WO 00/03004 PCT/EP99/04$04 Presenilin 2 specific rib~ozyme Technical Field of the invention s The present invention belongs to the field of presenilins and neurodegenerative diseases. The invention relates to a substance capable of inhibiting presenilin 2 expression in neurodegenerative diseases and in Alzheimer's disease. The invention is furthermore concerned with ribozymes capable of cleaving presenilin Z-specific RNA. Additionally, the invention pertains to recombinant vectors comprising specified ribozyme sequences andl microorganisms comprising such ~o recombinant vectors.
Background Art Neurodegenerative diseases are characterized by neuronal and synaptic cell loss. Neuronal cell ~s loss is caused at least in part by apoptotic cell death. Ne:urodegenerative diseases include the chronic forms as Alzheimer's disease (AD), Parkinson's disease, Huntington's chorea and the acute form 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 IO%) of cases belonging to the subgroup of familiar Alzheimer's disease (FAD) ?o are earlier in onset and segregate strongly within families sul;gesting a genetic etiology.
AD is a neurodegenerative disorder marked by the gradu~,al formation of extracellular neuritis plaques in the brain, particularly in the hippocampus and the adjoining cortex. In AD research, one small peptide has long claimed a large share of attention. Known as ~i amyloid (A~i), it is the major constituent of the abnormal structures called 'amyloiid plaques' that stud the brain of AD
Zs patients. Mutations in the gene that encodes the amyloid precursor protein (APP), which is cleaved by two secretases (j3- and y-secretase) to release A~3, account for some inherited cases of the disease (Chartier-Harlin et al., 1991; Goate et al., 1991:; Murreil et aL, 1991; Hendriks et al., I992; Mullan et al., 1992).
The discovery in 1995 that a new gene family - the presenilins - is responsible for the majority of 3o early-onset autosomal dominant cases of familial AD has led to the expectation that a fundamental understanding of the disease mechanism may not be far off' (Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1995). It has been shown, botlh in vivo, in fibroblasts and plasma of FAD patients as well as in transgenic animals and cell lines that the presenilin (PS) mutations cause a specific increase in the production of extracellular A(342, the long form of A~i ending at residue 42 (Borchelt et al., 1996, 1997; Duff et al., 1996; Scheuner et al., 1996). A(342 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(3 ending at residue 40, termed A~340 (Jarret et al., 1993; Mann et al., 1996).
Several reports described the proapoptotic behaviour of PS based on data observed in cells transiently or stably overexpressing PS (Deng et al., 1996; 'Vito et al., 1996; Wolozin et al., 1996;
Janicki et al., 1997; Kim et al., Science 1997, 277: 373-376; Loetscher et al., 1997).
ro Overexpression of PS2 increases the susceptibility of neurons to apoptotic stimuli and thus lead to neuronal death (Kim et al., Science 1997, 277: 373-376).
The presenilins undergo regulated proteolytic cleavage into the normal N-terminal (NTF) and C-terminal fragments (CTF), 22-30 kDa and 18-26 kDa, respectively, in size (Thinakaran et al., 1996; Kim et al., J Biol Chem 1997, 272, 11006-11010; P~odlisny et al., 1997).
Furthermore, the ~s PS proteins constitute substrates of a member of the caspa,se 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). C)ne of these alternative fragments is CTF 16.
If these normal or alternative PS fragments played an active role in the apoptotic process, then one would expect that, by inhibiting the generation of these fragments, apoptosis would be largely reduced. It could be demonstrated that inhibition of caspase 3 activation using specific peptide inhibitors leads to both, an inhibition of the formation of CTF16 and apoptotic cell death (Kim et al., Science 1997, 277: 373-376; Loetscher et al., 1997).
Currently there exists only symptomatic treatment of neurodegenerative diseases and in particular of AD. However, there is no disease-modifying treatment to cope with the pathology of said diseases. At present, there is no therapeutic way of preventing the neuronal cell death due to apoptosis. As described supra, PS are involved in apoptosis and thus also a cause for neuronal cell death.
The problem underlying the present invention therefore is 1:o provide agents to decrease neuronal 3o cell death due to apoptosis which thus can be used to treat neurodegenerative diseases, in particular AD.
Summary of the invention The above-captioned technical problem is solved by the embodiments characterized in the claims.
It was surprisingly found that the expression of PS2 whnch renders cells more vulnerable to apoptotic neuronal cell death in neurodegenerative diseases and in particular in AD can be selectively reduced or eliminated by using substances of the present invention which are capable of inhibiting preseniiin 2 expression. According to the present invention, these substances are in particular ribozymes capable of cleaving presenilin 2-specific RNA.
Preferably, said ribozymes are fusion ribozymes comprising a presenilin 2-specific ribozynne and an autocatalytical hammerhead ,o ribozyme. Furthermore, it is an object of the present invention to provide recombinant DNA
molecules coding for said ribozymes, a recombinant vector' comprising the cDNA
corresponding to said ribozymes and a host cell comprising said recombinant vector.
Additionally, this invention pertains to pharmaceutical compositions comprising said substances or said ribozymes or said recombinant vector and a pharmaceutically acceptable Garner. Advantageously, said substances or ,s said ribozymes or said recombinant vector can be used for the treatment of neurodegenerative diseases and preferably for the treatment of Alzheimer's disease. Said treatments are also embraced by this invention.
Brief description of the figures ar Figure 1 a) Sequence and structure of a general hammerhead ribozyme-target RNA complex.
The hammerhead ribozyme contains two domains, the substrate binding domain through which it recognizes and binds its target RNA via base pairing (marked by asterisks), and its catalytic =5 domain that possesses the catalytic activity to cleave its target RNA at the 3'end of the trinucleotide GUX [X=C, A, U].
b) Secondary structure of PS2 mRNA. The secondary structure of a part of the PS2 mRNA, starting with nucleotide 1 in the 5' untranslated region and ending at position nt 1236 in the translated region, was predicted by "mfold" (described infira in Example I ).
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 arrovrs. The numbering of the nucleotides corresponds to the sequence of human PS2 in the EMBL Data Bank, Accession No.
