WO1996020280A1 - Isolated polysialyl transferases, nucleic acid molecules coding therefor, methods of production and use - Google Patents

Isolated polysialyl transferases, nucleic acid molecules coding therefor, methods of production and use Download PDF

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WO1996020280A1
WO1996020280A1 PCT/US1995/016664 US9516664W WO9620280A1 WO 1996020280 A1 WO1996020280 A1 WO 1996020280A1 US 9516664 W US9516664 W US 9516664W WO 9620280 A1 WO9620280 A1 WO 9620280A1
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seq
nucleic acid
isolated nucleic
acid molecule
pst
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PCT/US1995/016664
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English (en)
French (fr)
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Rita Gerardy-Schahn
Minoru Fukuda
Jun Nakayama
Matthias Eckhardt
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Boehringer Mannheim Gmbh
La Jolla Cancer Research Foundation
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Priority claimed from US08/503,133 external-priority patent/US5747326A/en
Application filed by Boehringer Mannheim Gmbh, La Jolla Cancer Research Foundation filed Critical Boehringer Mannheim Gmbh
Priority to AU46870/96A priority Critical patent/AU692355B2/en
Priority to JP52051696A priority patent/JP2002509423A/ja
Priority to EP95944508A priority patent/EP0871743A1/en
Publication of WO1996020280A1 publication Critical patent/WO1996020280A1/en

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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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the isolation and cloning of nucleic acid molecules which encode polysialyl transferases, cell lines and vectors containing such molecules, and the uses of these. Further the invention relates to the isolated enzyme and its uses. Various diagnostic and therapeutic applications are included as part of the field of the invention, including stimulation of neurite outgrowth by appropriate cells.
  • Polysialic acid is added to the neural cell adhesion molecule, NCAM in a dynamically regulated, postranslational process (S. Hoffman and G.M. Edelman, Proc . Natl. Acad. USA 80: 5762-5766 (1983) , U. Rutishauser et al . , Science 240: 53-57 (1988)) .
  • the glycan structure is unusual in vertebrates and has been shown to be attached to the fifth immunoglobulin domain (K.L. Crossin et al . , J. Cell Biol . 99: 1848-1855 (1984) ) .
  • the presence of the large anionic carbohydrate structure that is PSA modulates NCAM binding 96/20280 PC17US95/16664
  • the subject matter of the invention is the isolation and molecular cloning of the key enzyme of eukaryotic PSA synthesis, i.e. , polysialyl transferase ("PST", hereafter) , and the isolation of nucleic acid molecules which encode this enzyme. More specifically, the invention relates to those nucleic acid molecules encoding mammalian PST enzymes, including hamster, human and other species. Especially preferred are nucleic acid molecules which hybridize to SEQ ID NOS: 1 and 7 and portions thereof, especially under stringent conditions, as set forth below.
  • An additional aspect of the invention is a method of detecting nucleic acid molecules which specifically code for PST proteins. Preferably detected are cDNA and mRNA species. Useful probes are nucleic acid molecules which bind specifically under the conditions of the test method applied, e.g. in situ hybridization, colony hybridization, Northern hybridization, or related techniques.
  • a further aspect of the invention is an oligonucleotide molecule which is complementary to a nucleic acid molecule encoding mammalian PST.
  • Such oligonucleotide molecules which are generally from 15 to 50 bases in length, are useful for inhibiting expression of PST, preferably on the DNA or mRNA level .
  • Such oligonucleotide molecules are useful as antisense agents in the therapy of pathological conditions involving a tumor, especially in the treatment and prevention of metastases.
  • isolated PST enzymes such as mammalian PST enzymes, which may be encoded by the isolated nucleic acid molecules of the invention, the production of said enzymes via recombinant methodologies, and the use of the enzymes in promoting neurite growth in nerve cells .
  • CHO-wt and CHO-1E3 cells were plated into cell culture dishes and cell fusion was induced by polyethylenglycol (50% PEG 1500 in 50 mM Hepes buffer pH 7.4) .
  • Double positive hybrids were selected with G418 and hygromycin. While the NCAM signal in hybrid cells was identical to that observed in parental cells, both fusion products expressed the 735 epitope.
  • FIG. 2 The PSA found on the surface of pEPST-ME7 transfectants is bound to NCAM.
  • Cells with the phenotype NCAM+/PSA- were transfected with pEPST-ME7 or pCDM8 alone, as described infra, harvested, and solubilized in lysis buffer, NCAM was immunoprecipitated from the lysates using an anti-NCAM serum, and the samples were analyzed by Western blot with mAb 735. Microheterogenous bands became visible in PST transfectants (lane 1: CHO-2A10; lane 3: NIH-3T3; lane 5 COS-hN-6) indicating that NCAM in these cells is polysialylated. In contrast, control transfection with pCDM ⁇ did not result in appearance of mab 735 epitopes (lane 2: CHO-2A10; lane 4: NIH-3T3; lane 6: COS-hN-6) .
  • Figure 3b FACS analysis of sublines isolated by limiting dilution from the small cell lung cancer cell line HTB119. While staining with mab MB2 (specifically directed against human NCAM) , indicates that NCAM expression on the 3 sublines is almost identical, the staining with mAb 735 reveals large differences in the amount of detectable PSA. HTB119-54.2 are about 100% PSA positive, in the subline HTB119-38 only 30% of the cells express PSA, and in HTB119-45 the number of PSA expressing cells is reduced to 2%. Immunostaining was performed as described in Fig. la.
  • RNA samples (3 ug each lane) were blotted onto nylon membranes and analyzed with a digoxigenin (Boehringer Mannheim) labeled RNA-probe that contained the entire coding region of pEPST-ME7. Final washing conditions were 0.1 x SSC, 0.1% SDS, and 65°C.
  • FIGS 4A-4G depict results obtained in various experiments wherein eukaryotic cells were cotransfected with pH ⁇ A-NCAM and one of pcDNAI-PST or pcDNAI . (These, and all other acronyms, are elaborated upon in the examples) .
  • COS-1 cells were co-transfected with pcDNAI-PST (panels C-F) or pcDNAI (panels A and B) and pH&A-NCAM.
  • the cells were fixed and examined by incubation with anti-PSA antibody 735 (panels B, D and F) or anti-NCAM antibody (Dako Co.
  • FIGS 7A-7F set forth the result of experiments designed to determine neurite outgrowth in confluent
  • FIG. 1 panes D, E and F were seeded on HeLa cell substrata and cultured for 15 hours. Neurons shown in panels A and D were grown on untransfected HeLa cells. Neurons shown in panels B and E were grown on HeLa cells stably transfected with N-CAM-encoding DNA only. Neurons shown in panels C and F were grown on
  • HeLa cells cotransfected with DNAs encoding N- CAM and human PST. Neurites were visualized by immunofluorescent neurofilament-staining. Bar 100 ⁇ m.
  • the nucleotide sequences according to the invention encode a protein that functions in the final step of PSA biosynthesis.
  • the close correlation between cell surface expression of PSA and the occurrence of the PST specific mRNA implies that the regulation of PST occurs predominantly on the mRNA level.
  • PST seems to be the primary factor involved in polycondensation of sialic acids during mammalian PSA synthesis and is unique in PSA positive cells. Confirmation for this assumption comes from the fact that expression in the NCAM positive cell lines tested resulted in biosynthesis of PSA.
  • Example 2 In order to isolate the defective gene that is in PST-C1 clones, the mutant clone CHO-2A10 was used for expression cloning. Samples of CHO-2A10 cells were transfected, via electroporation, with a complementary DNA (cDNA) library prepared from RNA obtained from CHO-wt cells (in pCDM ⁇ ) and vector pPSVEl-PyE carrying the polyoma large T-antigen (M.F.A.
