
  • the NOD mouse is a well characterized model of type I diabetes mellitus where the T helper cell response is critical (Hultgren et al . , 1996) .
  • the EAE model is an excellent model of multiple sclerosis (Krakowski et al . , 1996) , and there are now several animal models of asthma (Corry et al . , 1996) .
  • TL4 production has been shown to be linked in animal models to treatment of both allergic and autoimmune diseases (Kuchroo et al . , 1995; Wilson et al . , 1998) . Therefore, compounds shown to be effective in the various animal models described above are likely to be effective for therapeutic control of immune response and immunological disease in humans.
  • the therapeutic compositions including agents to interrupt or enhance CP2 activity for treatment of conditions such as those described above may be administered orally, topically, or parenterally, (e.g., intranasally , subcutaneously , intramuscularly, intravenously, or intra-arterially) by routine methods in pharmaceutically acceptable inert carrier substances. Topical administration at an affected site is preferred.
  • the therapeutic compositions of the invention may be administered by on-site delivery using micelles, gels or liposomes.
  • a sustained release formulation using a biodegradable biocompatible polymer is preferred.
  • the therapeutic agents can be administered in a dosage of 0.25 ⁇ g/kg/day to 5 mg/kg/day. Optimal dosage and modes of administration can readily be determined by conventional protocols .
  • Gavett et al . "Interleukin 12 inhibits antigen- induced airway hyperresponsiveness, inflammation and Th2 cytokine expression in mice, " The Journal of Experimental Medicine 182 : 1527-1536 (1995) . Goebel et al . , Adenovirus-mediated gene therapy for head and neck squamous cell carcinomas," Ann. Otol . Rhinol . Laryngol . 105:562-567 (1996) .
  • Hemoglobin switching in man and chicken is mediated by a heteromeric complex between the ubiquitous transcription factor CP2 and a developmentally specific protein," EMBO J. 1_4:97-105 (1995) .
  • B7-1 and B7-2 costimulatory molecules activate differentially the Thl/Th2 developmental pathways: application to autoimmune disease therapy," Cell 8 . 0:707-718 (1995).
  • NF-ATp A transcription factor required for the co- ordinate induction of several cytokine genes
  • the Drosophila tissue-specific factor Grainyhead contains novel DNA-binding and dimerization domains which are conserved in the human protein CP2 , " Mol. Cell. Biol. 14:4020-4031 (1994) .

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Abstract

L'invention a pour objet un système de transport d'implant dentaire qui comprend un flacon (100) dans lequel est logé un implant (114) ainsi qu'un support de guidage (116) possédant une collerette amovible (142).
PCT/US1998/003049 1997-02-20 1998-02-19 Systeme de transport d'implant dentaire muni d'un support de guidage a collerette amovible Ceased WO1998036641A1 (fr)

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EP1113081A1 (fr) * 1999-12-28 2001-07-04 Institut Pasteur De Lille Implication du gene connu cp2/lsf/lbp-1 dans la maladie d' alzheimer
US7511131B2 (en) * 2002-11-13 2009-03-31 Genzyme Corporation Antisense modulation of apolipoprotein B expression
US7888324B2 (en) 2001-08-01 2011-02-15 Genzyme Corporation Antisense modulation of apolipoprotein B expression
WO2012050985A1 (fr) 2010-10-13 2012-04-19 Trustees Of Boston University Inhibiteurs du facteur sv40 tardif (lsf) en tant qu'agents chimio-thérapeutiques anticancéreux
US8735364B2 (en) 2001-08-01 2014-05-27 Genzyme Corporation Antisense modulation of apolipoprotein B expression
US8916694B2 (en) 2004-05-05 2014-12-23 Genzyme Corporation SNPs of apolipoprotein B and modulation of their expression
US9045754B2 (en) 2006-05-05 2015-06-02 Isis Pharmaceuticals, Inc. Short antisense compounds with gapmer configuration
US9107933B2 (en) 2009-03-16 2015-08-18 Isis Pharmaceuticals, Inc. Compositions and methods of targeting apolipoprotein B for the reduction of apolipoprotein C-III
US9347061B2 (en) 2007-03-24 2016-05-24 Genzyme Corporation Administering antisense oligonucleotides complementary to human apolipoprotein B
US9802948B2 (en) 2010-10-13 2017-10-31 Trustees Of Boston Univeristy Inhibitors of late SV40 factor (LSF) as cancer chemotherapeutics
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WO2018044012A1 (fr) * 2016-08-30 2018-03-08 한양대학교 산학협력단 Peptide ayant une activité anticancéreuse, composition pharmaceutique pour le traitement et la prévention du cancer, composition alimentaire de santé fonctionnelle et composition cosmétique fonctionnelle le contenant en tant que principe actif
US11242353B2 (en) 2020-01-24 2022-02-08 Trustees Of Boston University Heterocyclic LSF inhibitors and their uses
US11420977B2 (en) 2018-08-02 2022-08-23 Trustees Of Boston University Late SV40 (LSF) inhibitors
US11458132B2 (en) 2020-09-01 2022-10-04 Trustees Of Boston University Quinolin-2(1H)-one inhibitors of Late SV40 Factor