L43964. The 4 ~ PCT/EP99104804 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).
c) Location of ribozymes and the corresponding substrate RNAs. Three different trinucleotides were chosen and the appropriate synthetic ribozymes designed for irr vitro ribozyme s cleavage studies and the exogenous use in cell cutture experiments. A
ribozyme targeted to a trinucleotide was designed with flanking substrate binding domains of various lengths, i.e. 5 ribozymes (rz1173/13.3, rz1173112, etc.) targeted to the GUCI173 trinucleotide (nucleotide numbering according to EMBL Data Bank, Accession 1'10. L43964). As substrate RNAs (target sites) for in viwo cleavage studies we routinely used short synthetic, 5' [32PJ-labeled {indicated by m asterisks), RNAs (shown as black bars with an arrow directing to the right site indicating sense RNA). In addition, as target sits GUCI I73 we generated a larger substrate RNA
(367 bp), iu vitro ttar~smibed from piasmid pBSK+lPS2.NcoI with T7 poiymerase (Fig. 4b) and [32P-CTP]-labeled (indicated by asterisks). The only ribozyme we used endogenously for further functional analyses was ribozyme rz1173 (indicated with "+"). In order to screen positive rz1173 fs overexpressing cell clones for PS2 mRNA levels, we used 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) as RNA probe for the RNase protection assay. This probe was generated by ira vitro transcription from plasmid pBSK+/PS2.NcoI with 'T3 palymerase {Fig. 4b) and [32P-CTP]-labeling.
zo Figure Z
In vitro cleavage studies of different synthetic ribozymes targeted to various regions in the PS2 mRNA.
a) Three synthetic ribozymes (rz1173, rz232, rz308, nucleotide numbering according to EMBL
Zs Data Bank, Accession No. L43964) were targeted to various regions in the PS2 mRN A and analyzed for their cleavage capacity in vitro with synthetic, 5' [32PJ-labeled RNA substrates containing the specific target trinucleotide. Each ribozyrne was used with different lengths of the flanking substrate binding region (i.e. rz1173i13.3, 12, 9, etc.). For rz1173 and rz232 so-called 'antisense ribozvmes', as-12 and as-15.1, respectively, were generated (described infra in Example 30 1 ). The ribozyme cleavage reaction was carried out under standard conditions and ribozymeaarget molar ratios were used as indicated. As controls substrate RNAs were used without riboz~~me 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).
b) Three different ribozymes, rz1173, rz232 and rz30E~, with a substrate binding domain in between I S-16 b were used for further detailed analyses concerning the required ribozymeaarget molar ratio for efficient cleavage in vitro. The ribozyme cleavage reaction was carried out under standard conditions (described infra in Example 1 ) with ribozymeaarget molar ratios as indicated.
s As controls substrate RNAs were used without ribozym~e treatment {lane "-").
Reactions were stopped and loaded onto a 20 % SDS-poiyacryiamide/ 6 M urea gel, dried onto filter paper and exposed on X-Omat AR films (Kodak).
Figure 3 a) In vitro efficiency of ribozyme ~z1I73 with substrate binding domains varying in length at different ribozymeaarget molar ratios. Ribozyme rz;1173 was used with flanking substrate binding domains in between 9-16 b in length (rz1173/13.3, I2, 9). The corresponding synthetic RNA substrate was S' [32P)-labeled. The ribozyme cleavage reaction was carried out under standard conditions (described infra in Example 1) with ribozymeaarget molar ratios as indicated.
if 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). Cleavage efficiencies were calculated with the Phospor Imaging System (BioRad).
b) Kinetics of the in vitro ribozyme cleavage of synthetic iz1173/13.3. The ribozyme cleavage reaction was carried out under standard conditions (described infra in Example 1) with a concentration of ribozymeaarget RNA of 100:1. As RNA substrate a 5' [32P]-labeled synthetic RNA was used containing the target trinucleotide GUCI 1'13 ~iquots were taken at the indicated time points and loaded onto a 20 % SDS-polyacrylamide/ 6 M urea gel. The cleavage kinetic of ribozyme rz1173/13.3 is shown in the upper figure. Ptibozyme cleavage in percentage was calculated with the Phosphor Imaging System (BioRad) anal is shown in the lower figure.
:s Figure 4 a) Sequence and structure of the ribozyme rzI173/13.3auto - PS2 mRNA complex.
The binding of hammerhead ribozyme rzl I73/13.3auto to a specific sequence of the PS2 mRNA is shown (nucleotide numbering according to EMBL Data. Bank, Accession No.
L43964). Base 3o pairing between the flanking regions of the substrate binding domain of rz1173 and the surrounding nucleotides of GUC1173 in the PS2 mRNA, is indicated by asterisks, the Wobble base pair "G-U" is marked by points. The ribozyme rzI173/13.3auto 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 S' end to the 3' end ofthe PS2-specific ribozyme rz1173113.3.
b) Vector constructs for in vitro and in vivo expression of rz1173/13.3 and the corresponding substrates. In vitro transcription from the plasmid pBSK+/pS2 rz1173.13.3 s yielded the biosynthetic ribozyme rz1173 that was then tested for in vitro cleavage activity in comparison to the synthetic ribozyme. Plasmid pBSK+/PS2 rz1173.13.3auto encodes the ribozyme construct box that was finally cloned into the :response plasmid (pLlHD i0-3) of the tetracycline-sensitive gene expression system (H. Bujard, Heidelberg) used for the PS2 'knock-down' in HeLa cells. This ribozyme construct box contained the PS2 speci~~c ribozyme m rz1173/13.3 and an autocatalytic ribozyme (see a). Tr<mscription from the T3 promotor of construct pBSK+/PS2.NcoI generated the sense PS2lNcol fragment containing the ribozyme rz1173 target sequence of PS2 that was used in irr vitro ribozyme cleavage reactions.
Transcription from the T7 promotor yielded the antisens~~ PS2/Ncol fragment that was used as probe in the RNaseI protection assay for quantification of PS2 mRNA levels in rz1173113.3 expressing HeLa cell clones.
Figure 5 In vitro transcribed ribozyme rz1173/13.3 cleaved 367 b long PS2 transcript.
The in vitro transcribed ribozyme rz1173 was incubated in increasing amounts (0,1; 0,3;
0,5; 1; 3; 5 ~tl of the :o total in vitro transcription reaction) together with the 367 b long, (32P]-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.
.s Figure 6 a) PS2 mRNA levels of various cell clones inducibly expressing rz1173/13.3. 49 clones that were stably transfected with the construct pLJHD 10-3/PSZ-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 ja 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 HtTAlPS2 rzl 173.40 was selected for further detailed analyses on the protein level.
b) PS2 protein levels in the selected cell line HtTA/PS2 rL1173.40. Extracts were made from the PS2 'knock-down' cell line at different time points after omission of doxycycline. For comparison with the endogenous PS2 protein level in H~eLa cells, 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/ palyacrylamide gels, blotted onto PVDF membranes and hybridized with the monoclonal antibody BLHFSC (1:2000 dilution). Both antibodies recognize the hydrophilic loop of PS2.
Figure 7 ro 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.
:o Figure 8 The PS2'knock-down' caused an inhibition of apoptosis.
The three transfected HeLa cell lines (the PS2 'knock-dawn' 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 Z 8 h under standard conditions z5 (described infra in Example 1 }. As a control, cells were not treated with staurosporine (lane "0").
