WO2000003248A1 - Method for identifying a presenilinase inhibitor - Google Patents
Method for identifying a presenilinase inhibitor Download PDFInfo
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- WO2000003248A1 WO2000003248A1 PCT/EP1999/004805 EP9904805W WO0003248A1 WO 2000003248 A1 WO2000003248 A1 WO 2000003248A1 EP 9904805 W EP9904805 W EP 9904805W WO 0003248 A1 WO0003248 A1 WO 0003248A1
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- presenilinase
- protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
Definitions
- the present invention belongs to the field of presenilins and neurodegenerative diseases. More particularly, the present invention provides methods for the identification of presenilinase inhibitors, substances identifiable with said methods, their use in the manufacture of a medicament for the treatment of neurodegenerative diseases and pharmaceutical compositions comprising said substances.
- Neurodegenerative diseases are characterized by neuronal and synaptic cell loss. Neuronal cell loss is caused at least in part by apoptotic cell death. Neurodegenerative diseases include the chronic forms as Alzheimer's disease (AD), Parkinson's disease, Huntington's chorea and acute forms as stroke. The majority of Alzheimer's disease cases are late in onset so far lacking an obvious genetic linkage and are characterized as sporadic, whereas a small percentage (approximately 10%) of cases belonging to the subgroup of familiar Alzheimer's disease (FAD) are earlier in onset and segregate strongly within families suggesting a genetic etiology.
- AD is a neurodegenerative disorder marked by the gradual formation of extracellular neuritic plaques in the brain, particularly in the hippocampus and the adjoining cortex.
- a ⁇ ⁇ amyloid
- a ⁇ 42 was shown to be deposited early and selectively in the disease process and to be more fibrillogenic in vitro than the more prevalent species of A ⁇ ending at residue 40, termed A ⁇ 40 (Jarret et al , 1993, Mann et al , 1996)
- the presenilins undergo regulated proteolytic cleavage into the normal N-terminal (NTF) and C- terminal fragments (CTF) These fragments are approximately 21-28 kDa (PS1 NTF), 28-30 kDa (PS2 NTF), 16-24 kDa (PS1 CTF) and approximately 20-25 kDa (PS2 CTF) respectively, in size (Okochi et al , 1997, Haas et al , 1998, Kim et al , Science, 1997, Thinakaran et al 1996, Podlisny et al , 1997)
- the PS proteins constitute substrates of a member of the caspase 3 protease family (CPP32) after its activation late in the course of apoptosis and are cleaved into alternative fragments (Kim et al , Science 1997, 277 373-376, Loetscher et al , 1997) According to Kim et al (Science 1997, 277 373-376), these alternative fragments of PS2 are a 20 kDa CTF and a 34 kDa NTF In our hands, this alternative CTF fragment of PS2 has the molecular weight of 16 kDa and thus is subsequently termed CTF ⁇ g
- the cleavage region of normal proteolytic PS2 cleavage is located within the distal region of exon 10 (originally called exon 9) between Met 298 and Ala 305 , whereas the suspected region of alternative cleavage is located within the PS2 loop encoded by exon 1 1 after Asp 326 or Asp 329 (Kim et al
- the presenilins may be cofactors for the activity of the ⁇ -secretase or even ⁇ -secretases itself by being autoactivated aspartvl proteases with ⁇ -secretase activity (Wolfe et al , 1999)
- presenilins may be trafficking proteins, and transport the substrates APP-C99 and C83 into cellular compartments where they get cleaved by the putative ⁇ -secretase.
- proteolytic fragments are the biologically active form of the presenilins (Thinakaran et al., 1996, Podlisny et al., 1997). Thus, if said proteolytic fragments constitute the active form of presenilins, they also may be either directly or indirectly involved in ⁇ -secretase activity and therefore in the generation of A ⁇ .
- the endogenous presenilin protein level only consists of these proteolytic fragments.
- the PS-protein does not exist in its full length indicating that immediately after expression the full-length presenilins get cleaved into stable fragments.
- the level of proteolytical fragments is increased until saturation level. When this saturation level is exceeded, no more proteolytical cleavage takes place and the full-length protein is detectable, i. e. in cells overexpressing presenilin, fragments are detected as well as, according to the degree of overexpression, the full-length protein.
- An increase in full-length PS may however, lead to increased degradation of said full-length PS (e.g.
- WO 97/41443 discloses the screening for the identification of compounds which inhibit or otherwise modulate the cleavage of PS1 comprising the measurement of PS1 into 18 kDa and 28 kDa species.
- WO 98/47917 discloses a 20 kDa PS2-CTF fragment resulting from alternative cleavage, an antibody specific for said fragment and a method for screening compounds that inhibit proteolytic processing of PS2 comprising providing a compound to a cell proteolytically processing PS2 and the measurement of said fragment in said cell.
- WO 97/27296 describes the presenilin-interacting proteins S5a, GT24, p0071, Rabl l, retionoid X receptor- ⁇ , cytoplasmic chaperonin, Y2H35, Y2H171 and Y2H41.
- the problem underlying the present invention therefore is to provide methods for identifying substances capable of reducing or eliminating the activity of the presenilinase comprising the measurement of full-length presenilin fused to a reporter and to provide said substances.
- the present invention pertains to methods for identifying a substance capable of reducing or eliminating the activity of the presenilinase wherein a cell or a cell line is cultivated expressing said presenilinase activity and a fusion-protein comprising the full-length presenilin 1 or presenilin 2 and a reporter is measured.
- the invention is furthermore concerned with substances identifiable with said methods, pharmaceutical compositions comprising said substances and the use of said substances in the manufacture of a medicament for the treatment of neurodegenerative diseases, preferably Alzheimer's disease.
- Figure 1 a) Sequence and structure of a general hammerhead ribozyme-target RNA complex.
- GUX trinucleotide
- the secondary structure of a part of the PS2 mRNA was predicted by "mfold" (described infra in example 1) Open loops that are good candidate regions suitable for targeting ribozymes are shown as black circles. The cleavage sites at the target trinucleotides are indicated with arrows. The numbering of the nucleotides corresponds to the sequence of human PS2 in the EMBL Data Bank, Accession No. L43964. The prediction for the secondary structure of the remaining part of the PS2 mRNA (nts 1001-2236) did not yield suitable open loop regions (data not shown).
- ribozymes and the corresponding substrate RNAs Three different trinucleotides were chosen and the appropriate synthetic ribozymes designed for in vitro ribozyme cleavage studies and the exogenous use in cell culture experiments A ribozyme targeted to a trinucleotide was designed with flanking substrate binding domains of various lengths, i.e 5 ribozymes (rzl 173/13 3, rzl 173/12, etc ) targeted to the GUC ⁇ 73 trinucleotide (nucleotide numbering according to EMBL Data Bank, Accession No L43964).
- RNAs As substrate RNAs (target sites) for in vitro cleavage studies we routinely used short synthetic, 5' [32p] -labeled (indicated by asterisks), RNAs (shown as black bars with an arrow directing to the right site indicating sense RNA)
- target site GUC1 173 we generated a larger substrate RNA (367 bp), in vitro transcribed from plasmid pBSK+/PS2 Ncol with T7 polymerase (Fig 4b) and [ ⁇ P-CTP]- labeled (indicated by asterisks)
- an antisense RNA containing the ribozyme target site of PS2 shown as black bars with an arrow directing to the left site indicating antisense RNA
- PS2 mRNA Three synthetic ribozymes (rzl 173, rz232, rz308, nucleotide numbering according to EMBL Data Bank, Accession No. L43964) were targeted to various regions in the PS2 mRNA and analyzed for their cleavage capacity in vitro with synthetic, 5' [32p]-labeled RNA substrates containing the specific target trinucleotide. Each ribozyme was used with different lengths of the flanking substrate binding region (i.e. rzl 173/13.3, 12, 9, etc.).
- ribozyme cleavage reaction was carried out under standard conditions and ribozyme: target molar ratios were used as indicated.
- substrate RNAs were used without ribozyme treatment (lane"-"). Reactions were stopped and loaded onto a 20 % SDS- polyacrylamide/ 6 M urea gel, dried onto filter paper and exposed on X-Omat AR films (Kodak).
- ribozymes Three different ribozymes, rzl 173, rz232 and rz308, with a substrate binding domain in between 15-16 b were used for further detailed analyses concerning the required ribozyme:target molar ratio for efficient cleavage in vitro.
- the ribozyme cleavage reaction was carried out under standard conditions (described infra in example 1) with ribozyme:target molar ratios as indicated.
- substrate RNAs were used without ribozyme treatment (lane "-"). Reactions were stopped and loaded onto a 20 % SDS-polyacrylamide/ 6 M urea gel, dried onto filter paper and exposed on X-Omat AR films (Kodak).
- Figure 3 a In vitro efficiency of ribozyme rzl 173 with substrate binding domains varying in length at different ribozyme:target molar ratios.