  • cDNA complementary DNA
  • PSA- positive transfectants were collected by panning (B. Seed and A. Aruffo, supra) on mAb 735, and plasmid DNA was subsequently extracted and amplified in E. coli . MC1061/p3 following Seed et al, supra. After 3 cycles of transfection and panning, the cDNA encoding PST was enriched to about 1 : 10' and could be finally isolated by sibling selection.
  • the cDNA clone pEPST-ME7 (for eukaryotic polysialyl transferase clone ME7 ) was isolated.
  • pEPST-ME7 contained an insert of 2026 base pairs (bp) and an open reading frame of 1080 bp (SEQ ID NO: 1) , potentially encoding a protein of 359 amino acids with a predicted Mr of 41.2 kDa.
  • the amino acid sequence (SEQ ID NO: 2) showed the characteristic features of the sialyltransferase family, including the two sialylmotifs (K. Drickamer, Glycobiol . 3: 2- 3 (1993) ) , as found at amino acids 141 to 183 and 258 to 300.
  • the requirements of a type II transmembrane protein P. Klein et al. , Biochem. Biophys .
  • a PST specific cDNA probe recognized two bands of about 2.8 kb and about 5.1 kb in
  • NCAM expression varied in the amount of PSA present.
  • FACS analysis 100% of the cells in subline HTB119-54.2, 30% in subline HTB119-38, and only 2% in subline HTB119-45 were positive with mAb 735 (Fig. 3b) .
  • Poly (A)+ RNA from these cell lines was isolated and analyzed in Northern blot with a probe containing the entire coding region of pEPST-ME7. The results are shown in Fig. 3a (lanes 5-7) . At least 6 bands were stained in highly polysialylated HTB119-54.2 and in HTB119-38 which express low amounts of PSA. Again, an extremely good correlation between surface expression and the level of PST mRNA was observed.
  • PSA can affect the metastatic potential of tumors
  • This approach is seen as being useful as a method for treating pathological conditions, such as tumor associated diseases characterized by excess or inappropriate expression of PST, and/or inappropriate level of PSA.
  • Three sets of experiments were carried out to test the ability of antisense oligonucleotides to inhibit expression of PST, and hence of PSA activity.
  • phosphothioate oligonucleotide molecules complementary to nucleotide sequences of SEQ ID NO: 1 were synthesized with phosphorothioate linkages on an automated nucleotide sequencer following M. Matsukara et al . , Proc . Natl. Acad. Sci. USA 84: 7705-7710 (1987) .
  • random oligonucleotide molecules having the same length were prepared in identical fashion. The purity of oligonucleotide molecules was determined by electrophoresis through 15% polyacrylamide gels stained with etidium bromide.
  • the oligonucleotide molecules prepared are complementary to sequence sections of SEQ ID NO: 1 as follows:
  • Oligo 1 Oligonucleotides 325-345 (21mer! (SEQ ID NO 3)
  • Oligo 2 Oligonucleotides 522-539 (18mer! (SEQ ID NO 4)
  • Oligo 3 Oligonucleotides 846-862 (17meri (SEQ ID NO 5)
  • Oligo 4 Oligonucleotides 1356-1373 (18mer) (SEQ ID NO: 6)
  • PSA-positive CHO-wt cells were incubated with 3 ⁇ mol of each of the oligonucleotides.
  • PSA activity was measured in a kinetic test, since PSA has a half-life longer than 24 hours.
  • Cells were analyzed by FACS analysis with mAb 735 according to Example 3 after 20, 40, 60 and 80 hours.
  • the oligodeoxynucleotides, and polylysine modified conjugates were prepared according to J.P. Leonetti et al . , Gene 72 (1988) 232-332, incorporated by reference, but summarized herein.
  • Cholesterol-modified CPG (controlled pore glass) was used as support.
  • the oligonucleotides were prepared from 2 ⁇ mol columns of these supports on an Applied Biosystems Model 394 DNA Synthesizer using the 1 ⁇ mol protocol supplied by the manufacture.
  • 3 ' -cholesterol-modified oligonucleotides with C ⁇ -linker were prepared from a commercially available 1 ⁇ mol CPG column (Clontech, Palo Alto, CA) . Standard reagents for ⁇ -cyanoethylphosphoramidite coupling chemistry were used.
  • the oligonucleotides were HPLC- purified, detritylated and precipitated from butanol as described in Reed, M.W. , et al . , supra.
  • the oligonucleotides were at least 90% pure (HPLC) .
  • PSA-positive CHO-wt cells (10 , 000/well) were plated in
  • an expression plasmid is transfected into a suitable E. coli strain using standard methodologies.
  • Such strains are, in the case of the use of an expression plasmid under the control of lac repressor such as the expression plasmid pll379, strains which possess a sufficiently high intracellular concentration of lac repressor.
  • These kinds of strains can be prepared by transfection of a second plasmid such as pREP4 (Diagen GmbH) , pUBS 500 or pUBS520 (Brinckmann et al., Gene 85 109-114 (1989)) .
  • the £. coli strains employed should preferably have low protease activity, as is the case, for instance, with E.
  • PST-containing protein aggregates from E. coli fermentation are solubilized in 6 M guanidinium hydrochloride, 100 mM Tris-HCl at pH 8, 1 mM EDTA, subsequently adjusted to a pH of 3 to 4 and dialyzed against 4 M guanidinium hydrochloride at pH 3.5.
  • the renaturing of the solubilized protein is then carried out in 1 M arginine at pH
  • PST (glutathione, oxidized) .
  • PST can be obtained, for instance, after addition of 1.4 M ammonium sulfate by adsorption to hydrophobic gel matrices such as Fractogel TSK Butyl and subsequent elution in 20 mM TrisHCl at pH 7.
  • DNA from a first mammalian species which encodes a PST is ligated into a vector and is then transcribed in cells of a second mammalian species.
  • This approach is used, e.g., to produce human PST in CHO cells, although it should be understood that a coding sequence from a mammalian species can also be transfected into cells of the same species.
  • a strong promoter-enhancer system can be used, and in the case of genomic PST fragments, this step is required because the promoters of PST are active only in certain cell types (e.g. melanomas) .
  • endogenous promoters may not be suitable for general recombinant expression in all cell types. Expression can also be accomplished by homologous recombination jj vitro. whereby a suitable exogenous promoter is used in combination with a PST coding region.
  • exogenous promoters and enhancers are generally from viruses such as SV40, hCMV, polyoma, or retroviruses .
  • promoter enhancer systems which are specific to a certain cell type or tissue type, such as WAP- or immune globulin promoters, or systems which are inducible, such as metallothionein and MMTV promoters .
  • pCMX-pLl (Umesono et al . , Cell 65: 1255-1266 (1991) ) is a suitable vector containing a CMV promoter.
  • the PST cDNA is provided with EcoRI linkers and then ligated into the vector's single EcoRI cleavage site and, by using the other known cleavage sites in the polylinker of this vector, the PST cDNA is oriented in the proper reading frame for promotion of expression by the CMV promoter.
  • An analogous procedure is applied when cloning into other vectors, such as pCDNA3 (Invitrogen, San Diego/USA) or pSG5
  • the DNA of these expression plasmids is from E. coli and may be transfected into a mammalian cell sample of choice, applying standard techniques. See, e.g. Methods of Enzymology 185 (Gene Expression Technology) , ed. David V. Goeddel, Academic Press 1991, section V. After transfection, the cells may be cultured in minimum essential medium (MEM) without addition of fetal calf serum, whereby PST is detectible in the cell culture supernatant after 48 hours.