(a) Apoptosis sensitivity. Analyses were carried out using a cell death detection ELISA
(Boehringer Mannheim; described infra in Example 1). 7~he degree of apoptosis was expressed directly as the absorbance at 405-490 nm. (b) Alamar blue reduction assay.
Cell viability was measured using the Alamar Blue reduction (see Example 1) and given in percentage of the 3o control. (c) LDH release. LDH release was determined (Etoehringer Mannheim;
described infra in Example 1 ) and given as optical densities.
Figure 9 The PS2 'knock-down' seemed to have no influence on the caspase 3 {CPP32) activation following an apoptotic stimulus. The three HeLa cell limes (the PS2 'knock-down' cells [PS2 k.d.] and cell lines overexpressing wildtype [PS2 wt] or mutant PS2 [PS2 mut]) were treated with s 1 uM staurasporine under standard conditions (describe;d infra in Example 1). As a control, extracts were made of cells not treated with staurosporine: (lane "c"). (a) Extracts were prepared at the indicated time points and identical protein amounts were loaded onto a 12 % SDS/
polyacrylamide gel. After blotting onto PVDF membranes, hybridization with a CPP32-specific antibody (Transduction Laboratories, 1:500 dilution) was carried out. This antibody recognizes the CPP32 holoenzyme and the 17 kDa active N-terminalf fragment (shown in a as an example) that is generated upon proteolytic cleavage. (b) The CPP32 holoenzyme is indicated by an arrow.
Figure 14 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 uM
staurosporine under standard conditions (described infra in Example 1 ). As a control, extracts were made of cells not treated with staurosporine (lane 'Nc"). (a) Extracts were prepared at the indicated time points and identical protein amounts were loaded onto a 12 %
SDS/
zo polyacrylamide gel. 'After blotting onto PVDF membranes hybridization with a PARP-specific antibody (Boehringer Mannheim, 1:2000 dilution) was carried out. This antibody recognizes the PAIRP holoenzyme and the proteolytic fragments (indicated by arrows). (b) PARP
hoioenzyme and the 85 kDa fragment are marked by arrows.
~s Figure i i Inhibition of apoptosis by PS2 'knock-down' - time-course experiments. The three HeLa cell lines (the 'PS2 knock down' cells [PS2 k.d.] and cell lines overexpressing wildtype [PS2 wt] or mutant PS2 [PS2 mutt) were treated with 1 uM staurosporine under standard conditions. As a control, extracts were made of cells not treated with staurosporine (lane "c").
30 a) No CTFIb could be detected during apoptosis in the PS2 'knock-down' cell line. Extracts were prepared at the indicated time points and identical protein amounts were used for immunoprecipitation with the polyclonal PS2lloop-speciific antibody 3711 After 12 % SDS/
polyacrylamide gel electrophoresis and blotting onto PVDF membranes, hybridization with the monoclonal antibody BLHFSC (1:2000 dilution), that was also raised against the loop region, was carried out.
b) The PS2 'knock-down' showed an inhibitory effect an apoptosis compared with the overexpression of wildtype or mutant PS2. In parallel to extract preparations (a) the cells were s 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.
Figure I2 No CTFI6 generation occured at subtoxic staurosporine concentration after 18 h. HeLa ro cells overexpressing wildtype (PSZ wt) or mutant PS2 (PS:? mut) were treated with staurosporine concentrations as indicated under standard conditions. As a control, cells were not treated with staurosporine (lane "c"). Extracts were prepared after 18 h and identical protein amounts were used for immunoprecipitation with the polyclonal PS2/Ioop-specific antibody 3711. After 12 SDS/ polyacrylamide gel electrophoresis and blotting onto PVDF membranes, hybridization with rs the monoclonal antibody BLHFSC (1:2000 dilution), that v~ras also raised against the loop region, was corned out.
Detailed description of the invention :o The invention pertains to a substance capable of inhibiting preseniiin 2 expression in neurodegenerative diseases. Neurodegenerative diseases. include, but are not limited to, Alzheimer's disease (AD}, Parkinson's disease, Huntington's chorea and stroke.
The gene encoding the transcript of presenilin 2 which maps to human chromosome 1 as well as the gene product presenilin 2 are known in the art. As used herein, the term "presenilin 2 gene" or a "PSZ 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 A1 (all herein incorporated by reference) including any allelic variant and heterospecific mammalian homologues. Additional human splice variants as described in WO 96/34099 A1 have been found in which a single colon or a region encoding thirty-three residues may be spliced-out in some 3a transcripts. The term "presenilin-2 gene" or "PS2 gene" primarily relates to a coding sequence, but can also include some or all of the flanking regulatory regions and/or introns. The term PSZ
gene specifically includes artificial or recombinant genes created from cDNA
or genomic DNA, including recombinant genes based upon splice variants. The presenilin 2 gene 'has also been referred to as the ES-I gene (e.g. US 5840540 A) or the STM2 gene (e.g., Levy-Lahad et ai., 1995).
The invention further comprises a substance capable c~f inhibiting presenilin 2 expression in Alzheimer's disease {AD). The invention particularly comprises a substance capable of inhibiting s presenilin 2 expression in familiar Alzheimer's disease. The term "AD"
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 (FAD)" 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.
The term "substance" as used herein means a chemical, pharmaceutical or biotechnological compound, preferably a nucleic acid molecule.
The invention particularly relates to a substance consisting of an anti-sense oligonucleotide.
fs Anti-sense oIigonucleotides are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990). In the cell, anti-sense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The anti-sense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA
that is double-stranded. The use of anti-sense methods to inhibit the in vitro or in vivo (also in the =o animal model) translation of genes is well known in the art (e.g. Marcus-Sekura, 1988). An anti-sense core nucleic acid may at least contain 10 nucleotides complementary to the target message.
Said anti-sense oligonucieotides also comprise peptide nucleic acids, phosphodiester anti-sense oligonucleotides and phosphorothioate oligonucleotides {Etoado R3 et al, 1998).
Anti-sense 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 I A I .
Similarly, the anti-sense oligonucleotide of the present invention may be used to block the PS2 30 expression in neurodegenerative diseases or preferably in AD or FAD.
The present invention further provides a ribozyme capable of cleaving presenilin 2-specific mRNA.
WO 00/03004 PCTlEP99/04804 The term "ribozyme" used in the present invention relates to an RNA capable of specifically interacting with a target RNA and of irreversibly cleaving it at a defined site. Preferably, 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 s complementary to two neighboring sequences of the tarl;et RNA (substrate binding domain or hybridization region (b)) so as to allow binding of the ribo~yme via base-pairing and thus selective cleavage of the target RNA.
Thus, according to the present invention, said ribozyme preferably comprises a catalytic region (a) and at least one hybridization region (b), with the hybridization region (b) essentially being .o complementary to a region of the mRNA that is transcribed from the presenilin 2 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 presenilin 2 gene in order to selectively cleave these RNAs ~s (see figure 1 for a hammerhead ribozyme according to the ;present invention).