- Ribozyme rzl 173 was used with flanking substrate binding domains in between 9-16 b in length (rzl 173/13.3, 12, 9).
- the corresponding synthetic RNA substrate was 5' [ 2p]-labeled.
- the ribozyme cleavage reaction was carried out under standard conditions (described infra in example 1) with ribozyme:target molar ratios as indicated.
- the ribozyme cleavage reaction was carried out under standard conditions (described infra in example 1) with a concentration of ribozyme target RNA of 100 1 As RNA substrate a 5' [32p]-l a beled synthetic RNA was used containing the target trinucleotide GUC i ⁇ 73 Aliquots were taken at the indicated time points and loaded onto a 20 % SDS-polyacrylamide/ 6 M urea gel The cleavage kinetic of ribozyme rzl 173/13 3 is shown in the upper figure Ribozyme cleavage in percentage was calculated with the Phosphor Imaging System (BioRad) and is shown in the lower figure
- FIG. 4 a) Sequence and structure of the ribozyme rzll73/13.3auto - PS2 mRNA complex.
- the binding of hammerhead ribozyme rzl 173/13 3auto to a specific sequence of the PS2 mRNA is shown (nucleotide numbering according to EMBL Data Bank, Accession No L43964) Base pairing between the flanking regions of the substrate binding domain of rzl 173 and the surrounding nucleotides of GUC1 173 in the PS2 mRNA is indicated by asterisks, the Wobble base pair "G-U" is marked by points
- the ribozyme rzl 173/13 3auto is an example for a fusion ribozyme comprising the PS2-specific ribozyme rzl 173/13 3 and the autocatalytical hammerhead- ribozyme directly fused with its 5' end to the 3' end of the PS2- specific ribozyme rzl 173/13.3
- the in vitro transcribed ribozyme rzl 173 was incubated in increasing amounts (0, 1 , 0,3, 0,5, 1, 3, 5 ⁇ l of the total in vitro transcription reaction) together with the 367 b long, [ 32 P]-labeled PS2 transcript under standard conditions (described infra in example 1 )
- the first lane shows the substrate RNA without treatment
- In lane "-" substrate RNA was incubated under standard conditions without ribozyme Marker RNA of known size was loaded onto the polyacrylamide gel for comparison
- FIG. 6 a) PS2 mRNA levels of various cell clones inducibly expressing rzll73/13.3. 49 clones that were stably transfected with the construct pUHD 10-3/PS2-rzl 173.13 3auto were tested for PS2 expression after omission of doxycycline with the RNase protection assay (RPA)
- the first two lanes are control reactions, in which tRNA is used for hybridization with the [32p]-labeled antisense RNA probe of PS2
- These hybridization reactions were carried out in the absence (-) or presence (+) of RNases mRNA from the control cell line HtTA was used in the RPA as standard and indicated the endogenous PS2 mRNA level
- Cell line HtTA/PS2 rzl 173 40 was selected for further detailed analyses on the protein level
- Extracts were made from the PS2 'knock-down' cell line at different time points after omission of doxycycline.
- extracts were prepared from cells growing in standard medium supplemented with doxycycline at day 0 and 14 Proteins were immunoprecipitated using antibody 3711, separated on SDS/ polyacrylamide gels, blotted onto PVDF membranes and hybridized with the monoclonal antibody BI.HF5C (1 2000 dilution) Both antibodies recognize the hydrophilic loop of PS2
- the PS2 'knock-down' HeLa cell line was less sensitive against an apoptotic stimulus as calculated by ethidiumbromide/ acridine orange staining.
- Three HeLa cell lines (the PS2 'knock-down' cells [PS2 k d ] and cell lines overexpressing wildtype [PS2 wt] or mutant PS2 [PS2 mut]) were used for determination of their apoptotic sensitivity to staurosporine (a) Overexpression of wildtype or mutant PS2 was demonstrated by immunofluorescence with antibody 2972 (1 300 dilution), that recognizes the N-terminus of PS2, in the presence (+Dox) or the absence (-Dox) of doxycycline (b) After treatment with staurosporine in concentrations as indicated for 18 h, the cells were fixed and incubated with ethidiumbromide and acridine orange as described infra in example 1 Figure 8
- the PS2 'knock-down' caused an inhibition of apoptosis.
- the three transfected HeLa cell lines (the PS2 'knock-down' cells [PS2 k.d.] and two cell lines overexpressing wildtype [PS2 wt] or mutant PS2 [PS2 mut]) as well as the original HeLa cell line were treated with indicated concentrations of staurosporine for 18 h under standard conditions (described infra in example 1). As a control, cells were not treated with staurosporine (lane "0").
- the PS2 'knock-down' seemed to have no influence on the caspase 3 (CPP32) activation following an apoptotic stimulus.
- the three HeLa cell lines (the PS2 'knock-down' cells [PS2 k.d.] and cell lines overexpressing wildtype [PS2 wt] or mutant PS2 [PS2 mut]) were treated with 1 ⁇ M staurosporine under standard conditions (described infra in example 1).
- As a control extracts were made of cells not treated with staurosporine (lane "c").
- Extracts were prepared at the indicated time points and identical protein amounts were loaded onto a 12 % SDS/ polyacrylamide gel.
- CPP32-specific antibody Transduction Laboratories, 1:500 dilution
- This antibody recognizes the CPP32 holoenzyme and the 17 kDa active N-terminal fragment (shown in a as an example) that is generated upon proteolytic cleavage, (b)
- the CPP32 holoenzyme is indicated by an arrow.
- the PS2 'knock-down' seemed to have no influence on PARP cleavage following an apoptotic stimulus.
- the three HeLa cell lines (the PS2 'knock-down' cells [PS2 k.d ] and cell lines overexpressing wildtype [PS2 wt] or mutant PS2 [PS2 mut]) were treated with 1 ⁇ M staurosporine under standard conditions (described infra in example 1).
- As a control extracts were made of cells not treated with staurosporine (lane "c").
- Extracts were prepared at the indicated time points and identical protein amounts were loaded onto a 12 % SDS/ polyacrylamide gel.
- PARP-specific antibody Boehringer Mannheim, 1 :2000 dilution
- This antibody recognizes the PARP holoenzyme and the proteolytic fragments (indicated by arrows), (b) PARP holoenzyme and the 85 kDa fragment are marked by arrows.
- Extracts were prepared at the indicated time points and identical protein amounts were used for immunoprecipitation with the polyclonal PS2/loop-specific antibody 371 1 After 12 % SDS/ polyacrylamide gel electrophoresis and blotting onto PVDF membranes, hybridization with the monoclonal antibody BI.HF5C (1 :2000 dilution), that was also raised against the loop region, was carried out.
- the PS2 'knock-down' showed an inhibitory effect on apoptosis compared with the overexpression of wildtype or mutant PS2.
- the cells were analyzed for apoptosis using the cell death detection ELISA (Boehringer Mannheim). The degree of apoptosis was expressed directly as the absorbance at 405-490 nm.
- PS2 Asp366Ala affects processing of ⁇ APP a) Expression of PS2 Asp366Ala results in a marked reduction of A ⁇ production Pooled clones stably expressing PS2 Asp366Ala or a subcloned cell line (clone 11) were metabolically labeled with 35 S-methionine for 2 h followed by a cold chase for additional 2 h Conditioned media were immunoprecipitated with antibody 3926 to synthetic A ⁇ Cells stably exspressing the PS2 Asp366Ala produce significantly reduced amounts of A ⁇ In addition reduced production of p3, which results from the combined action of ⁇ - and ⁇ -secretase (Haass and Selkoe, 1993) is observed The effect of the dominant negative PS2 mutation on amyloidogenesis appears to be enhanced in the subcloned cell line b) Expression of PS2 Asp366Ala results in the accumulation of C-terminal proteolytic fragments of ⁇ APP Cell lysates from poole
- the presenilin-luciferase fusion-protein is schematically depicted.
- the presenilin proteins probably contain eight transmembrane domains (TM), with both the N- and C-terminus, as well as the large hydrophilic loop between TM6 and TM7 oriented toward the cytoplasm (Haass, 1997).
- the putative presenilinase cleaves in the loop region
- the gray box indicates the published cleavage site (Podlisny et al, 1997; Shirotani et al., 1997; Shirotani et al., 1999; Wisniewski et al, 1997).
- the luciferase fused into the N-terminus of presenilin with or without amino acid deletions in the presenilin protein is shown.
- A Stably transfected cell line expressing the presenilin (PS1 or PS2)-luciferase fusion-protein.
- the fusion-protein is cleaved due to the activity of the presenilinase into fragments of regulated proteolytic cleavage
- the NTF is fused to the reporter luciferase.