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WO2001048240A3 (fr) * 1999-12-28 2001-12-06 Pasteur Institut Implication d'un gene connu appele cp2/lsf-lbp-1 dans la maladie d'alzheimer
EP1113081A1 (fr) * 1999-12-28 2001-07-04 Institut Pasteur De Lille Implication du gene connu cp2/lsf/lbp-1 dans la maladie d' alzheimer
US7888324B2 (en) 2001-08-01 2011-02-15 Genzyme Corporation Antisense modulation of apolipoprotein B expression
US8735364B2 (en) 2001-08-01 2014-05-27 Genzyme Corporation Antisense modulation of apolipoprotein B expression
US7511131B2 (en) * 2002-11-13 2009-03-31 Genzyme Corporation Antisense modulation of apolipoprotein B expression
US7803930B2 (en) 2002-11-13 2010-09-28 Isis Pharmaceuticals, Inc. Antisense modulation of apolipoprotein B-expression
USRE44760E1 (en) 2002-11-13 2014-02-11 Genzyme Corporation Antisense modulation of apolipoprotein B-expression
US8916694B2 (en) 2004-05-05 2014-12-23 Genzyme Corporation SNPs of apolipoprotein B and modulation of their expression
US9045754B2 (en) 2006-05-05 2015-06-02 Isis Pharmaceuticals, Inc. Short antisense compounds with gapmer configuration
US9347061B2 (en) 2007-03-24 2016-05-24 Genzyme Corporation Administering antisense oligonucleotides complementary to human apolipoprotein B
US9107933B2 (en) 2009-03-16 2015-08-18 Isis Pharmaceuticals, Inc. Compositions and methods of targeting apolipoprotein B for the reduction of apolipoprotein C-III
WO2012050985A1 (fr) 2010-10-13 2012-04-19 Trustees Of Boston University Inhibiteurs du facteur sv40 tardif (lsf) en tant qu'agents chimio-thérapeutiques anticancéreux
US9597325B2 (en) 2010-10-13 2017-03-21 Trustees Of Boston University Inhibitors of late SV40 factor (LSF) as cancer chemotherapeutics
US9802948B2 (en) 2010-10-13 2017-10-31 Trustees Of Boston Univeristy Inhibitors of late SV40 factor (LSF) as cancer chemotherapeutics
US9815845B2 (en) 2010-10-13 2017-11-14 Trustees Of Boston University Inhibitors of late SV40 factor (LSF) as cancer chemotherapeutics
US10392398B2 (en) 2010-10-13 2019-08-27 Trustees Of Boston University Inhibitors of Late SV40 Factor (LSF) as cancer chemotherapeutics
WO2018044012A1 (fr) * 2016-08-30 2018-03-08 한양대학교 산학협력단 Peptide ayant une activité anticancéreuse, composition pharmaceutique pour le traitement et la prévention du cancer, composition alimentaire de santé fonctionnelle et composition cosmétique fonctionnelle le contenant en tant que principe actif
KR101926918B1 (ko) 2016-08-30 2018-12-07 한양대학교 산학협력단 항암 활성을 갖는 펩티드, 이를 유효성분으로 함유하는 암 예방 및 치료용 약학 조성물 및 건강기능식품 조성물
US10654889B2 (en) 2016-08-30 2020-05-19 Industry-University Cooperation Foundation Hanyang University Peptide having anticancer activity, and pharmaceutical composition, health functional food composition and functional cosmetic composition for preventing and treating cancer comprising the same as active ingredient
US11420977B2 (en) 2018-08-02 2022-08-23 Trustees Of Boston University Late SV40 (LSF) inhibitors
US11242353B2 (en) 2020-01-24 2022-02-08 Trustees Of Boston University Heterocyclic LSF inhibitors and their uses
US11458132B2 (en) 2020-09-01 2022-10-04 Trustees Of Boston University Quinolin-2(1H)-one inhibitors of Late SV40 Factor

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