The term "essentially complementary" as used in the present invention is to be understood such that the complementarity between ribozyme and target nucleic acid region is so high that it allows the specific binding of the ribozyme via hybridization and selective cleavage of the target nucleic acid region under those conditions under which the ribozyme is used. The ribozymes are preferably completely complementary to the target nucleic acid region.
The term "selective cleavage" as used in the invention is to be understood such that the expression of the PS2 gene is suppressed to such an extent that the desired therapeutical effect is achieved.
The selective inhibition of the gene expression in cells by the ribozyme according to the invention =s does therefore not mean that the target gene will be irreversibly damaged or eliminated. Rather, the use of the ribowmes 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; Morg<~n and Anderson, 1993;
Yamada et al., so 1994).
In a preferred embodiment of the ribozyme according to the invention, said ribozyme can be presented by the following general formula:
(b) (a) (b) 5' [N3_ao) [CUGANGARNo_3oSGAAA] [N3-ao] 3', s wherein N is G, C, A or U, R is a purine, and S is a pyrimidine, and wherein the central region No_ 30 of sequence (a) can be replaced by 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 end Altman, 1994) such that ribozyme e;ffectivity and selectivity is favorably influenced. The length of the hybridization region (b) (1~I3-zo) 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 ef~'icient cleavage, but, if the difference between the target RNA and non-target RNA does not comprise the ~s 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.
Preferably, 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 zo hybridization region (b) is 12 to 30 nucleotides.
In a particularly preferred embodiment of the present invention, the ribozyme contains the following nucleotide sequence (5'to 3'} in the catalytic domain:
CUGAUGAX3CXXYYYYZZZZGAAAC wherein:
CUGAUGA and GAAAC are conserved nucleotide sequf,nces;
X is any nucleotide selected from A, G, C and U which is complementary to Z, so that G is paired with C and A is paired with U or C is paired with G and U is paired with A;
Y is any nucleotide selected from A, G, C and U;
Z is any nucleotide selected from A, G, C and U which is complementary to X, so that G is paired with C and A is paired with U or C is paired with G and U is paired with A.
3o 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. ~.. Svmons ( 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 s effectively cleave target RNAs in trans, the mechanism of action being similar to that of the hammerhead r7bozymes. 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). On the basis of the requirements with. respect to the structure and sequence m of the hairpin ribozyme necessary for an effective cleavage .and with respect to the cleavage motif on the target RNA explained Fn 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. Here, too, it was possible to determine the minimum sequence and/or rs structure necessary for a cleavage in trans, and, as described above for hammerhead and hairpin ribozymes, the person skilled in the art can construct axehe;ad ribozymes on the basis of the data described in the art which exhibit the properties required for the purpose according to the invention (see, e.g. Been, 1994, and Wu et al., I993).
Determined target sequences with the pertaining, highly specific ribozyme were observed to allow zo a considerably increased catalytic activity by adapting the catalytic region (Koizumi et al., 1989, Koizumi and Ohtsuka, 1992). If the kinetic data show a too low ribozyme efficiency, the person skilled in the art can optimize the ribozyme structure by well-established in vitro evolutionary processes (3oyce, 1992; Yuan and Altman, 1994).
According to the present invention, the ribozyme may be modified such that resistance to a 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-efli'ects can be reduced.
Examples of such modifications are the substitution of the 2'-OH groups of the ribose by 2'-H, 2'-O-methyl, 2'-O-ally!, 2'-fluoro or 2'-amino groups (Pa~olella et al., 1992, and Pieken et al., 30 1991 } or the modification of phosphodiester compounds, t>y, e.g., replacing one or two oxygen atoms by sulphur atoms (phosphorous thioate or phosphorous dithioate;
Eckstein, 1985, and Beaton et al., in: Eckstein, F. (ed.) Oligonucleotides and analogues - A
practical approach -Oxford, JRL Press {1991); 109-135 or by a methyl group (methyl phosphonate;
Miller, loc. cit., WO 00/03004 PCTlEP99/04804 137-1 S4). Further modifications include conjugation of the RNA with poly-L-lysine, polyaikyl derivatives, cholesterol or PEG. Preferably, 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.
s The invention preferably comprises a ribozyme that cleaves downstream of the GUU232 site of presenilin 2-specific RNA.
The invention also preferably comprises a ribozyme that cleaves downstream of the GUC3pg site of presenilin 2-specific RNA.
The invention also preferably comprises a ribozyme that cleaves downstream of the GUC 1173 site ra of presenilin 2-specific RNA.
The numbering of the nucleotides of said GUU or GUC sites corresponds to the PS2 sequence in the EMBL Data Bank, Accession No. L43964.
The invention more preferably pertains to a ribozyme which is a fusion-ribozyme comprising a presenilin 2-specific ribozyme and an autocatalytical hamrraerhead-ribozyme fused with its S ' end ~s to the 3 ' end of the presenilin 2-specific ribozyme (see figure 4).
The invention preferably pertains to a ribozyme wherein :.aid ribozyme comprises the following nucleotide sequence (S ' to 3 ') or a bioequivalent thereof:
UUCUUUGGCUGAUGAGGCCGUGAGGCCGAAACA(:AGCG
Furthermore, the invention preferably pertains to a ribozyme wherein said ribozyme comprises the 7a following nucleotide sequence (S' to 3') or a bioequivalent thereof:
CUUUGGCUGAUGAGGCCGUGAGGCCGAAACACACi Furthermore, the invention preferably pertains to a ribozyme wherein said ribozyme comprises the following nucleotide sequence (S' to 3') or a bioequivalent thereof:
UUGGCUGAUGAGGCCGUGAGGCCGAA.A.CAC
=s Furthermore, the invention preferably pertains to a ribozyme wherein said ribozyme comprises the following nucleotide sequence (S' to 3'} or a bioequivalent thereof CUUUGGCUGAUGAGGCCGUGAGGCCGAAACACAA
Furthermore, the invention preferably pertains to a ribozyme wherein said ribozyme comprises the following nucleotide sequence (S' to 3') or a bioequivalent thereof:
3o UGGUUUUUCUGAUGAGGCCGUUAGGCCGAAACACGUCG
Furthermore, the invention preferably pertains to a ribozyme wherein said ribozyme comprises the following nucleotide sequence (S ' to 3 ') or a bioequivalent thereof:
GUUUUUCUGAUGAGGCCGUUAGGCCGAAACACGLJ
WO 00/03004 PCT/EP99104$04 IS
Furthermore, the invention preferably pertains to a ribozyme wherein said ribozyme comprises the following nucleotide sequence (S ' to 3 ') or a bioequivalent thereof:
UUUUCUGAUGAGGCCGUUAGGCCGAAACACGU
Furthermore, the invention preferably pertains to a ribozyme wherein said ribozyme comprises the s following nucleotide sequence (S ' to 3 ') or a bioequivalent thereof GAAUCCCGCUGAUGAGGCCGUUAGGCCGAAACCU~L1GG
Furthermore, the invention preferably pertains to a ribozyme wherein said ribozyme comprises the following nucleotide sequence (S ' to 3 ') or a bioequivalent thereof GAUCCCGCUGAUGAGGCCGUUAGGCCGAAACCUU~'G
Furthermore, the invention preferably pertains to a riibozyme wherein the autocatalytical hammerhead-ribozyme comprises the following nucleotide sequence (S ' to 3 ') or a bioequivaient thereof GAUCCGUCGACGGACUCGAGUCCGUCCUGAUGAGUCCGUGAGGACGAAACGGAUC
The term "bioequivalent" as used herein means, that a ribozyme with a nucleotide sequence ~s different from the before-mentioned sequences carries out the same desired biological function.