  • MEM minimum essential medium
  • PEPST-ME7 cDNA in the mutant clone CHO-2A10, in NCAM-positive subclones of NIH-3T3 cells, and in COS-hN-6.
  • surface expression of PSA was shown by immunofluorescence using mAb 735 as is now explained.
  • the presence of PSA in these cells indicates that PST is being expressed, because the cells normally do not produce PSA.
  • Tissue culture dishes were seeded with freshly trypsinized cells 18 hours before transfection with 2mg of pEPST-ME7 , or an equal amount of the base vector pCDM ⁇ described supra, using lipofectamin (Gibco BRL) . 62 hours later, transfected cells were incubated with the PSA specific mab 735 for 30 minutes at room temperature, and any mab bound to target was detected with fluorescein conjugated, anti- mouse Fab fragments.
  • a nucleic acid fragment to be employed for hybridization is cloned in a suitable transcription vector (with a T3 , T7 or SP6 promoter) .
  • a suitable transcription vector with a T3 , T7 or SP6 promoter
  • 1 to 2 ⁇ g of the plasmid were linearized, purified by means of phenol/chloroform extraction and precipitated with ethanol.
  • This preparation was incubated for two hours at 37°C.
  • RNA or D ⁇ A immobilized on nylon membranes was hybridized with DIG-labelled R ⁇ A probes. Hybridization was carried out under identical conditions, except that lower hybridization temperatures were used for D ⁇ A-RNA hybridizations.
  • the membranes were first pre-hybridized in hybridizing buffer (50% formamide, 50 mM sodium phosphate pH 7.0, 7% SDS, 0.1% ⁇ -lauroylsarcosine, 5xSSC, 2% blocking reagent) for 1 to 2 hours (65°C in the case of R ⁇ A-R ⁇ A and 50°C in the case of D ⁇ A-R ⁇ A hybridizations) .
  • Hybridization with DIG-labelled probe was carried out for 16 to 90 hours and, at 65°C and 50°C, respectively. Immediately beforehand, the probe was heated to 98°C for 5 minutes and subsequently cooled on ice. After hybridization, the membrane was washed, twice for 5 minutes in 2xSSC, 0.1% SDS (at ambient temperature) and twice for 15 minutes in 0. lxSSC, 0.1% SDS (at 65°C) .
  • the detection of the hybridized probe was carried out applying the following procedure:
  • COS and CHO cells are used, frequently, as recipients for eukaryotic DNA, and were tested to determine if they were appropriate .
  • Example 10 A human fetal brain cDNA library (Invitrogen, San Diego,
  • CA (40 ug) constructed in plasmid pcDNAI was cotransfected into 2.4x10 COS-1 cells, together with an equal amount of pH ⁇ A-NCAM, using lipofectamine.
  • plasmid DNA in the positive cells was itself isolated, following the classic procedure of Hirt, J. Mol. Biol. 26: 365-369 (1967) , incorporated by reference.
  • the isolated plasmids were then amplified in host bacteria MC 1061/P3, using both ampicillin and tetracycline. This was possible because starting plasmid pcDNAI contains supF suppressor tRNA, and thus confers resistance to both antibiotics. In contrast, the pH ⁇ -A-NCAM plasmid confers resistance to ampicillin, but not to tetracycline.
  • the isolated plasmids were divided into 23 plates, each plate containing about 500 colonies of the bacteria. Plasmid DNA was prepared from each plate. As a result, the procedure results in rescue of positive library clones, but not others.
  • the recovered plasmid DNA was transfected into COS-1 cells with pH ⁇ A-NCAM and then subjected to sibling selection, which is a standard technique, resulting in sequentially smaller, active pools, activity being determined in an immunoassay, using mAb 735, described supra, and mAb 1263, the latter of these being used only in the analysis following cloning.
  • sibling selection which is a standard technique, resulting in sequentially smaller, active pools, activity being determined in an immunoassay, using mAb 735, described supra, and mAb 1263, the latter of these being used only in the analysis following cloning.
  • HeLa cells were cotransfected, using lipofectamine, with pH ⁇ A-NCAM alone, or pH ⁇ A-NCAM and pcDNAI- PST, in the same manner described supra, on COS-1 cells. Following transfection and incubation, the HeLa cells were subjected to Western Blotting. To perform the blotting, the lysates obtained from 2xl0 fc cells (untransfected parent or cotransfected cells) , were subjected to SDS gel electrophoresis, transferred to nitrocellulose, and incubated with anti-NCAM antibody (Becton-Dickinson) .
  • PSA polysialic acid
  • lysates of untransfected HeLa cells showed only barely detectable cross-reactivity of an ⁇ 100-kDa protein with anti-NCAM antibody (lane 1) .
  • lysates of HeLa cells co-transfected with pcDNAI-PST and pH ⁇ A-NCAM displayed multiple bands of immunoreactivity corresponding to species of 140 kDa and greater molecular mass (see smear in lane 2) .
  • Lysates of the co-transfected cells that were treated with endo-N prior to electrophoresis displayed a single sharp band of immunoreactivity corresponding to a 140- kDa protein.
  • sialyl motif "L” (amino acids 141 to 185 in SEQ ID NO: 8) shared by all sialyl transferases cloned to date (Sasaki, et al, supra: Wen, et al . , J. Biol. Chem. 267: 21011-21019 (1992) ; Datta, et al . , J. Biol. Chem. 270: 1497-1500 (1995) . Datta, et al . , identify this motif as being the binding site for enzyme substrate CMP-NeuNAc.
  • the predicted sialyl motif "S" is located at amino acids 288-300 in SEQ ID NO: 8. Upstream from motif L, the predicted amino acid sequence shows a cluster of basic amino acids (Arg Arg Arg) , at residues 114-116, and one at residues 137-140, i.e., Arg Arg Phe Lys . These clusters are either completely absent or incomplete in the corresponding sequences of GD3 synthase and STX. These basic amino acid clusters in PST may be critical for PST' s binding to the acceptor containing multiple negatively charged sialic acid residues.
  • the distribution of PST mRNA in human tissues was determined via Northern Blotting.
  • RNA Human fetal and adult brain poly(A) 4 RNA was obtained, and electrophoresed in 1.2% agarose gel containing 2.2 M formaldehyde, followed by transfer to a nylon filter. Similarly, Northern blots of human multiple tissue poly(A) " RNAs were purchased (Clontech, Palo Alto CA) . The RNAs were hybridized with a gel purified cDNA insert of pcDNAI-PST, which had been labelled with ⁇ [ 32 P]-dCTP via random oligonucleotide priming (Feinberg, et al . , Anal. Biochem. 132: 6-13 (1983)) , in accordance with Bierhuizen, et al . , Genes and Dev.
  • Results, set forth in figure 5 show a strong, 6.5 kb band and a weak, 3.7 kb band in poly(A) * RNA from fetal brain. The size difference is probably due to alternative usage of polyadenylation sites .
  • the signal in adult brain was weaker.
  • the signal from fetal lung and fetal kidney was strong, while the fetal liver signal was not.
  • PST transcripts were detected strongly in heart, spleen, and thymus tissue, and moderately in brain, placenta, lung, large intestine, small intestine, and peripheral blood leukocytes .
  • samples of tissue were fixed for 24 hours in cold, 4% paraformaldehyde in 0.1M phosphate buffer, (pH 7.4) , followed by embedding in paraffin, and sectioning (3 u thickness) .
  • Tissue sections were deparaffinized, hydrated, and then immersed in absolute methanol containing 0.3% H 2 0- for 30 minutes.