- the presenilinase-luciferase fusion-protein is cleaved by the endogenous presenilinase, the C-terminus is bound to the microtiter plates via antibody-antigen interaction and the luciferase fused to the N-terminus is washed away during the washing steps In this case, no signal is detected
- the invention pertains to a method for identifying a substance capable of reducing or eliminating the activity of the presenilinase
- a method according to the present invention comprises cultivating a cell or a cell line to express said presenilinase activity and a fusion-protein, said fusion-protein comprising the full-length presenilin 1 or presenilin 2 and a reporter, incubating said cell or cell line with a test substance, measuring the quantity of the full-length fusion-protein employing the reporter and comparing the quantity of full-length fusion-protein obtained by the before-mentioned step to the quantity of full-length fusion-protein measured for a control.
- the presenilin is cleaved into fragments and thus no full-length protein coupled to the reporter can be detected (see figure 15).
- Two particular examples of the method according to the present invention which should not be construed as limiting the present invention are disclosed in examples 2 and 3, infra.
- the activity of the presenilinase as used herein may be the activity of any enzyme (e.g. a protease) or of a protein or of a chemical substance capable of cleaving said presenilin.
- Said activity of the presenilinase is thus capable of generating fragments of regulated or alternative proteolytic cleavage.
- said presenilinase activity leads to cleavage of presenilin into fragments of regulated proteolytic cleavage, e.g. cleavage of PS2 is within the distal region of exon 10 (originally called exon 9) between Met 298 and Ala 305 .
- said activity of the presenilinase may also be the autocatalytical cleavage activity of the presenilin wherein the presenilin itself leads to the generation of fragments without the activity of another enzyme or substance.
- presenilin may have the activity of the ⁇ -secretase
- said activity of the presenilinase may also be the activity of the ⁇ -secretase.
- a suitable cell or cell line preferably an eukaryotic cell or a cell line, to be transformed with nucleic acid constructs to express said fusion-protein may be any cell or cell line known to the expert in the field, in particulary cells or cell lines used in neurological and neurobiology research.
- Examples of such cells or cell lines useful for producing the transformed cell lines of the invention include mammalian cells or cell lines (e.g., the cell lines H4, U373, NT2, human embryonic kidney (HEK) 293, PC12, COS, CHO, fibroblasts, myelomas, neuroblastomas, hybridomas, oocytes, embryonic stem cells), insect cells lines (e.g., using baculovirus vectors such as pPbac or pMbac (Stratagene, La Jolla, CA)), yeast (e.g., using yeast expression vectors such as pYESHIS (Invitrogen, CA)), and fungi.
- mammalian cells or cell lines e.g., the cell lines H4, U373, NT2, human embryonic kidney (HEK) 293, PC12, COS, CHO, fibroblasts, myelomas, neuroblastomas, hybridomas, oocytes, embryonic stem cells
- insect cells lines e.g.,
- a wide variety of vectors have been developed and are widely available which allow inducible (e.g., LacSwitch expression vectors, Stratagene, La Jolla, or the tTA-response plasmid pUHD 10-3, Gossen and Bujard, 1992) or cognate (e.g., pcDNA3 vectors, Invitrogen, Chatsworth, CA) expression of presenilin nucleotide sequences under the regulation of an artificial promoter element.
- promoter elements are often derived from CMV or SV40 viral genes, although other strong promoter elements which are active in eukaryotic cells can also be employed to induce transcription of presenilin nucleotide sequences.
- these vectors also contain an artificial polyadenylation sequence and 3' UTR (untranslated region) which can also be derived from exogenous viral gene sequences or from other eukaryotic genes.
- artificial, non-coding, spliceable introns and exons are included in the vector to enhance expression of the nucleotide sequence of interest (in this case, presenilin sequences).
- These expression systems are commonly available from commercial sources and are typified by vectors such as pCDNA3 and pZeoSV (Invitrogen, San Diego, CA). Both of the latter vectors have been successfully used to cause expression of presenilin proteins in transfected COS, CHO, and PC12 cells (Levesque et el.
- Vectors may be introduced into the recipient or "host" cells by various methods well known in the art including, but not limited to, calcium phosphate transfection, strontium phosphate transfection, DEAE dextran transfection, electroporation, lipofection (e.g., Dosper Liposomal transfection reagent, Boehringer Mannheim, Germany), microinjection, ballistic insertion on micro-beads, protoplast fusion or, for viral or phage vectors, by infection with the recombinant virus or phage.
- methods well known in the art including, but not limited to, calcium phosphate transfection, strontium phosphate transfection, DEAE dextran transfection, electroporation, lipofection (e.g., Dosper Liposomal transfection reagent, Boehringer Mannheim, Germany), microinjection, ballistic insertion on micro-beads, protoplast fusion or, for viral or phage vectors, by infection with the recombinant virus or phage.
- a fusion-protein according to the present invention is a protein encoded by the nucleic acid encoding the full-length presenilin 1 (PS1) or presenilin 2 (PS2) linked to the nucleic acid encoding the reporter (described infra) and is expressed in a cell line as described supra according to standard methods known to the expert in the art (see also e.g. Sambrook et al., 1989).
- "Full- length” as used herein relates to the entire presenilin and the respective gene as described infra and comprises also mutations of said gene.
- full-length also relates to presenilin or the respective gene with N-terminal deletions of e.g.
- fusion-protein may also comprise one or several linker or spacer molecules e.g. located between the presenilin (PS) and the reporter and /or at the 5' or 3' end of the construct.
- PS presenilin
- Said linker or spacer molecules are peptides, preferably 2-50 amino acids long, or chemical substances capable of linking the presenilin to the reporter or capable of maintaining a certain space between presenilin and reporter to enable the proper function of both molecules and to avoid steric hindrance
- the reporter is fused to the N-terminus of the PS in one of the following ways
- fusion-proteins according to the present invention are preferably encoded by one of the following constructs
- the DNA encoding the reporter is fused to the N-terminus of the DNA encoding the full-length PS in one of the following ways' (i) direct fusion to the N-terminus of PS with or without nucleic acid deletions in the presenilin gene or (ii) fusion to the N-terminus via a spacer (6-150 base pairs) between the reporter gene and the presenilin N-terminus
- Presenilin 1 gene or "PS 1 gene” means the mammalian gene first disclosed and described in Sherrington et al (1995)
- Presenilin 2 gene or "PS2 gene” means the mammalian gene first disclosed and described in US 5840540 A, and later described in Rogaev et al (1995) and Levy-Lahad et al (1995), and WO 96/34099 Al (all herein incorporated by reference) including any allelic variant and heterospecific mammalian homologues Additional human splice variants as described in WO 96/34099 Al have been found in which a single codon or a region encoding thirty-three residues may be spliced-out in some transcripts
- Presenilin-2 gene or "
- a control may also be the quantity of reporter bound to presenilin fragments measured in the supernatant. Said control may then be compared to the quantity of full- length fusion-protein of presenilin and reporter bound to a solid support.
- a non-limiting example of such a control is disclosed in example 2, infra. Said control is used for calibration of the method of the present invention. Further methods to provide controls or standards for the method of the present invention are known to the expert in the field and are embraced by the present invention.
- any of the well-known reporter genes can be operatively linked to the full-length presenilin gene and may be expressed in the cell line according to the present invention.
- reporter genes include, but are not limited to E. coli ⁇ -galactosidase ( ⁇ -gal, Luban and Goff, 1995), xanthine-guanine phosphoribosyl transferase (Chu and Berg, 1985), galactokinase (Schumperli et al, 1982), interleukin-2 (Cullen, 1986), thymidine kinase (Searle et al., 1985), alkaline phosphatase (Toh et al., 1989; Henthorn et al., 1988), secretory alkaline phosphatase (SEAP) or secreted placental alkaline phosphatase (Berger et al., 1988) and chloramphenicol-acetyltransferase (CAT, Alton and Vapnek, 1979, Gorman et al., 1982; Tsang et al., 1988) green fluorescent protein (GFP) produced by the bio
- reporter genes such as reporter enzymes
- bioassays can be carried out for biologically active proteins such as interleukin-2.
- Enzyme assays can be performed when the reporter gene product is a reporter enzyme such as alkaline phosphatase or ⁇ -galactosidase.
- various types of immunoassays such as competitive immunoassays, direct immunoassays and indirect immunoassays may be used.
- Such immunoassays involve the formation of immune complexes containing the reporter gene product and a measurable "reporter" or a "label".
- reporter includes moieties that can be detected directly, such as fluorochromes and radiolabels, and moieties such as enzymes that must be reacted or derivatized to be detected.
- the term “employing the reporter” or “detection of the reporter” as used herein relates to direct or indirect detection of said expression products of the reporter genes with standard methods known in the art.
- Examples for said detection of the reporter include, but are not limited to the measurement of the substrate or the substrate reaction product of a reporter enzyme or the detection of light emitted by the reporter gene product such as luminescence, the detection of a coloured substrate or substrate reaction product which is due to the activity of a reporter enzyme and the detection of radioactivity due to the activity of the reporter gene product.