The skilled practitioner can synthesize a ribozyme or modify the ribozymes disclosed in the present invention using standard techniques and examine the obtained ribozymes with the test systems disclosed in the examples of the present application in order to establish the bioequivalent function of said ribozymes. A "bioequivalent" ribozyme may contain additional nucleotides at the :o S' end (in particular the PS-2 specific ribozymes) or at the 3' end (in particular the autocatalytical ribozyme) which do not hybridize with the target RNA. Said additional nucleotides may be added during to the cloning process of the ribozyme. Such additional nucleotides are exemplified in fig.
4a) with the ribozyme rz1173/13.3auto: see the PS2-specific ribozyme rz1173/13.3 (AAG at the ' end) and the autocatalytical ribozyme (UCUAG at the 3 ' end).
:5 In a preferred embodiment, the present invention relates to a ribozyme having the sequence WCUUUGGCUGAUGAGGCCGUGAGGCCGAAACAC:AGCG or CUUUGGCUGAUGAGGCCGUGAGGCCGAAACACAGor jo UUGGCUGAUGAGGCCGUGAGGCCGAAACACor CUUUGGCUGAUGAGGCCGUGAGGCCGAAACACAA.or UGGUULJUUCUGAUGAGGCCGUUAGGCCGAAACAI~GUCG or GUUUUUCUGAUGAGGCCGUUAGGCCGAAACACGjJor UUUUCUGAUGAGGCCGUUAGGCCGAAACACGUor GAAUCCCGCUGAUGAGGCCGUUAGGCCGAAACCCTUGGor GAUCCCGCUGAUGAGGCCGUUAGGCCGAAACCULJGor m GAUCCGUCGACGGACUCGAGUCCGUCCUGAUGACTUCCGUGAGGACGAAACGGAUC
or UUCUUUGGCUGAUGAGGCCGUGAGGCCGAAACACAGCGGAUCCGUCGACGGACUC
~s GAGUCCGUCCUGAUGAGUCCGUGAGGACGAAACGGAUCor CUUUGGCUGAUGAGGCCGUGAGGCCGAAACACA(JGAUCCGUCGACGGACUCGAGU
CCGUCCUGAUGAGUCCGUGAGGACGAAACGGAUC:or zo UUGGCUGAUGAGGCCGUGAGGCCGAAACACGAUt:CGUCGACGGACUCGAGUCCGL' CCUGAUGAGUCCGUGAGGACGAAACGGAUCor CUUUGGCUGAUGAGGCCGUGAGGCCGAAACACAAGAUCCGUCGACGGACUCGAGU
CCGUCCUGAUGAGUCCGUGAGGACGAAACGGAUC: or as UGGLJUUUUCUGAUGAGGCCGUUAGGCCGAAACACGUCGGAUCCGUCGACGGACUC
GAGUCCGUCCUGAUGAGUCCGUGAGGACGAAAC(JGAUC or GUWUUCUGAUGAGGCCGUUAGGCCGAAACACGIJGAUCCGUCGACGGACUCGAGL~
3o CCGUCCUGAUGAGUCCGUGAGGACGAAACGGAUCor UUUUCUGAUGAGGCCGUUAGGCCGAAACACGUGAUCCGUCGACGGACUCGAGUCC
GUCCUGAUGAGUCCGUGAGGACGAAACGGAUCor GAAUCCCGCUGAUGAGGCCGUUAGGCCGAAACCiJUGGGAUCCGUCGACGGACUCG
AGUCCGUCCUGAUGAGUCCGUGAGGACGAAACGIJAUCor s GAUCCCGCUGAUGAGGCCGUUAGGCCGAAACCUUGGAUCCGUCGACGGACUCGAG
UCCGUCCUGAUGAGUCCGUGAGGACGAAACGGA1:JC.
The invention additionally pertains to a recombinant DN,A molecule coding for any one of the ribozymes according to the present invention.
The invention further relates to a recombinant cDNA molecule coding for any one of the m ribozymes according to the present invention.
A recombinant vector comprising the cDNA corresponding to any one of the ribozymes is also included in the invention.
More particularly, a recombinant vector comprising the c:DNA corresponding to any one of the ribozymes fused to the cDNA sequence corresponding; to the autocatalytical hammerhead rf ribozyme is included in the invention.
Suitable vectors comprise plasmids, viruses {including phage) and integratable DNA fragments (i.e., integratable into the host genome by recombination). Tn the present specification, "vector" is generic to "plasmid"; but plasmids are the most commonly used form of vectors at present.
However, all other 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.
The present invention additionally pertains to a host cell comprising the recombinant vector comprising the cDNA corresponding to any one of the ribozymes.
Suitable host cells are any prokaryotes, yeasts or higher eukaryotic _cells which have been a transformed or transfected with the nucleic acids of the present invention so as to cause clonal propagation of those nucleic acids andlor expression of the; proteins or peptides encoded thereby.
Such cells or cell lines will have utility both in the propagation and production of the nucleic acids and proteins of the present invention but also, as further described herein, as model systems for diagnostic and therapeutic assays. As used herein, the term "transformed cell"
is intended to 3o embrace any cell, or the descendant of any cell, into which has been introduced any of the nucleic acids of the invention, whether by transformation, tra:nsfection, infection, or other means.
Methods of producing appropriate recombinant veci:ors, transforming cells with those recombinant vectors, and identifying transformants are well known in the art and are only briefly reviewed here (see, for example, Sambrook et al. 1989).