  • biotinylated mAb 735 was contacted to the sections, followed by avidin-peroxidase complexes, and then a peroxidase substrate which yields a color upon reaction with the enzyme.
  • Figures 6A to 6D show strong presence of polysialic acid in neurons of fetal cerebral cortex, bronchial epithelia of fetal lung (arrows in figure 6B) , Hassall's corpuscles of the thymus (see the arrows in figure 6C) , and epithelial cells of the thymus (arrowheads in figure 6C) .
  • tissues lacking PST mRNA were also negative for polysialic acid staining.
  • Cells were trypsinized (0.5% trypsin) , counted, seeded at low density over monolayers of HeLa cells, and cultured in Dulbecco' s modified Eagle's medium containing 10% fetal bovine serum. The sensory neuron culture had nerve growth factor added to it as well. Cultures were grown up for 15 hours, after which they were fixed with 4% formaldehyde, in phosphate buffered saline. 28
  • polysialic acid is a critical regulator of neurite outgrowth on living cells.
  • cDNAs were prepared using RNA isolated from neonatal brain of BALB/c mice as the template, following standard procedures.
  • the cDNAs were subjected to nucleic acid amplification (performed according to standard procedures) using oligonucleotides based on the human PST cDNA (see Sequence ID NO: 7) as primers.
  • the first primer corresponded to nucleotides 206-225 in SEQ ID NO: 7
  • the second primer corresponded to the complement of nucleotides 1284-1299 in SEQ ID NO: 7.
  • the product of this amplification procedure which is similar in length to the cDNA encoding human PST, is purified, ligated into a plasmid such as pBluescript II, and characterized by DNA sequence analysis.
  • Genomic DNA was isolated from mouse cell line I29SVJ according to standard procedures and ligated into the Lambda FIX II cloning vector. The resulting genomic DNA library was screened using conditions as described supra for hybridization to an oligonucleotide probe corresponding to nucleotides 1-557 of SEQ ID NO: 7. Hybridizing plaques were purified and characterized by DNA sequence analysis.
  • DNA sequence analysis of the genomic DNA thus isolated revealed that it contains at least two exons : one corresponding to an extreme 5 'domain of the human PST cDNA (i.e., nucleotides 1-325 of SEQ ID NO: 7) and a second corresponding to a more downstream location of the human PST cDNA (i.e., nucleotides 458-715 of SEQ ID NO: 7) .
  • the genomic structure of the gene encoding mouse PST can be elucidated.
  • the resulting information is particularly useful in designing gene "knock-out" experiments in mice, wherein the mouse PST gene in mouse embryos is rendered nonfunctional .
  • the resulting transgenic mice are then analyzed for the effects of a lack of PST activity.
  • the transfected COS-1 cells were subjected to immunoflourescent staining, using murine monoclonal antibodies M6703 (Nakayama, et al, J. Histochem Cytochem 41:1563-1572 (1993) ) 12E3 (Seki, et al . Anat. Embryol 184: 395-401 (1991) ) , and 735 (Frosch, et al, Proc. Natl . Acad. Sci . USA 82: 1194- 1198 (1985) ) . All of these references are incorporated by reference.
  • the mAb M6703 is known to react with oligosialic acids which contain 2 or 3 ⁇ -2, 8 linked sialic acid residues, 12E3 reacts with polysialic acids containing 6 or more residues of ⁇ -2, 8 linked sialic acid, and 735 with polysialic acids containing 8 or more of these residues.
  • fluoresein isothiocyanate (FITC) conjugated goat-antimouse IgG antibody F(ab' ) fragments were added to detect M6703 and 735, while FITC conjugated goat-antimouse IgM antibody F(ab') 2 fragments were used to detect 12E3.
  • the IFA protocol was identical to that described supra.
  • N-CAM was absent, because there was no staining of the transfectants with an anti N-CAM antibody.
  • PST does not require N-CAM in order to produce PSAs.
  • HeLa cells were cotransfected with pcDNAI-PST and pSV 2 neo, and selected with antibiotic G418, using standard protocols, i.e., those described in the prior examples. Following the selection, immunofluorescent staining was carried out in the same manner described for the COS-1 cells.
  • Transfected HeLa cells were stained with mAb 735.
  • the transfectants were treated with the enzyme endo-N, which is known to cleave PSA, no staining was detected by antibody 735.
  • endo-N which is known to cleave PSA
  • a fusion protein was produced, using a putative catalytic domain for PST, a signal peptide, and an IgG binding domain.
  • the signal peptide was derived from human granulocyte colony stimulating factor (G-CSF) , and the IgG binding domain was that of the S. aureus protein A.
  • the fusion protein was prepared using cDNA sequences, and the well known polymerase chain reaction ("PCR" hereafter) .
  • PCR polymerase chain reaction
  • the pcDNAI-PST plasmid was used as a template.
  • the upstream and downstream primers used were:
  • amino acids 40-359 of SEQ ID NO: 8 constitute a soluble, PST active molecule.
  • pAMoA-GD3 the plasmid pAMoA-GD3 , described by Sasaki, et al . , J. Biol. Chem 22:15950-15956, (1994) incorporated by reference, was digested with BamHI and Kpnl, with the result that pAMoA was formed, which contained only DNA encoding the human G-CSF signal sequence and a sequence encoding i _ aureus protein A.
  • the PCR product referred to herein was then digested with the same two endonucleases, and cloned into pAMoA, using standard methods.
  • the resulting plasmid has been named pAMoA- PST.
  • the plasmid pAMoA-PST was then digested with Sail and Asp718, resulting in excision of cDNA encoding a fusion protein consisting of the human G-CSF signal peptide, S. aureus protein A, and the PST sequence referred to, supra.
  • the released cDNA insert was filed in using Klenow fragment of DNA polymerase I, and cloned into pcDNAI which had been digested previously with EcoRV.
  • the resulting plasmid was named pcDNAI-A PST.
  • a control vector was also prepared, consisting of only signal peptide and protein A cDNA. This vector was named pcDNAI-A. Standard methodologies were used to confirm that the vectors were in proper 5 '-3' orientation.
  • the vectors pcDNAI-A PST and pcDNAI-A were transfected, separately, into COS-1 cells, using the lipofectamine method described, supra.
  • the transfected cells were cultured for 62 hours, after which any fusion protein secreted into culture medium was adsorbed to IgG-Sepharose 6FF, essentially following the methods of Kukowska-Latallo, et al., Genes Dev 4: 1288-1303 (1990) , and Bierhuizen, et al . , Proc. Natl. Acad. Sci USA 89:9326-9330 (1992) , both of which are incorporated by reference.
  • Resin was collected via centrifugation, washed nine times with 50mM Tris-HCl, pH7.5 containing 1% bovine serum albumin followed by two washes with 20mM Tris-HCl, pH7.5, containing 7.5 mM CaCl,, and 0.05% Tween 20. Finally, the resin was suspended in an equal volume of serum free medium opti-MEMl. This entire procedure is akin to that described by Kukowska - Latallo, et al . , supra Sasaki, et al . , J. Biol. Chem 22:15950-15956 (1994) , incorporated by reference.
  • a substrate solution was prepared, which contained lOO ⁇ g of a substrate sialylated glycoprotein (elaborated upon infra) , dissolved in 50 ⁇ l of 0.1M sodium cacodylate buffer (pH 6.0) , which contained 20mM MnCl 2 , 1% Triton CF-54, and 2.4 nmoles of CMP-[ 14 C] NeuNAc, which served as a donor substrate.
  • the products contained in the sample buffer were subjected to SDS polyacrylamide gel electrophoresis (7.5% acrylamide gel) , and any sialic acids incorporated therein were determined fluorographically.