- competitive immunoassays samples from induced cultures (following cell disruption if the reporter gene product is not secreted) are incubated with an antibody against the reporter gene product and a known amount of labeled reporter gene product. Any unlabeled product produced by the cells competes with the labeled material for binding to the antibody. The resulting immune complexes are separated and the amount of labeled complex is determined.
- the reporter gene product produced by the cells can be quantified by comparing observed measurements to results obtained from standard curves.
- Direct immunoassays involve incubating culture samples with a labeled antibody against the reporter gene product and separating any immune complexes that form. The amount of label in the complexes is determined and can be quantified by comparison to standard curves.
- Enzyme-linked immunosorbant assays ELISAs
- ELISAs Enzyme-linked immunosorbant assays
- reporter used will depend upon the type of immunoassay used.
- reporter examples include, e.g., radiolabels such as 32 P, 125 I, 3 H and 14 C; fluorescent reporters such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone; chemiluminescers such as the various luciferin compounds; and enzymes such as horseradish peroxidase, alkaline phosphatase, lysozyme and glucose-6-phosphate dehydrogenase.
- the antibody or reporter gene product can be tagged with such labels by known methods.
- coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bisdiazotized benzadine and the like may be used to tag the antibodies with fluorescent, chemiluminescent or enzyme labels.
- the genetic control elements used in this invention can be inserted into many reporter gene- containing vectors, including but not limited to plasmids pSV2Apap, pMAMneo-CAT, pMAMneo-LUC, pSVOCAT, pBCO, pBLCAT2, pBLCAT3, pONl, pCHHO, pCH126 and various plasmids described by De Wet et al, 1987.
- the invention pertains to a method as described wherein the presenilinase is specific for presenilin 1.
- the invention pertains to a method as described wherein the presenilinase is specific for presenilin 2.
- the invention pertains to a method as described wherein the substance capable of reducing or eliminating the activity of the presenilinase reduces or eliminates the autoproteolytical cleavage of the presenilin.
- the invention pertains to a method as described wherein the substance capable of reducing or eliminating the activity of the presenilinase reduces or eliminates the activity of ⁇ -secretase.
- the invention pertains to a method as described wherein the substance prevents cleavage of the presenilin 1 into fragments of regulated endoproteolytic cleavage wherein the N-terminal fragment (NTF) is approximately 21-28 kDa in size and the C- terminal fragment (CTF) is approximately 16-24 kDa in size (Haas et al., 1998; Okochi et al.,
- the invention pertains to a method as described wherein the substance prevents cleavage of the presenilin 2 into fragments of regulated endoproteolytic cleavage wherein the N-terminal fragment (NTF) is approximately 28-30 kDa (NTF) kDa in size and the C-terminal fragment (CTF) is approximately 20-25 kDa in size (Haas et al., 1998; Kim et al., J Biol Chem 1997, 272, 11006-11010; Podlisny et al., 1997).
- NTF N-terminal fragment
- CTF C-terminal fragment
- the sizes of said fragments can not be exactly determined, as known to the skilled artisan, and vary slightly (1 up to 4 kDa) according to the method used (e.g. SDS polyacrylamide gels and marker proteins used thereon). Thus, only approximate sizes are indicated.
- fragments of regulated proteolytic cleavage are products of presenilin cleavage by the presenilinase (see definition infra), not by a member of the caspase 3 protease family (CPP32).
- the NTF fragment of regulated or normal proteolytic cleavage has a smaller molecular weight than the NTF of alternative cleavage (e.g. for
- PS2 approximately 30 kDa (regulated) versus 34 kDa (alternative) according to WO 98/47917) and the CTF of regulated or normal cleavage has a higher molecular weight.
- the invention pertains to a method as described wherein the reporter is fused to the N-terminus of the presenilin.
- the present invention is concerned with a method as described supra wherein
- measuring the quantity of the full-length protein employing the reporter comprises immobilizing antibodies specific for the C-terminal portion, preferably the loop region of the C-terminal portion, of presenilin 1 or presenilin 2 to a solid surface, extracting the protein from said cell or cell line after cultivation, incubating said protein extracts with said antibodies, and measuring the amount of full-length fusion-protein bound by said antibodies by detection of the reporter Even more particularly, the invention pertains to a method as described wherein any unbound fusion-protein fragments are measured by detection of the reporter after incubating said protein extract according to the above-mentioned step
- the invention pertains to a method as described wherein the cell or cell line is expressing said fusion-protein at such a level that little or no full-length fusion-protein is detected when no substance capable of reducing or eliminating the activity of the presenilinase is present More particularly, the invention pertains to a method as described wherein the reporter is luciferase
- the invention pertains to a method as described wherein the method is a high throughput screening assay (HTS) HTS relates to an experimental setup wherein a large number of substances is tested simultaneously
- HTS setup may be carried out in microplates, may be partially or fully automated and may be linked to electronic devices such as computers for data storage, analysis, and interpretation using bioinformatics.
- HTS also comprises ultra high throughput screening formats (UHTS)
- UHTS formats may be carried out using 384 or 1536 well microplates, sub-microliter or sub-nanoliter pipettors, improved plate readers and procedures to deal with evaporation HTS methods are described e g in US 5876946 A or US 5902732 A
- the expert in the field can adapt the above-described method to a HTS or UHTS format without the need of carrying out an inventive step
- said method may be an immunological or a molecular biology or biochemical method Immunological methods are known to the expert in the field and include, but are not limited to ELISAs (enzyme-linked immuno-sorbent assay) or Sandwich-E IS As, dot
- the invention is further concerned with a substance capable of reducing or eliminating the activity of the presenilinase, identifiable with a method as described above.
- substance means a chemical, pharmaceutical, biochemical or biotechnological substance.
- the invention is further concerned with a substance identifiable with said methods, wherein the substance is selected from the group consisting of: a) a substance capable of reducing or eliminating the enzymatic activity of the presenilinase or b) a substance capable of reducing or eliminating the expression of the presenilinase at the translational or transcriptional level or c) a substance capable of reducing or eliminating the expression of the substrate of the presenilinase or d) a substance capable of reducing or eliminating the formation of complexes between presenilin fragments.
- the substance is selected from the group consisting of: a) a substance capable of reducing or eliminating the enzymatic activity of the presenilinase or b) a substance capable of reducing or eliminating the expression of the presenilinase at the translational or transcriptional level or c) a substance capable of reducing or eliminating the expression of the substrate of the presenilinase or d) a substance capable of reducing or eliminating the formation of complexes between prese
- a substance according to a) identifiable with said methods is directly inhibiting the presenilinase by blocking the enzymatic activity of the presenilinase.
- a substance may be another enzyme capable of cleaving the presenilinase e.g. a protease or a so-called anti-enzyme or antizyme.
- a substance further includes a biochemical substance, e.g. a polypeptide capable of blocking the substrate-binding site of the presenilinase such as a substrate analogue.
- chemical substances preferably substances which chemically modify the presenilinase in a way that the presenilinase cannot carry out its enzymatic activity any more, such as derivatizing agents.
- derivatizing agents preferably substances which chemically modify the presenilinase in a way that the presenilinase cannot carry out its enzymatic activity any more.
- Other substances known in the art capable of carrying a similar action are also included in the present invention.
- a substance according to b) identifiable with said methods may be any inhibitor of the presenilinase translation or transcription. Examples include, but are not limited to transcription terminators or repressors or translation inhibitors such as ribozymes, rifampicin or chloroamphenicol .
- a substance according to c) identifiable with said methods may be a substance blocking the translation or transcription of the substrate of the presenilinase.
- Such substances include, but are not limited to substances inhibiting e.g. PS 1 or PS2 at the RNA or DNA level, e.g. a ribozyme as disclosed in example 1 (RNA level, see also e.g. figure 1, figure 4) or a mutation of PS1 or PS2 as disclosed in example 4 (DNA level, see also figures 13 and 14).
- a substance according to d) identifiable with said methods may be a substance capable of reducing or eliminating the formation of complexes between presenilin fragments.
- PS fragments which may be the biologically active form of the presenilins and subsequently preventing the deposition of A ⁇
- the suppression of the generation of said PS fragments which may be the biologically active form of the presenilins and subsequently preventing the deposition of A ⁇ may be achieved by reducing or eliminating the activity of the presenilinase. Reducing or eliminating the activity of the presenilinase should go along with a concomitant increase in the full-length protein, a decrease in the biologically active fragments and thus may lead to the reduction of A ⁇ and/or also may prevent apoptotic cell death.
- the invention is concerned with a substance as described, wherein the substance is capable of reducing the amount of presenilin fragments and preventing neuronal cell death and/or capable of reducing the deposition of A ⁇ and/or reducing the formation of amyloid plaques.