Prokaryotic cells useful for producing the transformed cells of the invention include members of the bacterial genera Escherichia (e.g., E. coli), Pseudomonas {e.g., P.
aeruginosa), and Bacillus s (e.g., B. subtillus, B. stearothermophilus), as well as many others well known and frequently used in the art. Bacterial cells (e.g., E. coIi) may be used with a variety of expression vector systems including, for example, plasmids with the T7 RNA polymeraselpromoter system, bacteriophage ~.
regulatory sequences, or M13 Phage mGPI-2. Bacterial hosts may also be transformed with fusion protein vectors which create, for ro example, lacZ, trpE, maltose-binding protein, poly-His tags, or glutathione-S-transferase fusion proteins. All of these, as well as many other prokaryotic expression systems, are well known in the art and widely commercially available (e.g., pGEX-27 (Amrad, USA) for GST
fusions).
Eukaryotic cells and cell lines useful for producing the transformed cells of the invention include mammalian cells and cell lines (e.g., PC12, COS, CHO, libroblasts, myelomas, neuroblastomas, ~s hybridomas, human embryonic kidney 293, 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.
To accomplish expression in eukaryotic cells, a wide variety of vectors have been developed and are commercially available which allow inducible (e.g., Lac;Switch expression vectors, Stratagene, :o La Jolla, CA) or cognate (e.g:, pcDNA3 vectors, Invitrogen, Chatsworth, CA) expression of presenilin nucleotide sequences under the regulation of an artificial promoter element. Such promoter elements are often derived from CMV of SV~40 viral genes, although other strong promoter elements which are active in eukaryotic cells can also be employed to induce transcription of presenilin nucleotide sequences. Typically, these vectors also contain an artificial =s polyadenylation sequence and 3' UTR which can also be derived from exogenous viral gene sequences or from other eukaryotic genes. Furthermore, in some constructs, artificial, non-coding, spliceable introns and exons are included in the vector to enhance expression of the nucleotide sequence of interest (in this case, PS2 sequences). These expression systems are commonly available from commercial sources and are typified by vectors such as pCDNA3 and 3o pZeoSV (Invitrogen, San Diego, CA}. Innumerable commercially-available as well as custom-designed expression vectors are available from commercial sources to allow expression of any desired presenilin transcript in more or less any desired cell type, either .constitutively or after exposure to a certain exogenous, stimulus (e.g., withdrawa.l of tetracycline or exposure to IPTG).
Recombinant vectors may be introduced into the recipient a~r "host" cells by various methods well known in the art including, but not limited to, calcium phosphate transfection, strontium phosphate transfection, DEAE dextran transfection, eiect;roporation, 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.
The invention also pertains to a pharmaceutical composition comprising a substance or a ribozyme or a DNA molecule or a recombinant vector as described above and a pharmaceutically acceptable corner therefor. The term "pharmaceutically acceptable carrier" as used herein refers to ro conventional pharmaceutic excipients or additives used in the pharmaceutical manufacturing art.
Said pharmaceutical composition of the present invention may contain said recombinant vector to be used for gene therapy and may contain a colloidal dispersion system or liposomes for targeted delivery of the pharmaceutical composition.
One example of a targeted delivery system for antisense polynucleotides is said colloidal rf dispersion system. Colloidal dispersion systems include ma<;romolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes ar liposome formulations. The preferred colloidal system of this invention is a liposame. 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 viva and ex vivo delivery methods. It has been shown that large unilamellar vesicles (LLJV), which range in size from 0.2-4.0 p.m can encapsulate a substantial percentage of an aqueous buffer ct>ntaining large macromolecules. 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). In addition to mammalian cells, liposomes have a been used far delivery of polynucleotides in plant, yeast and bacterial cells. In order far a liposome to be an efficient gene transfer vehicle, the follovring 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 t:o 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 3o efficiency; and (4) accurate and effective expression of ,genetic information (Mannino et al., 1988).
The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence ofdivalent cations.
The pharmaceutical composition of the present invention may contain said recombinant vector as a naked "gene expression vector". This means that the con:>truct is not associated with a delivery s vehicle (e.g. Iiposomes, colloidal particles and the like). Once of the principal advantages of naked DNA vectors is the lack of a immune response stimulated by the vector itself.
The present invention comprises the use of a substance or a ribozyme or a DNA
molecule or a recombinant vector as described above in the manufacture of a medicament for the treatment of neurodegenerative diseases.
ro The present invention more particularly comprises the use of a substance or a ribozyme or a DIv'A
molecule or a recombinant vector as described above in the manufacture of a medicament for the treatment of Alzheimer's disease.
The present invention most particularly comprises the use of a substance or a ribozyme or a DNA
molecule or a recombinant vector in the manufacture of a medicament for the treatment of ~s familiar Alzheimer's disease.
The invention also pertains to a process for the production of a ribozyme characterized in that a DNA molecule is expressed in a host.
The invention more specifically pertains to a process for the production of a ribozyme, characterized that a DNA molecule is synthesized in an automatic synthesizer.
Zo The following examples serve to further illustrate the present invention; but the same should not be construed as limiting the to the scope of the invention disclosed herein.
The examples illustrate specific substances/ribozymes as claimed in the present invention, corresponding nucleic acid molecules and recombinant vectors and the use of said :5 substances/ribozymes for the inhibition of apoptosis in neurodegenerative diseases and thus for the treatment of said diseases.
Example 1 - General Methods cDlVA constructs 3o Human wildtype (W) PSZ cDNA (Science 269: 973-977, 1995). The N 141 V
mutation in the human PS2 cDNA was generated by site-directed mutagenesis (Stratagene). Both full length PS?
cDNA sequences were cloned into the EcoRI restriction site of the tTA-response plasmid pLJHD
10-3 (Gossen and Bujard, 1992) to generate the tetracycline-regulated expression vectors pUHD 10-3/PS2wt and pUHD 10-3/PS2mut for inducible expression in cells. A 297 by PS2.NcoI
cDNA fragment (nts. 960-1257 according to the EMBL Data Bank, Accession No.
L43964) was cloned into the pBluescriptIl/SK+ plasmid (pBSK+/PS2.NcoI) and used for in vitro transcription.
s Oligoribonucleotide sequences Riboryme sequences.
rz 1173113.3 5'-UUCUUUGGCUGAUGAGGCCGUGA~GGCCGAAACACAGCG-3 ;
rx 1173/12 S'-CUUUGGCUGAUGAGGCCGUGAGGCCGAAACACAG-3';
rL 117319 5'-UUGGCUGAUGAGGCCGUGAGGCCGAAA.CAC-3';
~o Wz 1173// 1.12 5'-CUUUGGCUGAUGAGGCCGUGAGGCCGAAACACAA-3';
as 1173112 S'-CUUUGGCUGAUUCGGCCGUGAGGCCGAUACACA-3';
ri 232/15.1 S'-UGGUUUULTCUGAUGAGGCCGUUAGGCCGAAACACGUCG-3 ;
cz 232/12 5'-GUUUUUCUGAUGAGGCCGUUAGGCCGAAACACGU-3 ;
n 232/i0 5'-UUUIJCUGAUGAGGCCGUUAGGCCGAAACACGU-3 ;
rs as 232/15.1 S'-UGGULJUUUCUGAWCGGCCGUUAGGCCGAUACACGUC-3';
rz 308/15 S'-GAAUCCCGCUGAUGAGGCCGUUAGGCCGAAACCUUGG-3 ;
rt 308/12 5'-GAUCCCGCUGAUGAGGCCGUUAGGCCGAAACCUUG-3'.