  • the fluorography was carried out by fixing the gels in 40% methanol, for 30 minutes, followed by two 15 minute soaks in dimethylsulfoxide followed by soaking in dimethylsulfoxide containing 2,5 - diphenyloxazole (18.5g in 88ml of DMSO) in accordance with Bonner, et al . , Eur. J. Biochem 46:83-88 (1974), incorporated by reference.
  • sialylated glycoproteins referred to supra were rat a ⁇ , - acid glycoprotein, fetuin, and huma ⁇ , - acid glycoprotein, all of which had been purchased from Sigma.
  • NANase III cleaves all of ⁇ -2, 3 linked sialic acids, ⁇ -2, 6 linked sialic acids, and ⁇ -2, 8 linked sialic acids.
  • reaction mixtures were digested for 36 hours with either of N-Glycanase, following the manufacturer's instructions, or with endoneuraminidase ("endo- N") , in accordance with Hallenback, et al . , J. Biol. Chem 262:3553-3561 (1987) , incorporated by reference. Following digestion, the materials were subjected to SDS-polyacrylamide gel electrophoresis and fluorography, as described herein.
  • NANase II digestion but were digested by NANase III, which differs from NANase II in its ability to cleave ⁇ -2, 8 - linked sialic acid. Further, the products were susceptible to cleavage by both endoneuriminadase, and N-Glycanase.
  • isolated nucleic acid molecules which encode polysialic acid transferases.
  • These transferases may be eukaryotic proteins, more preferably mammalian, and most preferably human.
  • non-human mammalian species embraced by the invention are various rodent species, such as mouse, rat, rabbit, and guinea pig "PSTs" .
  • isolated nucleic acid molecules comprising or consisting essentially of SEQ ID NOS: 1 or 7 , as well as their complementary sequences, as well as isolated nucleic acid molecules which encode proteins consisting of SEQ ID NO: 2 or SEQ ID NO: 8.
  • isolated nucleic acid molecules consisting of nucleotides 301-1377 of SEQ ID NO: 1, and consisting of nucleotides 213-1289 of SEQ ID NO: 7. Also included are isolated nucleic acid molecules which (i) code for PST proteins as described herein and which hybridize with one or both of nucleotides 721-1200 of SEQ ID NO: 1, and nucleotides 633 to 1112 of SEQ ID NO: 7 under stringent conditions. The term stringent conditions is discussed below.
  • Proteins such as those coded by the sequences set forth supra, and all isolated protein having PST activity are also a feature of the invention. These proteins may have particular amino acid sequences, such as that of SEQ ID NO: 2 or SEQ ID NO: 8.
  • the PST proteins can occur in natural allelic variations which differ from individual to individual. Such variations of the amino acids are usually amino acid substitutions. However, they may also be deletions, insertions or additions of amino acids to the total sequence.
  • the PST protein according to the invention depending, both in respect of the extent and type, on the cell and cell type in which it is expressed - can be in glycosylated or non- glycosylated form.
  • polysialyl transferase activity denotes the protein's capability of catalyzing the polycondensation of ⁇ -2,8-linked sialic acids in vivo and/or in vitro.
  • the number of sialic acids that are being condensed is dependent on the surrounding conditions, including, e.g., the cell type in which condensation takes place, and on the CMP-activated substrate (sialic acid or derivatives, e.g. N-glycolylneuraminic acid or N-acetylneuraminic acid) .
  • the number of condensed sialic acids may vary widely and ranges between but a few (e.g. 10) and several hundreds or thousands of such monomers.
  • N-acetylneuraminic acid residues are condensed.
  • PST activity may also include induction of PSA synthesis in NCAM expressing cell lines as well as cell-free synthesis of oligosaccharides .
  • Expression of PST in tumorigenic cells can influence metastatic efficiency of the cells, and expression of PST in substrate cells and/or nerve cells can also regulate neurite outgrowth, as was shown, supra.
  • polysialyltransferases of the invention catalyze the polycondensation of sialic acids on acceptor structures like NCAM but do not use isolated poly- or oligomeric N- acetylneuraminic acid substrates like colominic acid or capsular polysaccharides from E.coli Kl, E.coli K92 or
  • Neisseria meningitidis sero group B Neisseria meningitidis sero group B.
  • the bacterial polysialyltransferases use such substrates from
  • E.coli K92 PST of S.M. Steenbergen et al . , J. Bacteriol .
  • polysialyltransferases according to the invention show a different acceptor specificity than bacteria polysialylic transferases .
  • isolated and/or purified indicates that the material modified thereby has been modified from its native, in vivo cellular environment.
  • the recombinant DNAs, RNAs, polypeptides and proteins of the invention are useful in ways that the DNAs, RNAs, polypeptides or proteins as they naturally occur are not, such as in the isolation of nucleic acids encoding related proteins and in the treatment of certain pathological conditions .
  • Stringent conditions refers to hybridization in the presence of 1M NaCl, 1% SDS, and 10% dextran sulfate, followed by two washes of a filter at room temperature for 5 minutes, in 2xSSC, and one final wash for 30 minutes. This final wash may be at 0.5xSSC, 0.1% SDS, more preferably at 0.2xSSC, 0.1% SDS, and most preferably at O.lxS ⁇ C, 0.1% SDS, final wash taking place at 65°C.
  • Those of ordinary skill in the art will recognize that other conditions will afford the same degree of stringency, and are encompassed by the phraseology "under stringent conditions", and are encompassed herein.
  • the PST gene or its variants in genomes of any mammalian cells and tissue may be isolated.
  • Such processes and suitable standard stringent hybridization conditions are known to a person skilled in the art and are described for example by J. Sambrook, Molecular Cloning: A Laboratory Manual (1989) and B.D. Hames, et al . , Nucleic Acid Hybridization: A Practical Approach (1985) . In this case the standard protocols described in these publications are usually used for the experiments.
  • Cells and tissue which are positive for PSA can be identified either by an antibody which is specific for polysialic acids and/or by the use of endoneuraminidase NE.
  • Such specific antibodies can be easily produced according to M. Frosch et al . , supra or according to Moolenaar, et al . , supra, both of which are incorporated herein by reference.
  • the isolation of endoneuraminidase NE is described in S. Tomlinson and P.W. Taylor, J. Virol. 55: 374-378 (1985) , which is incorporated herein by reference.
  • Endoneuraminidase NE degrades ⁇ -2,8-linked sialic acids with at least 8 sialic acid residues and releases in the cell or tissue supernatant monomeric sialic acids.
  • nucleic acids encoding mammalian proteins having PST activity may be isolated by screening suitable cDNA or genome libraries under suitable hybridization conditions with nucleic acids disclosed herein (including nucleic acids derived from any of SEQ ID NOS: 1 or 7) .
  • the library can be screened with a portion of the disclosed nucleic acids including substantially the entire coding sequence thereof or with a suitable oligonucleotide probe based on a portion of the nucleic acids.
  • a probe is a single- stranded DNA or RNA that has a sequence of nucleotides that includes at least about 10-50 contiguous bases, preferably 16- 40, most preferably 25-30, that are the same as (or the complement of) any contiguous bases set forth in any of SEQ ID NOS: 1 or 7.
  • Preferred regions from which to construct probes include sequences predicted to encode transmembrane domains, catalytic domains, sialylmotifs and the like.
  • Either the full-length cDNA clones, fragments thereof or oligonucleotides based on portions of the cDNA clones can be used as probes, preferably labeled with suitable label means for ready detection. Non-radioactive labels are preferred. These probes can be used for identification and isolation of additional nucleic acids encoding proteins having PST activity.