- the invention is concerned with a substance identifiable with said methods, wherein the presenilinase is specific for presenilin 1.
- the invention is concerned with a substance identifiable with said methods, wherein the presenilinase is specific for presenilin 2.
- the invention is concerned with a substance as described, wherein the substance reduces or eliminates the autoproteolytical cleavage of the presenilin. It was surprisingly found that presenilin itself may have autoproteolytic activity. It was further found that presenilin 1 and 2 itself may be capable of cleaving the C-terminal fragments of APP leading to the deposition of A ⁇ and therefore may be a cofactor for the ⁇ -secretase or the ⁇ -secretase itself.
- a substance capable of reducing or eliminating the autoproteolytic cleavage of the presenilin can prevent neuronal cell death due to increased apoptosis because of the presence of presenilin fragments and also reduce the deposition of A ⁇ and subsequently reduce the formation of amyloid plaques.
- the invention is concerned with a substance as described, wherein the substance reduces or eliminates the activity of ⁇ -secretase.
- the invention is concerned with a substance as described, wherein the substance prevents cleavage of presenilin 1 into fragments of regulated cleavage wherein the N-terminal fragment (NTF) is approximately 21-28 kDa in size and the C- terminal fragment (CTF) is approximately 16-24 kDa in size.
- NTF N-terminal fragment
- CTF C- terminal fragment
- the invention is concerned with a substance as described, wherein the substance prevents cleavage of presenilin 2 into fragments of regulated endoproteolytic cleavage wherein the N-terminal fragment (NTF) is approximately 28-30 kDa
- NTF N-terminal fragment
- the invention comprises a substance identifiable with a method as described, wherein the substance is an antisense-oligonucleotide or a ribozyme.
- Antisense oligonucleotides are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. The use of antisense methods to inhibit the in vitro or in vivo (also in the animal model) translation of genes is well known in the art (e.g. Marcus-Sekura, 1988). An antisense core nucleic acid may at least contain 10 nucleotides complementary to the target message.
- Said antisense oligonucleotides also comprise peptide nucleic acids, phosphodiester antisense oligonucleotides and phosphorothioate oligonucleotides (Boado RJ et al, 1998).
- Antisense nucleic acids have been described in the art to inhibit the expression of proteins associated with toxicity or gene products introduced into the cell, such as those introduced by an infectious agent (e. g. a virus). They furthermore are useful to block expression of a mutant protein or a dominantly active gene product such as amyloid precursor protein in AD as described in WO 981881 1 Al . Similarly, the antisense oligonucleotide of the present invention may be used to block the presenilinase or presenilin expression in neurodegenerative diseases or preferably in
- the ribozyme has a central sequence not complementary to the target RNA that is responsible for its catalytic activity (catalytic domain or region (a)), and two flanking sequences essentially complementary to two neighboring sequences of the target RNA (substrate binding domain or hybridization region (b)) so as to allow binding of the ribozyme via base-pairing and thus selective cleavage of the target RNA
- said ribozyme may comprise a catalytic region (a) and at least one hybridization region (b), with the hybridization region (b) essentially being complementary to a region of the mRNA that is transcribed from the presenilinase or presenilin gene
- the ribozyme according to the invention is preferably characterized in that the hybridization region (b) consists of two domains flanking the catalytic region (a) and being essentially complementary to the target nucleic acid region so as to be capable of selectively binding to all mRNAs that are transcribed by the presenilinase or presenilin gene in order to selectively cleave these RNAs (see also figure 1 for a hammerhead ribozyme).
- ribozymes are preferably completely complementary to the target nucleic acid region
- selective cleavage as used in the invention is to be understood such that the expression of the target gene, e g. the presenilinase or presenilin gene is suppressed to such an extent that the desired therapeutical effect is achieved
- ribozyme The selective inhibition of the gene expression in cells by the ribozyme according to the invention does therefore not mean that the target gene will be irreversibly damaged or eliminated Rather, the use of the ribozymes advantageously only leads to the selective inhibition of the translation of said gene
- the property of ribozymes to specifically bind target RNA and to inactivate them by cleavage has been successfully demonstrated several times for the case of specific inhibition of
- HIV-RNA (Lisziewicz et al , 1993, Yu et al 1993, Morgan and Anderson, 1993, Yamada et al ,
- Said ribozyme can also be presented by the following general formula
- N3.20 [CUGANGARNc oSGAAA] [N3.20] 3', wherein N is G, C, A or U, R is a purine, and S is a pyrimidine, and wherein the central region N 0 . 3 0 of sequence (a) can be replaced by a linker which is different from nucleic acid, e.g., a hydrocarbon chain (Thomson et al, 1993).
- a linker which is different from nucleic acid, e.g., a hydrocarbon chain (Thomson et al, 1993).
- the conserved nucleotides within the catalytic region are essential for the catalytic effect but can be optionally modified by the person skilled in the art with the below-mentioned method (Joyce, 1992; Yuan and Altman, 1994) such that ribozyme effectivity and selectivity is favorably influenced.
- the length of the hybridization region (b) (N 3-2 o) depends on many factors and is selected such that a sufficient hybridization to the RNA to be cleaved is achieved under the selected conditions (such as temperature, ion environment) in order to allow efficient cleavage, but, if the difference between the target RNA and non-target RNA does not comprise the cleavage motif per se, there is no sufficient hybridization to the non-target RNA.
- the choice of the length of the hybridization region thus depends on, e.g., the GC content of the RNAs and the number of nucleotides differing between target RNA and non-target RNA.
- the lengths of the 5' hybridization region and the 3' hybridization region are equal, but they can be asymmetrical, e.g., a combination of three and 20 nucleotides.
- the overall length of the hybridization region (b) is 12 to 30 nucleotides.
- the ribozyme according to the invention can be a hammerhead, hairpin or axehead ribozyme.
- the structure of hammerhead ribozyme in general is known to the person skilled in the art and is described in, e.g., Symons (1992), and Rossi (1993). As outlined below, the skilled practitioner may modify the catalytic structure such that it yields optimum results for the projected use in terms of effectivity and substrate specificity.
- Hairpin ribozymes were originally identified to be part of the minus strand of the TRSV (tobacco ringspot virus) satellite RNA. In the meantime, it has been shown that these ribozymes can effectively cleave target RNAs in trans, the mechanism of action being similar to that of the hammerhead ribozymes. The regions being responsible for substrate binding and catalytic effect were determined and the invariable structure or sequence motifs characterized.
- the cleavage motif of the target RNA is N'GNPy (N is G, C, U or A, Py is C or U) (see, e.g., Rossi, 1993, and Hampel et al., 1990).
- ribozyme On the basis of the requirements with respect to the structure and sequence of the hairpin ribozyme necessary for an effective cleavage and with respect to the cleavage motif on the target RNA explained in the art, the skilled practitioner can construct a ribozyme using standard techniques that possesses the desired properties.
- Axehead ribozymes were originally defined to be part of the genomic and antigenomic RNA of the hepatitis delta virus.
- Said ribozyme may also be a fusion-ribozyme comprising a presenilinase-or presenilin-specific ribozyme and an autocatalytical hammerhead-ribozyme fused with its 5' end to the 3' end of the presenilinase-or presenilin-specific ribozyme.
- the ribozyme may be modified such that resistance to nucleases is achieved, increasing the retention time and thus the effectivity of the ribozyme at the target site, e.g., in certain cells of a patient. Furthermore, the amount of ribozyme to be applied and, if any, related side-effects can be reduced.
- the ribozymes according to the invention contain at least one of the above-described phosphate modifications and/or at least one of the above- described ribose modifications.
- the invention pertains to a pharmaceutical composition
- a pharmaceutical composition comprising a substance as described and a pharmaceutically acceptable carrier therefor.
- pharmaceutically acceptable carrier refers to conventional pharmaceutic excipients or additives used in the pharmaceutical manufacturing art (see e.g. Remington's Pharmaceutical Sciences (1980)).
- Said pharmaceutical composition of the present invention may contain a vector comprising the substance of the present invention to be used for gene therapy and may contain a colloidal dispersion system or liposomes for targeted delivery of the pharmaceutical composition.
- Suitable vectors comprise plasmids, viruses (including phage) and integratable DNA fragments (i e , integratable into the host genome by recombination) All forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host
- colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome formulations
- the preferred colloidal system of this invention is a liposome Liposomes are artificial membrane vesicles which are useful as delivery verhicles in vitro and in vivo These formulations may have net cationic, anionic or neutral charge characteristics are useful characteristics with in vitro, in vivo and ex vivo delivery methods.
- RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al , 1981)
- liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells
- the following characteristics should be present (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity, (2) preferential and substantial binding to a target cell in comparison to non- target cells, (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al , 1988).