Autoriboryme sequence. S'-GAUCCGUCGACGGACUC~GAGUCCGUCCUGAUGAGUC
CGUGAGGACGAAACGGAUC-3'.
RNA substrate sequences. 1173 S'-CGCUGUGUCCCAA,AGAA-3'; 232 S'-CGACGUGUUAAAAACCA-3'; 308 S'-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 ~s phosphodiester bond is cleaved. The numbering of the nucleotides correponds to the PS2 sequence in the EMBL Data Bank, Accession No. L4396~1. 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. rz1173/13.3).
3o 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. Oiigoribonucleotide purification was done either by HPLC
(reversed phase, trityl on) or on denaturing SDS-PAGE/ 8 M urea.
Synthesis of synthetic riborymes and RNA substrates. Oligoribonucleotides were synthesized using standard phosphoramidite chemistry (Boehringer Ingelheim Pharma KG, Department of s Chemical Reseach, Biberach; Interactiva inc., Ulm). The ribozymes contained stabilizing 2' methylnucleosides and 3'-terminal modifications, namely 3',3'-inverted termini and 2',3'-dideoxynucleosides, making them suitable for the cell transfections. 2'-0-methyl-ribonucleotides were introduced in order to prevent endonucleoly~tic degradation, while 3'-terminal dideoxynucleotides (ddA and ddC) or 3',3'-inverted dG residues protected the sequence from exonucleolytic degradation. These modifications have been reported to increase stability several thousand fold over native ribozymes, while the catalytic activity is only minimally impaired. The ribozymes were synthesized with flanking substrate binding regions of varying length (between 6-8 nts} which hybridize to the PS2 mRNA by base pairing. The catalytic domain of the designed hammerhead ribozymes contained the minimal set of conserved ribonucleotides.
~s Cloning of ribozyme DNA into plasmids and in vitro transcription. The DNA
coding for the ribazyme rz1173/13.3 was cloned into pBluescriptII/SK+ (Stratagene). The resulting plasmid pBSK+/pS2-rz1173.13.3 (see Fig. 4b) 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). For the =o tetracycline-regulated expression in HeLa cells, 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 rzI173/I3.3auto DNA sequence 'was cloned into the tTA-responsive plasmid pUHD 10-3 to generate plasmid pI1HD10-3/PS2-rzl 173.13.3auto (Fig.
4b).
t The prediction of secondary structure of the PSZ mRNA
The mast 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.}
~32p~ labeling of substrate and ribozyme RNA
As 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 ZO
p.l containing 20 pmol synthetic substrate RNA, 3 pl (lOpCi/ul) [y-32P]-ATP, 2 pl 10x phosphorylation buffer, 13 pl H20 and 10 U polynucleotide kinase (Boehringer Mannheim) by incubation at 37°C for 1 hour. For in vitro transcription, the plasmid pBSK+/PS2.NcoI (Fig. 4b}
was linearized with Xhol, phenol/ chloroform-extracted, and ethanol-precipitated. In vitro s transcription was carried out in 20 pl of a mixture containing 1 ul 10 rnM
GTP, 1 ltl 10 mM ATP, 1pl i0 mM UTP, 2 ul 10 x transcription buffer, 1 pl 0.2 M DTT, 1 pl RNase inhibitor (20 U), 5 pl -32P-CTP (10 mCi/ml), 1 pl 0.1 mM CTP, 5 pl H20, 1 pl (10 U) T7 RNA
Polymerise (in vitro transcription kit, Clontech). The reactions were incubated for 45 min at room temperature (RT). To degrade the template DNA, 1 pl RNase-free DNase I was added and incubated at 37°C
for 30 min. After phenollchloroform extraction, both, the _'i'[32P]-labeled synthetic as well as the ra in vitro transcribed, [32P]-labeled RNA substrates were purified by 20% SDS-PAGE/ 6M urea, eluted from the gel, precipitated and resuspended in DEP~C water. An aliquot was counted in a scintillation counter (Amersham} to determine the specific radioactivity.
rs In vitro ribozyme cleavage assay [32p]-labeled substrate RNA (20.000cpm/reaction) and ribozyme RNA were incubated in 50 mM
Tris-HCl (pH 7.5) and 10 mM MgCi2 for S 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, x 0 mM
EDTA, pH 8.0, 0,002 % bromphenol blue and xylene cyanol). Substrates and cleavage products) :o were separated by electrophoresis on a 20 % SDS-polyacrylamide/ 6 M urea denaturing gel and detected by autoradiography (X-GMAT AR films, Kodak).
Cell culture, cell lines and DNA transfection Cell culture. Control cells and all transfected HeLa cell tines were cultured in Dulbecco's modified a Eagle's medium (DMEM} supplemented with 10 % heat-inactivated FCS, 100 units/ml penicillin, {100 pg) streptomycin and 1 mM L-glutamine at 37°C in humidified air atmosphere with 5 C02.
Cell lines. 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 30 (Gossen, M. and Bujard, H. 1992). The HtTa cell line was transfected with the plasmids pUHDIO-3/PS2 wt {wildtype PS2), pUHDlO-3/PS2 mut (mutant (N141V) PS2) or pUHDlO-3/PS2-rz1173.13.3auto (ribozyme rz1173/13.3) to give rise to the double stable cell lines HtTA/PS2-wt.l3, HtTA/PS2-mut.5, and HtTA/PS2-rz1173.40, respectively.
DNA transfectioir. Stable DNA transfections were performed by the calcium-phosphate precipitation method with 50 lxg of purified DNA (Qiagen., Hilden) and S Itg of pCEP4 plasmid DNA for hygromycin selection. Hygromycin-.resistant clones were obtained after 2 weeks of selection in medium containing 200 pg/ml hygromycin. Individual cell clones were isolated, s expanded, and tested for either wildtype or mutant PS2 overexpression by Western blotting and immunocytochemistry using PS2-specific antibodies. PSZ'knock-down' cell clones were identified by quantitation of the PS2 mRNA level (RNase protection assay; Boehringer Mannheim) and the PS2 protein level {immunoprecipitationl Western blotting).
Antibodies, immunoprecipitation and Western blotting Antibodies. To recognize PS2, the following three antibodies were used:
polyclonal antibodies 3711 and 2972 raised against PS2lloop- and PS2/N-terminus-GST fusion proteins, respectively;
monoclonal antibody BLHFSC raised against the same PS;? loop - GST fusion protein as 3711.