  • a method for identifying nucleic acids encoding proteins having PST activity comprising: contacting mammalian DNA with a nucleic acid probe as described above, wherein said contacting is carried out under conditions favorable to the hybridization of the probe to its complement, and identifying nucleic acid molecules which hybridize to the probe.
  • cDNA clones can be incorporated into expression vectors and expressed in suitable host cell lines as described herein to determine if the corresponding protein product displays PST activity as also described herein.
  • nucleic acid amplification techniques which are well known to those of skill in the art, can be used to isolate nucleic acids encoding proteins with PST activity. This is encompassed by employing oligonucleotides based on the sequences disclosed herein in SEQ ID NOS: 1, or 7 as primers for amplifying mammalian RNA or DNA.
  • ribonuclease (RNase) protection assays and in situ hybridization assays can be employed to determine which cells and tissues express mRNA encoding the protein. These assays provide a sensitive means for detecting and quantifying an RNA species in a complex mixture of total or cellular RNA.
  • RNase ribonuclease
  • the use of recombinant DNA technology enables the production of numerous PST protein derivatives. Such derivatives can for example be modified in individual or several amino acids by substitution, deletion or addition.
  • the derivatization can for example be carried out by means of site directed mutagenesis. Such variations can be easily carried out by a person skilled in the art (J. Sambrook, supra, B.D. Hames, supra) . It merely has to be ensured that the characteristic properties of the PST protein (polysialyl transferase activity) are preserved.
  • PST is an intracellular (Golgi-resident) enzyme which may be present in the cell in a dimeric form usually linked via the N-terminus, dimerization in the cell is essentially carried out via the transmembrane region (approximately amino acid Nos. 8 to 20 of SEQ ID NOS: 2 and 8, e.g.) . Even when in monomeric form, however, the enzyme will display activity.
  • a soluble enzyme which is monomeric and in which the transmembrane region and, thus, approximately the first 20 to 30 amino acids, preferably 25 amino acids, are absent, is preferred.
  • a soluble enzyme of this type is also active in vitro and catalyzes the polycondensation of ⁇ -2,8-linked sialic acids.
  • the invention thus also comprises an isolated PST protein which is a product of prokaryotic or eukaryotic expression of an exogenous DNA as described herein.
  • the invention additionally concerns PST proteins and nucleic acid molecules from other cells and tissue such as mammalian cells and tissues, including mouse, rat, bovine, sheep, etc., which exhibit polysialyl transferase activity in an essentially analogous manner.
  • These proteins can be obtained in a manner well known to the art.
  • a library such as a cDNA library of a particular mammal or other eukaryote, for sequences which hybridize with the complements of disclosed sequences.
  • the proteins according to the invention can be obtained in a reproducible manner and in large amounts.
  • the nucleic acid molecules are integrated into suitable expression vectors, such as exogenous nucleic acid molecules, according to methods familiar to a person skilled in the art, and are introduced into a prokaryotic host cell or a eukaryotic host cell.
  • suitable expression vectors such as exogenous nucleic acid molecules, according to methods familiar to a person skilled in the art, and are introduced into a prokaryotic host cell or a eukaryotic host cell.
  • Such an expression vector preferably contains a regulatable/inducible promoter to which the coding sequence is operably linked.
  • suitable host cells such as, e.g. , E.
  • coli prokaryote
  • Saccharomyces cerevisiae Terato carcinoma cell line PA-1 sc 9117 (B ⁇ ttner et al . , Mol. Cell Biol. 11: 3573-3583 (1991) )
  • insect cells such as Sf9
  • all insect cells transfected with baculovirus vector and also CHO or COS cells.
  • the transformed or transduced host cells are cultured under conditions which allow for expression of the heterologous or homologous gene.
  • the isolation of the protein can be carried out according to known methods from the host cell or from the culture supernatant of the host cell . Such methods are described for example by Ausubel, et al . , Curr. Prof. Mol. Biol.
  • the invention provides methods of producing recombinant PST that can be used, for example, in cell-free synthesis of oligosaccharides, in the study of PST structure and function, and in screening compounds as potential inhibitors of PST enzymatic activity. Soluble PST enzyme is preferred for these uses.
  • a DNA molecule comprising or consisting of PST coding sequences (i.e.
  • nucleotides 301-1377 in SEQ ID NO: 1 or nucleotides 213-1289 in SEQ ID NO: 7) lacking nucleotides which code for the first 20-30 or more amino acids of the N- terminus .
  • DNA molecules comprising nucleotides 330-1289 of SEQ ID NO: 7 and the equivalent section of SEQ ID NO: 1.
  • Expression of recombinant PST may be further enhanced by including in the PST DNA at positions immediately upstream of the first codon and down of the translation termination codon, respectively, 5' and 3' untranslated sequence (e.g., 5' untranslated sequence approximating a consensus sequence [see, e.g. , Kozak (1991) J. Biol. Chem. 266: 19867-19870] such as nucleotides 207-212 in SEQ ID NO: 7 and 3' untranslated sequence comprising nucleotides 1293-1304 in SEQ ID NO: 7) .
  • nucleic acid encoding soluble PST is linked, at the 5 ' end, to nucleic acid encoding a signal peptide capable of directing secretion of proteins in the selected host cell.
  • nucleic acids encoding the protein A signal sequence can be linked to the 5 ' end of nucleic acid encoding PST for secretion of PST from appropriate host cells, in a manner well known to the skilled artisan.
  • test based upon nucleic acid molecules encoding PST protein, which are then used to detect nucleic acid molecules which code specifically for PST proteins.
  • a test can be carried out, e.g., in cells or cell lysates.
  • Such a test can be carried out in accordance with standard nucleic acid diagnostic methods. In such cases, the sample to be examined is brought into contact with a probe which hybridizes with the nucleic acid molecules coding for the PST protein of interest such as nucleotides 301-720 of SEQ
  • Hybridization between the probe and nucleic acid molecules from the sample indicates the presence of expressed PST proteins.
  • Such methods are known to a person skilled in the art and are for example described in WO 89/06698, EP-A 0 200 362, USP 4,915,082, EP-A 0 063 879, EP-A 0 173 251, EP-A 0 128 018.
  • the nucleic acid molecule of the sample which codes for a PST protein is amplified before testing, e.g. by the well-known PCR technique. A derivatized (labelled) nucleic acid probe is normally used.
  • This probe is brought into contact with a carrier-bound denatured DNA or RNA from the sample and in this process the temperature, ionic strength, pH value and other buffer conditions are selected in such a way that - depending on the length of the nucleic acid molecule sample and the resulting melting temperature of the expected hybrid - the labelled DNA or RNA can bind to homologous DNA or RNA (hybridization, see also J. Mol. Biol. 98: 503 (1975) , ; Proc. Natl. Acad. Sci. USA 76: (1979) .
  • Suitable carriers are membranes or carrier materials based on nitrocellulose reinforced or bound nitrocellulose in a powder form or nylon membranes derivatized with various functional groups (e.g. nitro group) .
  • the hybridized DNA or RNA is then detected by incubating the carrier, after thorough washing and saturation to prevent unspecific binding, with an antibody or antibody fragment.
  • the antibody or antibody fragment is directed towards the substance incorporated into the nucleic acid probe during the derivatization .
  • the antibody is in turn labelled. It is, however, also possible to use a directly labelled DNA. After incubation with the antibodies, it is washed again in order to only detect specifically bound antibody conjugates. The determination is then carried out via the label of the antibody or antibody fragment according to well-known methods.