- composition of the liposome is usually a combination of phosphohpids, particularly high- phase-transition-temperature phosphohpids, usually in combination with steroids, especially cholesterol Other phosphohpids or other lipids may also be used
- the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations
- the pharmaceutical composition of the present invention may contain said vector as a naked "gene expression vector" This means that the construct is not associated with a delivery vehicle (e g liposomes, colloidal particles and the like)
- a delivery vehicle e g liposomes, colloidal particles and the like
- the invention also relates to the use of a substance as described in the manufacture of a medicament for the treatment of neurodegenerative diseases
- the invention relates to the use of a substance as described in the manufacture of a medicament for the treatment of Alzheimer's disease.
- the invention relates to the use of a substance as described in the manufacture of a medicament for the treatment of familiar Alzheimer's disease.
- Neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease, Huntigton's chorea and stroke.
- AD Alzheimer's disease
- Parkinson's disease Parkinson's disease
- Huntigton's chorea Huntigton's chorea and stroke.
- Alzheimer's disease refers to a neurodegenerative disorder marked by the gradual formation of extracellular neuritic plaques in the brain, particularly in the hippocampus and the adjoining cortex. The majority of Alzheimer's disease cases are late in onset lacking an obvious genetic linkage and are characterized as sporadic. The term "familiar Alzheimer's disease
- AD refers to a subgroup of AD comprising a small percentage (approximately 10%) of cases which are earlier in onset and segregate strongly within families suggesting a genetic etiology.
- Example 1 exemplifies a method of reducing or eliminating the presenilinase activity by reducing or eliminating the presenilinase substrate at the RNA level with ribozymes.
- Example 2 illustrates a method for identifying a substance or substances capable of reducing or eliminating the activity of the presenilinase.
- Example 3 illustrates a method for identifying a substance or substances capable of reducing or eliminating the activity of the presenilinase wherein said substance is capable of reducing or eliminating the formation of stable complexes between N-terminal and C-terminal PS- fragments.
- Example 4 demonstrates methods of reducing or eliminating the generation of presenilin fragments and A ⁇ by mutagenizing the substrate of the presenilinase.
- Example 1 Method of reducing or eliminating the presenilinase activity by reducing or eliminating the presenilinase substrate at the RNA level with ribozymes
- ribozymes which cleave the PS2-specific RNA
- said ribozymes reduce or eliminate the presenilinase substrate, the full-length PS2 and therefore reduce or eliminate the presenilinase activity and the occurrence of presenilin fragments which are linked to the pathology of neurodegenerative diseases, preferably AD
- Fusion ribozymes comprising a PS2 specific ribozyme and the autoribozyme: rz 1173/13.3auto 5'-UUCUUUGGCUGAUGAGGCCGUGAGGCCGAAACACAGCGG
- RNA substrate sequences 1173 5'-CGCUGUGUCCCAAAGAA-3', 232 5'-CGACGUGUUAAAAACCA-3', 308 5'-CCAAGGUCCGGGAUUC-3'
- Ribozyme numbering corresponds to the nucleotide position in the PS2 mRNA of the guanidine in the target GUX (indicated in the RNA substrate sequences in bold), after which the phosphodiester bond is cleaved
- the RNA substrates, which represent partial sequences of the PS2 mRNA are named accordingly The number of base pairs formed by hybridization of the substrate binding domain of the ribozyme to the target mRNA is indicated in numbers, wobble base pairs in numbers behind the point (i e rzl 173/13 3)
- Synthetic and in vitro transcribed ribozymes and RNA substrates were strictly handled under RNase free conditions DEPC (diethylpyrocarbonate) water or nuclease free water (Promega, Heidelberg) was used Oligoribonucleotide purification was done either by HPLC (reverse
- the DNA coding for the ribozyme rzl 173/13 3 was cloned into pBluescriptII/SK+ (Stratagene)
- the resulting plasmid pBSK+/PS2-rzl l73 13 3 was transcribed in vitro using T7 polymerase according to manufacturer's instructions (Clontech) and the purity of the ribozyme RNA was controlled by OD260/280 measurement and gel electrophoresis (20 % SDS-PAGE/ 8 M urea)
- the DNA encoding a self-splicing ribozyme was attached directly at the 3' end of rzl 173/13 3 cDNA to generate pBSK+/PS2-rz 1173 13 3 auto (see Figs 4a, b)
- the rzl 173/13 3auto DNA sequence was cloned into the tTA-responsive plasmid pUHD 10-3
- the most probable secondary structure of the PS2 mRNA was determined by the method of Zuker et al (1989) by using the SQUIGGLES software included in the Wisconsin Sequence Analysis Package (Genetic Computer Group Inc ) [32 P] -labeling of substrate and ribozyme RNA
- RNA substrates for the in vitro cleavage reaction we used either short 16-17 base (b), 5'[32p]- labeled synthetic oligoribonucleotides or a 367 b long RNA substrate that was radioactively labeled upon in vitro transcription.
- the phosphorylation reaction was carried out in a total of 20 ⁇ l containing 20 pmol synthetic substrate RNA, 3 ⁇ l (lO ⁇ Ci/ ⁇ l) [ ⁇ - 32 P]-ATP, 2 ⁇ l lOx phosphorylation buffer, 13 ⁇ l H2O and 10 U polynucleotide kinase (Boehringer Mannheim) by incubation at 37°C for 1 hour.
- the plasmid pBSK+/PS2.NcoI (Fig. 4b) was linearized with Xhol, phenol chloroform-extracted, and ethanol-precipitated.
- In vitro transcription was carried out in 20 ⁇ l of a mixture containing l ⁇ l 10 mM GTP, l ⁇ l 10 mM ATP, l ⁇ l 10 mM UTP, 2 ⁇ l 10 x transcription buffer, 1 ⁇ l 0.2 M DTT, 1 ⁇ l RNase inhibitor (20 U), 5 ⁇ l - 32 P-CTP (10 mCi/ml), 1 ⁇ l 0.1 mM CTP, 5 ⁇ l H 2 O, 1 ⁇ l (10 U) T7 RNA Polymerase (in vitro transcription kit, Clontech).
- [ 3 2p]_iabeled substrate RNA (20.000cpm/reaction) and ribozyme RNA were incubated in 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl 2 for 5 min at 95°C followed by a 60 min incubation at 37°C. Reactions were stopped by addition of formamide gel-loading buffer (80 % formamide, 10 mM EDTA, pH 8.0, 0,002 % bromphenol blue and xylene cyanol). Substrates and cleavage product(s) were separated by electrophoresis on a 20 % SDS-polyacrylamide/ 6 M urea denaturing gel and detected by autoradiography (X-OMAT AR films, Kodak).
- formamide gel-loading buffer 80 % formamide, 10 mM EDTA, pH 8.0, 0,002 % bromphenol blue and xylene cyanol
- DMEM Eagle's medium
- the HtTa cell line stably stably transfected with the pUHD 15-1/neo DNA plasmid encoding the tetracycline-sensitive transactivator (tTA) of the "Tet-off' expression system (Gossen, M and Bujard, H 1992)
- the HtTa cell line was transfected with the plasmids pUHD10-3/PS2 wt (wildtype PS2), pUHD10-3/PS2 mut (mutant (N141V) PS2) or pUHDlO- 3/PS2-rzl l73 13.3auto (ribozyme rzl 173/13 3) to give rise to the double stable cell lines HtTA/PS2-wt 13, HtTA/PS2-mut 5, and HtTA/PS2-rzl 173 40, respectively DNA transfection Stable DNA transfections were performed by the calcium-phosphate precipitation method with 50 ⁇ g of purified DNA (Qiagen, Hilden) and 5 ⁇
- Cells were grown in 6 cm ⁇ -dishes to confluence. Cell lysis were carried out in a buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.6, 2 mM EDTA, 0,2 % (v/v) NP40, 1 mM PMSF, and 5 ⁇ g/ml Leupeptine. Triton X-100 and Nonidet P-40 were added to a final concentration of 1 %. 30 ⁇ g of protein extract was loaded onto a 10-12 % SDS-PAGE and electrophoresed.
- RNA isolation was carried out as described by the manufacturer's instructions (Boehringer Mannheim). Cells were grown in 75 cm ⁇ -culture flasks and washed twice with ice cold phosphate-buffered saline (PBS) (1,7 M KH 2 PO 4 , 5 mM Na 2 HPO 4 , 0,15 M NaCl, pH 7,4). Cells were trypsinized, pelleted by centrifugation, and lysed in 3 ml lysis buffer (0.1 M Tris-HCl, pH7.5, 0.3 M LiCl, 10 mM EDTA, 1 % lithium dodecylsulfate, 5 mM DTT).
- PBS ice cold phosphate-buffered saline
- the DNA was mechanically sheared by passing the extracts six times through a 21 gauge needle.
- 1.5 ml of a biotin-labeled oligo (dT) 2 rj probe was added and mixed with pre- washed 150 ⁇ l streptavidine magnetic particles.