  • the detection of the PST expression can be carried out for example as: in situ hybridization with immobilized whole cells using immobilized tissue smears and isolated metaphase chromosomes, colony hybridization (cells) and plaque hybridization (phages and viruses) ,
  • RNA detection RNA detection
  • serum analysis e.g. cell type analysis of cells in serum by slot-blot analysis
  • amplification e.g. PCR technique
  • the invention therefore includes a specific method for the detection of nucleic acid molecules which code for a PST protein which is characterized in that the sample to be examined is incubated with a nucleic acid probe which may be selected from the group comprising nucleic acid molecules or specific oligo- nucleotides which hybridize with the nucleotides 301 to 720 and/or 1201 to 1377 of SEQ ID NO: 1, 633 to 1112 of SEQ ID NO: 7 and their complementary sequences.
  • the nucleic acid probe is incubated with the nucleic acid molecules from the sample and the hybridization of the nucleic acid molecules in the sample and nucleic acid probe is detected, if desired, via a further binding partner.
  • PST and its expression is a valuable prognostic marker in tumor diagnostics (metastasis, progress) .
  • oligonucleotide molecules such as SEQ ID NOS: 3, 4, 5, and 6, as well as (a) nucleotides 237-257, (b) nucleotides 434-451, (c) nucleotides
  • nucleotides 1268-1285 of SEQ ID NO: 7 and their complementary sequences which hybridize to PST coding sequences and may specifically inhibit the expression of PST in mammalian cells. It has been found that when an oligonucleotide molecule reaches a length which is more than about 15 to 17 nucleotides the sequence is unique relative to the entire human genome. Thus, these "15 * meres" are specific to particular regions of the genome. It has been shown that short antisense oligo- nucleotides can be imported into cells and inhibit expression of a gene to which they are targeted (Zamecnik et al . , Proc. Natl. Acad. USA 83: 4143 (1986) ) . In particular, oligonucleotide molecules having 15 to 50, preferably 15 to 25, bases are suitable for use.
  • oligonucleotide molecules which is complementary to, and designed on the basis of portions of the PST genes described herein are useful for inhibiting the expression of PST.
  • a further embodiment of the invention is an oligonucleotide molecule which hybridizes in a manner which is specific for nucleic acid molecules of mammalian polysialyl transferase, e.g. with a part or all of SEQ ID NO: 1, or SEQ
  • hybridizes in a manner which is specific for a nucleic acid molecule of mammalian polysialyl transferase means that such a nucleic acid molecule or oligonucleotide molecule, when transfected into mammalian cells such as human cells, binds to the nucleic acid molecules which code for a polysialyl transferase in said cells. Specific binding occurs if these nucleic acid molecules inhibit the expression of PST in a considerable manner (more than 50%, preferably more than 80%, or more than 90%) and in such fashion that the other metabolism processes of the cell are not impaired.
  • an oligonucleotide molecule or a nucleic acid molecule which is to hybridize specifically with the PST nucleic acid molecule will at least in its essential part be complementary to sequences in the other regions of SEQ ID NOS: 1, or 7 especially in the coding region of nucleotides 301 to 720 and 48
  • oligonucleotides which bind to the PST gene at or in the vicinity of the start codon or the promoter region. Standard hybridization conditions are described in Sambrook et al . , supra.
  • a preferred oligonucleotide molecule interferes in a sequence-specific manner with processes such as the translation of PST mRNA into the protein by binding to PST mRNA.
  • Further oligonucleotide molecules which are suitable for use are oligo- nucleotide molecules which are complementary to genomic DNA which can interact in forming a triple helical structure (M. Cooney et al . , Science 241: 456 (1988) and Duvall-Valentine et al. , Proc. Natl. Acad. Sci. USA 89: 504-508 (1992) ) . Formation of a triple helix prevents expression of the gene normally encoded by the double helix.
  • Preferred oligonucleotide molecules include oligonucleotide derivatives, such as phosphotriester, methylphosphonates, phosphorothioates or substituted oligonucleotides, such as acridine, interchalating coupled oligonucleotides, or ⁇ -anomers or ⁇ -anomers (J.J. Toulme and C. Helene, Gene 72: 51-58 (1988) ) . Phosphorothioates and methylphosphonates are specifically preferred.
  • Such oligo derivatives can be synthesized according to the state of the art, for example by automated technology (S. Beaucage and M. Caruthers, Tetrahedron Lett. 37: 3556 (1981) ; G.
  • Another aspect of the invention is a soluble complex for targeting an antisense oligonucleotide molecule according to the invention which hybridizes in a manner which is specific for nucleic acid molecules encoding mammalian polysialyl transferase with, e.g., a part or all of SEQ ID NO: 1, or SEQ ID NO: 7 complexed with an oligonucleotide molecule binding agent, which complexes the oligonucleotide molecule under extracellular conditions and releases said oligonucleotide molecule under intracellular conditions as an oligonucleotide molecule specifically bindable to polysialyl transferase encoding nucleic acid molecule.
  • oligonucleotides are covalently coupled to polycations, such as polylysine (M. Lemaitre et al . , Proc. Natl. Acad. Sci. USA 84: 648-652 (1987)) .
  • polycations such as polylysine (M. Lemaitre et al . , Proc. Natl. Acad. Sci. USA 84: 648-652 (1987))
  • polycations such as polylysine (M. Lemaitre et al . , Proc. Natl. Acad. Sci. USA 84: 648-652 (1987))
  • polycation conjugates e.g.
  • transferrin are described in WO 92/20316, USP 5,166,320, WO 92/19281, WO 92/13570, EP-A 0 388 758, WO 93/07283, WO 92/17210, WO 91/17773, WO 93/04701 and transfer peptides as specified in PCT/EP94/01147 are also suitable.
  • the efficacy of the internalization of the nucleic acid molecules according to the invention in cells can be improved by binding the nucleic acid molecule to amphiphilic molecules, such as polyethylene glycol in a complex. Further preferred are the use of transfection reagents, such as DOTMA (WO 91/06309 and WO 91/17424) . Liposomes and dendromers are also useful .
  • the nucleic acid molecules according to the invention can be coupled non-covalently to conjugates from a DNA-binding substance (e.g. polycations) and/or a cell-specific ligand (e.g. protein, preferably PSA specific antibody) (Wu, et al . J. Biol. Chem. 4429-4432 (1987) ; Wu et al . (1988)) .
  • a DNA-binding substance e.g. polycations
  • a cell-specific ligand e.g. protein, preferably PSA specific antibody
  • internalization of the nucleic acid molecules in cells is accomplished by means of a soluble DNA carrier system consisting of a chemically synthesized conjugate comprising mannose and lactose as the ligands (P. Midoux et al . , Nucl. Acids Res.
  • EP-A 0 388 758 discloses chemically synthesized transferrin polycation conjugates which form complexes with polyanionic nucleic acid molecules . By means of the binding to the transferrin receptor these complexes can be internalized in the target cells.
  • WO 93/07283 also describes, with regard to non-viral gene transfer, a "2-ligand-system" comprising a DNA-binding (polycationic) portion (substance with affinity towards nucleic acid molecules) and an internalization factor for the take-up of DNA in the cell .
  • a socalled endosomolytic agent which corresponds, for example, to a virus or a virus component (e.g. adenovirus or influenza hemagglutinin) .
  • the carrier for the oligonucleotide molecule is a conjugate from a cell-specific ligand and a DNA binding substance, these substances are preferred to be covalently linked and the linkage typically is a peptide bond.
  • This can be formed, for example, with a water-soluble carbodiimide as described by G. Jung et al . , Biochem. Biophys . Res. Comm. 101: 599-606 (1981) .
  • An alternative linkage is a disulfide bond.