- the poly (A+)-selected mRMA was eluated with 25 ⁇ l H 2 O and its concentration measured.
- RNAse protection assay RPA
- RPA RNAse protection assay
- Radioactively labeled antisense RNA probes (5x10 ⁇ cpm) were coprecipitated with 1 ⁇ g isolated mRNA in 30 ⁇ l hybridization buffer (40 mM Pipes (1,4-Piperazindiethane-sulfoneacid), 400 mM NaCl, 1 mM EDTA, 80 % formamid, pH 6.4) and incubated at 45°C overnight. The same amount of mRNA was incubated with 1 ⁇ l yeast tRNA (5 ⁇ g/ ⁇ l) as control reaction.
- RNA hybrid- fragments were extracted by phenol/chloroform/isoamyl-alcohol (25:24: 1). After ethanol precipitation the fragments were resolved on 5 % SDS-polyacrylamide/8 M urea gels and exposed overnight at -80°C to X-Omat AR films (Kodak). Induction and analysis of apoptosis
- Apoptosis was induced in HeLa cells that were 80 % confluent Staurosporine was added at different concentrations for various periods After incubation, cells were tested for viability and apoptotic parameters
- the hammerhead ribozymes consist of two domains, the substrate binding domain (I e the hybridization region (b)) and the catalytic domain or region (a) (Fig la) (Haseloff and Gerlach,
- the PS2 mRNA was searched for potential GUX consensus sites (Fig lb) Several factors were taken into consideration when designing the most suitable PS2-cleaving ribozymes (i) the accessibility of the mRNA target sites for the ribozyme, (ii) the strength of the ribozyme-target
- the optimum length of the substrate binding domain has been reported to be in the order of 12-16 nucleotides.
- ribozymes targeted to the same site in the PS2 mRNA, but with flanking regions of various lengths (Fig. lc).
- a so-called 'antisense ribozyme' i.e. rz232/as-15.1 that comprises the exact sequence of the respective ribozyme, but carries mutations of conserved bases in the catalytic domain, thus disabling this ribozyme to cleave its target RNA.
- RNA substrate containing the GUC (rz308, rzl 173) or the GUU (rz232) trinucleotide was targeted by appropriate ribozymes varying in the length of the flanking substrate binding domain (Fig. la, c, 2a; length in bases indicated in numbers, i.e 13.3, 12, 9).
- ribozyme rzl 173 very effectively cleaved the PS2 mRNA in the coding region, we selected this ribozyme for the PS2 'knock-down' in cultured cells and investigated the optimal length of its substrate binding domain in more detail (described supra under general methods). Whereas rzl 173/13.3 (substrate binding domain: 13 bases and 3 bases forming wobble base pairs with the target mRNA) produced significant amounts of the expected cleavage product within 1 h at a molar ribozyme: target ratio of 1: 1, much higher molar ratios were required when the binding domain was shortened to 9 bases (Fig. 3a).
- In vitro transcribed ribozyme rzl 173/13.3 also cleaves longer substrate RNAs
- ribozyme rzl 173 also cleaves longer substrate RNA molecules that might already adopt a secondary structure
- we in vitro transcribed plasmid pBSK+/PS2.NcoI into a 367 b RNA and studied the cleavage of this substrate RNA by the in vitro transcribed ribozyme rzl 173/13.3auto (Fig. 5).
- the autocatalytic ribozyme should be able to splice itself out of the initial transcript to generate ribozyme rzl 173 with a defined 3' end.
- Apoptosis sensitivity is decreased in PS 2 'knock-down' cells and increased in cells overexpressing wildtype or mutant PS2
- the level of induced PS2 expression was determined by immunocytochemistry (Fig. 7a) and biochemical analyses (data not shown). Various methods were used to assess apoptosis, including fluorescence microscopy, cell death detection ELISA, caspase 3 activation, and proteolytic cleavage of PARP and PS2. For determination of cell viability, Alamar Blue reduction and LDH release was measured.
- Apoptosis was induced by increasing concentrations of staurosporine.
- Cells were incubated with an ethidiumbromide/ acridine orange mixture that staines living cells green.
- Apoptotic cells showed a characteristic chromatin condensation, nuclear fragmentation and the generation of apoptotic bodies, and their chromatin was stained orange upon ethidiumbromide intercalation (Fig. 7b).
- no apoptotic cells could be detected in the PS2 'knock-down' cells (PS2 k.d.) and in the wildtype (PS2 wt) or mutant PS2 (PS2 mut) overexpressing cells (Fig. 7b, lane K).
- the sensitivity of HeLa cells to the apoptotic stimulus, staurosporine is dependent on the PS2 expression level.
- the N141V PS2 mutation caused an earlier onset, rather than an increase in the extent of cell death.
- the PS2 'knock-down' resulted in a significant reduction of apoptosis sensitivity. Similar data were obtained when cells were stained with Hoechst 33258 to visualize apoptotic cells (data not shown).
- the PS2 expression level does not affect the kinetics of caspase 3 activation and PARP cleavage
- PS2 'knock-down' or overexpression changes the kinetics of processes characteristic of the execution phase of apoptosis
- PARP poly(ADP)ribose polymerase
- the caspase 3, or CPP32 is activated by cleavage into two proteolytic fragments (17 and 10 kDa in size).
- the antibody used for immunoprecipitation of caspase 3 recognizes the uncleaved CPP32-holoenzyme and the 17 kDa fragment, but not the 10 kDa C-terminal fragment (example shown in Fig. 9a). No difference in caspase 3 activation following induction of apoptosis could be detected between PS2 wt, PS2 mut and PS2 k.d. cells (Fig. 9b).
- PARP constitutes one downstream target of activated caspase 3 and is cleaved into two proteolytic fragments, 85 and 27 kDa in size (Kim et al., Science 1997, 277: 373-376). Therefore, PARP is quite often used as marker for apoptosis (example shown in Fig. 10a).
- Fig. 10a The kinetics of PARP cleavage in the three HeLa cell lines. Again, there was no significant difference in the time-course of the appearance of the proteolytic PARP fragments (Fig. 10).
- Example 2 Method for identifying substances capable of reducing or eliminating the activity of the presenilinase
- Example 2 is schematically depicted in figure 15.
- the cell line used for this test is selected in a way that it is expressing the fusion-protein comprising PS1 and luciferase or PS2 and luciferase at a level that only little or no full-length protein is detected (i.e. slightly overexpressing said fusion-protein) which would otherwise increase the background of the test.
- This can be done by either selecting an appropriate cell clone or, alternatively, by applying the inducible expression systems, e.g.
- tTA-response plasmid pUHD 10-3 (Gossen and Bujard, 1992), with which it is possible to vary the expression level of the exogenous protein, as the presenilin-luciferase fusion-protein, by using a specific doxycyclin (tetracyclin) concentration in the culture medium.
- tetracyclin doxycyclin
- the C-terminal PS-fragment and a fragment consisting of the PS-N-terminus and luciferase are detectable. Normally, these fragments are bound to each other or interact with each other in a stable complex, but they can be separated from each other by using either 0.5% SDS or 1% Triton in the extraction buffer (Capell et al., 1998).
- This full-length fusion-protein can also bind via its C-terminus to the coated plate and can, due to the presence of luciferase, give a signal after addition of the luciferase substrate.
- a substance capable of reducing or eliminating the activity of the presenilinase reduces or inhibits the cleavage of the presenilin- luciferase fusion-protein and thus causes an increase of the full-length fusion-protein which binds to the coated microtiter plate via antigen-antibody interactions and causes a signal or a signal increase, respectively.
- the DNA encoding the reporter luciferase is fused to the N-terminus of the DNA encoding the full-length PS in one of the following ways:
- fusion nucleic acid constructs are prepared according to standard procedures for cloning and sub-cloning (e.g.
- H4/Luc-PS-l H4/Luc-PS-2
- Said cell lines are plated onto 96-well microtiter plates
- the stably transfected cells are grown to confluence and incubated with the test substances for 8-
- Microtiter plates are pre-coated with the monoclonal antibodies specific for the C-terminus of
- PSl loop region
- PS2 loop region
- BI.HF5C PS2 (loop region) termed BI.HF5C, respectively (Steiner et al, 1999; the following antibodies may be used instead: SI 82 (C-20), cat # scl244 and STM2 (C-
- the bound material is tested for luciferase activity according to the protocol provided by
- test substance i e a substance according to the present invention
- the test substance i e a substance according to the present invention
- the luciferase fused to the N-terminus is bound to the microtiter plates and gives rise to a signal due to the presence of full-length presenilin protein
- the presenilinase-luciferase fusion-protein is cleaved by the endogenous presenilinase, the C-terminus is bound to the microtiter plates via antibody-antigen interaction and the luciferase fused to the N-terminus is washed away during the washing steps. In this case, no signal is detected
- wash (the unbound material) is tested for luciferase activity as a control according to the protocol provided by Boehringer Mannheim GmbH, Mannheim, Germany
- Example 3 Method for identifying substances capable of reducing or eliminating the formation of stable complexes between N-terminal and C-terminal PS-fragments
- This example illustrates a method for identifying a substance or substances capable of reducing or eliminating the activity of the presenilinase wherein said substance is capable of reducing or eliminating the formation of stable complexes between N-terminal and C-terminal PS-fragments.
- the assay is essentially carried out as disclosed in example 2, however, the following modifications are made'
- the stably transfected cells as disclosed in example 2 are grown to confluence.
- the cells are washed with phosphate-buffered saline (PBS) and then protein extracts are made from said cells Since the interactions between N-terminal and C-terminal fragments are SDS- and Triton-labile, CHAPS extracts are made according to a published protocol (Capell et al , 1998). This extraction method does not disturb complex generation
- Microtiter plates are pre-coated with the monoclonal antibodies specific for the C-terminus of PSl (loop region) termed BI 3D7 or PS2 (loop region) termed BI.HF5C, respectively (Steiner et al., 1999; the following antibodies may be used instead S182 (C-20), cat # scl244 and STM2 (C- 20), cat # scl456, both Santa Cruz Biotechnology, Inc , Santa Cruz, California/ USA) Said protein extracts are added to the pre-coated microtiter plates and incubated according to standard procedures (Immunochemistry 1, A Practical Approach, A P Johnstone and M W Turner, loc.
- the luciferase fused to the N- terminus is washed away leading to a reduction or inhibition of the luciferase activity and thus a smaller signal is measured.
- the wash is preserved and also tested for luciferase activity as a control according to the protocol provided by Boehringer Mannheim GmbH, Mannheim, Germany.
- Example 4 Methods of reducing or eliminating the generation of presenilin fragments and thereby A ⁇
- This example exemplifies methods for the reduction or elimination of presenilinase activity at the DNA-level by mutagenizing the PS gene and thus modifying the substrate of the presenilinase.
- the mutated PS gene leads to the reduction of PS fragments and also A ⁇ which are linked to the pathology of neurodegenerative diseases, preferably AD.
- AD Alzheimer's disease
- PSl Presenilin-1
- a deletion of the PS2 gene can not be used to investigate the normal function of PS2 in amyloidogenesis, since endogenous PSl expression will abolish effects on ⁇ APP processing. Indeed, a gene knock out of PS2 causes no dramatic phenotype (Boeve et al, 1998), whereas a knock out of PSl leads to embryonic lethality (Shen et al, 1997; Wong et al, 1997). In order to investigate a functional role of PS2 in A ⁇ generation, we now generated a dominant negative mutation, which not only inhibits the function of overexpressed PS2, but also blocks accumulation of endogenous presenilins.
- CTF C-terminal fragment
- the mutation inhibits endoproteolytic processing of PS2, and simultaneously reduces accumulation of both, endogenous PSl and PS2 fragments.
- HEK293 cells stably expressing PS2 Asp366Ala were generated by transfection of the previously described cell line expressing the Swedish mutation (Steiner et al, 1999). Mutagenesis.
- the cDNA encoding PS2 Asp366Ala was constructed according to a previously described protocol (Steiner et al, 1999).
- the mutant cDNA was cloned into the pcDNA3.1/Zeio(-) expression vector (Invitrogen) and sequenced to verify successful mutagenesis.
- Antibodies The polyclonal and monoclonal antibodies against amino acids 263-407 of PSl (3027, BI.3D7) and amino acids 297-356 of PS2 (3711, BI.HF5C) were described previously (Steiner et al, 1999). Antibody 3926 to synthetic A ⁇ (Leimer et al, submitted), or antibody 6E10 (product 300-10, Senentek Pic.) specific for amino acids 1-16 of A ⁇ , or antibodies specific for A ⁇ 40 or A ⁇ 42 (catalogue nos.
- Presenilins genes for life and death. Neuron, 18, 687-690.
- RNA enzymes distinguishing a single base mutation in RNA. Nucleic Acids Res 17, 7059-7071. Koizumi M, and Ohtsuka E (1992). In: Murray J.A.H. (Ed.), Antisense RNA and DNA, Wiley- Liss, New York, 373-381.
- Presenilins are processed by caspase-type proteases. J Biol Chem 272, 20655-20659.
- PS-1 presenilin- 1
- Presenilin proteins undergo heterogenous endoproteolysis between Thr291 and Ala299 and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue. Neurobiol Dis 3, 325-337.
- Alzheimer's disease-associated presenilin- 1 is controlled by proteolytic degradation and complex formation. J Biol Chem 273, 32322-32331.
- Firefly luciferase gene 20 structure and expression in mammalian cells. Mol Cell Biol 7, 725-737.
- Presenilin-1 is associated with Alzheimer's disease amyloid. Am J Pathol 151, 601-610.
Abstract
Description
Claims
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AU50340/99A AU5034099A (en) | 1998-07-09 | 1999-07-08 | Method for identifying a presenilinase inhibitor |
CA002332344A CA2332344A1 (en) | 1998-07-09 | 1999-07-08 | Method for identifying a presenilinase inhibitor |
EP99934634A EP1095279A1 (en) | 1998-07-09 | 1999-07-08 | Method for identifying a presenilinase inhibitor |
JP2000559432A JP2002520031A (en) | 1998-07-09 | 1999-07-08 | Method for identifying presenilinase inhibitor |
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WO2001049871A2 (en) * | 2000-01-06 | 2001-07-12 | Boehringer Ingelheim Pharma Kg | Process for finding a protease inhibitor |
WO2001075435A2 (en) * | 2000-04-03 | 2001-10-11 | Bristol-Myers Squibb Company | Fluorescence assay for gamma-secretase activity and inhibitors |
US20210108186A1 (en) * | 2018-05-22 | 2021-04-15 | Raymond J. Kelleher, III | Gene therapy for alzheimer`s disease |
Citations (3)
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WO1997027296A1 (en) * | 1996-01-26 | 1997-07-31 | Hsc Research And Development Limited Partnership | Nucleic acids and proteins related to alzheimer's disease, and uses therefor |
WO1997041443A2 (en) * | 1996-04-26 | 1997-11-06 | Smithkline Beecham Plc | Method for screening compounds |
WO1998047917A2 (en) * | 1997-04-24 | 1998-10-29 | The General Hospital Corporation | A PURIFIED 20 kDa PRESENILIN 2 C-TERMINAL FRAGMENT AND METHODS OF SCREENING FOR COMPOUNDS THAT INHIBIT PROTEOLYSIS OF PRESENILIN 2 |
-
1999
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- 1999-07-08 EP EP99934634A patent/EP1095279A1/en not_active Withdrawn
- 1999-07-08 JP JP2000559432A patent/JP2002520031A/en active Pending
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WO1997027296A1 (en) * | 1996-01-26 | 1997-07-31 | Hsc Research And Development Limited Partnership | Nucleic acids and proteins related to alzheimer's disease, and uses therefor |
WO1997041443A2 (en) * | 1996-04-26 | 1997-11-06 | Smithkline Beecham Plc | Method for screening compounds |
WO1998047917A2 (en) * | 1997-04-24 | 1998-10-29 | The General Hospital Corporation | A PURIFIED 20 kDa PRESENILIN 2 C-TERMINAL FRAGMENT AND METHODS OF SCREENING FOR COMPOUNDS THAT INHIBIT PROTEOLYSIS OF PRESENILIN 2 |
Non-Patent Citations (1)
Title |
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A. TAKASHIMA: "Biochemistry of presenilin 1", CLINICAL NEUROLOGY, vol. 37, no. 12, 1 December 1997 (1997-12-01), Tokyo JP, pages 1097 - 1098, XP002086912 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001049871A2 (en) * | 2000-01-06 | 2001-07-12 | Boehringer Ingelheim Pharma Kg | Process for finding a protease inhibitor |
WO2001049871A3 (en) * | 2000-01-06 | 2002-03-28 | Boehringer Ingelheim Pharma | Process for finding a protease inhibitor |
WO2001075435A2 (en) * | 2000-04-03 | 2001-10-11 | Bristol-Myers Squibb Company | Fluorescence assay for gamma-secretase activity and inhibitors |
WO2001075435A3 (en) * | 2000-04-03 | 2002-08-08 | Bristol Myers Squibb Co | Fluorescence assay for gamma-secretase activity and inhibitors |
US6713248B2 (en) * | 2000-04-03 | 2004-03-30 | Bristol-Myers Squibb Company | Methods for detection of gamma-secretase activity and identification of inhibitors thereof |
US20210108186A1 (en) * | 2018-05-22 | 2021-04-15 | Raymond J. Kelleher, III | Gene therapy for alzheimer`s disease |
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