  • a cell-specific agent which can be a natural or synthetic ligand (for example, a protein polypeptide, glycoprotein, etc.) or it can be an antibody or an analogue thereof which specifically binds to a cellular surface structure which then mediates internalization of the bound complex can be, and is preferably used in these systems.
  • a natural or synthetic ligand for example, a protein polypeptide, glycoprotein, etc.
  • an antibody or an analogue thereof which specifically binds to a cellular surface structure which then mediates internalization of the bound complex can be, and is preferably used in these systems.
  • Such antibodies are described in Frosch et al . , Proc. Natl. Acad. Sci. USA 82: 1194-1198 (1985) and in Molenaar, et al . , Cane. Res. 50: 1102-1106 (1990) .
  • the antibodies described in Molenaar recognize epitopes on all NCAM isoforms . After binding, these antibodies are internalized.
  • the cell-specific binding agent is a ligand which binds to a cell-surface receptor.
  • receptors are employed which are specific for such tissue cells from which the tumor to be treated originates .
  • the antisense oligos for the treatment of tumor diseases involving a high metastasis potential (small cell lung carcinoma, medulloblastoma, Wilms ' tumor and lymphoid tumor. See Kern, et al . , Leukemia and Lymphoma 12: 1-10 (1993)) .
  • Tumor antigens that are suitable for use as surface receptors are, for instance, the tumor antigens.
  • the employed cell-specific ligand acts as a substance which facilitates the internalization of the oligonucleotide molecules according to the invention.
  • Such internalization factors are, for example, transferrin or anti-CD4-antibody.
  • the optimal ratio of the cell-specific binding agent to the oligonucleotide molecule and the oligonucleotide binding agent in the complexes can be determined empirically. When polycations are used, the molar ratio of the components will vary depending on the size of the polycation and the size of the oligonucleotide.
  • the oligonucleotide molecules and carriers are mixed and incubated under conditions conductive to complexation. For example, the oligonucleotide molecule and carrier can be mixed at the appropriate ratio in 2 mol/1 NaCl and the solution can be diluted to 0.15 mol/1 and filtered to provide an administerable composition.
  • oligonucleotide molecules or the molecular complexes of this invention can be administered parenterally.
  • the complex is administered intravenously.
  • the complex is administered in solution in a physiologically acceptable vehicle .
  • a further object of the invention therefore is the use of a nucleic acid molecule or oligonucleotide molecule or complex according to the invention, for the production of a therapeutic agent for the treatment of tumor therapy, especially for prevention or inhibition of metastasis tumors .
  • a further aspect of the invention provides methods of retarding or preventing tumor metastasis comprising inhibition of PST activity within the tumor cells.
  • Preferred methods of retarding or preventing tumor metastasis comprise introducing into tumor cells oligonucleotides that specifically bind to all or a portion (s) of nucleic acid encoding proteins having PST activity, most preferably oligonucleotides complementary to all or portions of the sequences set forth in SEQ ID NOS: 1 or 7.
  • the tumor chosen is melanoma, small cell lung cancer, or lymphoma.
  • Other tumors which are neuro- ectodermal in nature which express inappropriate levels of PST may also be so treated.
  • the invention involves methods of modulating cellular interactions and adhesion comprising recombinant expression of exogenous PST in appropriate host cells expressing adhesion proteins, e.g., N-CAM.
  • adhesion proteins e.g., N-CAM.
  • it is preferably to utilize nucleic acids comprising or consisting of PST coding sequences (i.e., nucleotides 301- 1377 in SEQ ID NO: 1 or nucleotides 213-1289 in SEQ ID NO: 7) .
  • the PST-encoding nucleic acid can be introduced into the cells through transfection of the cells with PST-encoding DNA or injection of the cells with PST mRNA.
  • such methods include methods of promoting neurite outgrowth on nerve cells and/or substrate cells on which nerve cells are grown. Accordingly, in a further aspect, the invention involves methods of promoting neuroregeneration comprising transplantation of recombinant PST-expressing neuroepithelial cells into tissues exhibiting neurodegeneration.
  • antibodies generated against the PST proteins disclosed herein can be employed for studying PST tissue localization, structure of functional domains, purification of PST as well as in diagnostic and therapeutic applications and the like.
  • the antibodies employed will be monoclonal antibodies.
  • the antibodies can be prepared employing standard techniques, as are well known to those of skill in the art, using the invention PST proteins or portions thereof as antigens for antibody production. Both anti-peptide and anti- fusion protein antibodies can be used [see, for example, Current Protocols in Molecular Biology (Ausubel et al . eds . ) John Wiley and Sons, N.Y. (1989)] . Factors to consider in selecting portions of the PST protein for use as immunogen (as either a synthetic peptide or a recombinantly produced bacterial fusion protein) include antigenicity, accessibility (i.e., cytoplasmic domains) , uniqueness, etc.
  • PST-specific antibodies makes possible the application of the technique of immunohistochemistry to monitor the distribution and expression density of the PST protein.
  • PST-specific antibodies can be employed in methods of treating as indicated supra.
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • CAA CAT TCT GTA CAA GGC TGG AGA ATC AAT TCT TCT TTA GTC CTG GAG
  • Val Val Lys Ser Ser Phe Lys Pro Gly Asp Val lie His Tyr Val Leu 100 105 ' 110 GAC AGA CGC CGG ACG CTA AAT ATT TCC CAT GAT CTG CAC AGC CTC CTG 684
  • ATC CCC GCT TTC ATG GTC AAA GGA GGA GAG AAG CAC GTG GAA TGG GTT 1020 lie Pro Ala Phe Met Val Lys Gly Gly Glu Lys His Val Glu Trp Val 225 230 235 240
  • Val Pro lie Lys Arg Pro Ser Thr Gly Leu Leu Met Tyr Thr Leu Ala 275 280 285
  • Thr Arg Phe Cys Asp Glu lie His Leu Tyr Gly Phe Trp Pro Phe Pro 290 295 300 AAG GAT TTG AAT GGA AAA GCT GTG AAA TAT CAT TAC TAC GAT GAC TTG
  • Val Pro lie Lys Arg Pro Ser Thr Gly Leu Leu Met Tyr Thr Leu Ala 275 280 285

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PCT/US1995/016664 1994-12-22 1995-12-21 Isolated polysialyl transferases, nucleic acid molecules coding therefor, methods of production and use WO1996020280A1 (en)

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US08/503,133 US5747326A (en) 1995-07-17 1995-07-17 Isolated nucleic acid molecules which encode mammalian α2,8 polysialyl transferases
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EP (1) EP0871743A1 (enrdf_load_stackoverflow)
JP (1) JP2002509423A (enrdf_load_stackoverflow)
AU (1) AU692355B2 (enrdf_load_stackoverflow)
CA (1) CA2208291A1 (enrdf_load_stackoverflow)
WO (1) WO1996020280A1 (enrdf_load_stackoverflow)

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WO2008025990A1 (en) * 2006-09-01 2008-03-06 Imperial Innovations Limited Biological materials and uses thereof

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ATE228565T1 (de) * 1992-03-09 2002-12-15 Univ California Nukleinsäure, expressionsvektor und zusammensetzungen zur identifizierung und herstellung von rekombinanten sialyltransferasen

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008025990A1 (en) * 2006-09-01 2008-03-06 Imperial Innovations Limited Biological materials and uses thereof

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JP2002509423A (ja) 2002-03-26
EP0871743A1 (en) 1998-10-21
CA2208291A1 (en) 1996-07-04
EP0871743A4 (enrdf_load_stackoverflow) 1998-12-02
AU4687096A (en) 1996-07-19

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