To recognize the C-terminus of PS 1, the polyclonal antibody 3875 raised against a PS 1/loop -ls GST fusion protein, and the monoclonal antibody BL3D7 raised against the same fusion protein were used. The polyclonal antibodies were a kind gift of C. Haass (Mannheim).
For the analysis of poly(ADP-ribose) polymerase (PARP) cleavage and caspase 3 activation, we used a poIyclonal antibody against recombinant full-length PARP (Boehringer Mannheim) and a monoclonal antibody against the N-terminus of CPP32 (Transduction Laboratories), respectively.
ao Immunoprecipitation. Cells that were grown in 6 cm2-dishes to confluence were lysed with a mixture of 50 mM Tris-HCI, pH 7.6, i 50 rnM NaCI, 2 mM EDTA, 0.2 % NP-40, 1 mM
PMSF
and 5 lcg/ml leupeptin (buffer A). Immunoprecipitations were done with 3 ul of antibody 37I 1 and 20 p.l pre-washed protein A-Sepharose (Pharmacia) for' 2 h at 4°C.
Immunoprecipitates were sequentially washed in buffer A, high salt buffer (buffer A with 500 mM NaCI) and in buffer A
Zs containing 0.1 % SDS. The precipitates were solubilizE;d with 2x SDS sample buffer and electrophoresed on I2 % SDS-PAGE/ 6 M urea. The proteins were blotted onto PVDF
membranes for 1 h at 400 mA, and membranes were treated with antibodies according to the Western blotting protocol.
Western blotting. Cells were grown in 6 cm2-dishes to confluence. CeII iysis were carried out in a 3o bui~er containing 150 mM NaCI, 50 mM Tris-HCI, pH 7.6, 2 mM EDTA, 0,2 %
{v/v) NP40, 1 mM PMSF, and 5 ug/ml Leupeptine. Triton X-100 and Nonidet P-40 were added to a final concentration of 1 %. 30 pg of protein extract was loaded onto a 10-12 % SDS-PAGE and electrophoresed. After blotting of proteins onto PVDF membranes for 1 h at 400 mA, filters were blocked with S% low-fat milk powder in 10 mM Tris-HCIf, 170 mM NaCI, pH 8.0 /
0.1 % Tween (TBST) at 4°C overnight. After washing the flters with T'BST, the membranes were probed with the primary antibodies in 5 % milk powder/ TBST at RT, Following a washing step with TBST, filters were incubated for 1 h at RT with the peroxidase-conjungated secondary antibodies (Amersham) in 5 % milk powder/ TBST. Chemiluminescence was detected using the ECL
detection system (Amersham) and exposition to X-ray films (BiolVIax MR, Kodak).
RNA isolation and quanh'fication by RNase protection assay RNA isolation. mRNA isolation was carried out as described by the manufacturer's instructions fo (Boehringer Mannheim). Cells were grown in 75 cm2-culture flasks and washed twice with ice cold phosphate-buffered saline (PBS) (1,7 M KH2P04, 5 mM Na2HPO4, 0,15 M NaCI, pH 7,4).
Cells were trypsinized, pelleted by centrifugation; and lyse;d in 3 ml lysis buffer (0.1 M Tris-HCI, pH7.5, 0.3 M LiCI, 10 mM EDTA, 1 % lithium dodecylsulfate, 5 mM DTT). The DNA
was mechanically sheared by passing the extracts six times through a 21 gauge needle. 1.5 ml of a m biotin-labeled oligo (dT)20 probe was added and mixed with pre-washed 150 pl streptavidine magnetic particles. After separating and washing of the l;enerated biotin-streptavidine complex, the poly (A+)-selected mRMA was eluated with 25 pl H20 and its concentration measured.
RNAse protection assay (RPA). For RPA the plasmid pBSK+/pS2.NcoI was linearized with Xhol and in vitro transcription was carried out as described before with the T3 polymerase. RPAs were performed according to manufacturer's instructions (Boehringer Mannheim).
Radioactively labeled antisense RNA probes (SxlOS cpm) were coprecipitated with 1 ltg isolated mRNA in 30 pl hybridization buffer (40 mM Pipes (1,4-Piperazindietha.ne-sulfoneacid), 400 mM NaCI, 1 mM
EDTA, 80 % formamid, pH 6.4) and incubated at 45°C overnight. The same amount of mRNA
was incubated with 1 pl yeast tRNA (S pg/pl) as control reaction. After digestion with RNase A
:5 (5 pg/pl) and 2,5 pl RNase T1 (lOU/pL) for 30 min at 37°C, the protected RNA hybrid-fragments were extracted by phenoUchloroform/isoamyi-alcohol (25:24:1). After ethanol precipitation the fragments were resolved on 5 % SDS-pol:yacrylamide/8 M urea gels and exposed overnight at -80°C to X-Omat AR films (Kodak).
3o Induction and analysis of apoptosis Induction of apoptosis- Apoptosis was induced in He.La cells that were 80 %
confluent.
Staurosporine was added at different concentrations for various periods. After incubation, cells were tested for viability and apoptotic parameters.
Cell viability. After apoptosis induction, cell viability was determined using Alamar BIue reduction assay. In addition, cell membrane integrity was determine by measuring the LDH-release (Boehringer Mannheim).
Qualitative analysis of morphological changes of apoptotic cells.
Morphological changes of s apoptotic cells were determined by labeling the cells with different dyes.
Cells were plated onto glass slides which were covered with poly-L-lysine (100 pg/ml, Sigma) and laminin {2 pl/ml, Sigma). After incubation with staurosporine; cells were rinsed with PBS and stained with 20 ~1 of a mixture of 100 pglml acridine orange {Sigma) and 100 ug/ml ethidium bromide (Sigma) and viewed by fluorescence microscopy. Apoptotic cells were scored based on characteristic changes m of chromatin condensation and nuclear fragmentation. Alternatively, staining with Hoechst 33258 (0.5 pg/ml) was earned out, and apoptotic cell nuclei were detected by fluorescence microscopy.
Quantitative analysis by ELISA. DNA fragmentation was measured by quantification of cytosolic oligonucleosome-bound DNA using a cell death detection ELISA kit (Boehringer Mannheim) following the manufacturer's instructions.
is Example 2 Ribozyme strategy for the 'knock-down' of PS2 mRNA
Requirement for cleavage activity of hammerhead ribozymes is the presence of a GUX
(X=C,A,U) triplet in the target RNA (Haselof~ and Gerlach, 1988; Ruffner et al., 1990}. 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. I a) (Haseloff and Gerlach, 1988). They represent an advanced class of antisense ol:igonucleotides since they combine the substrate specificity of complementary nucleic acids with the potential not only to hybridize to but a also to degrade susceptible substrate RNAs catalyticaily. Due to their catalytic activity less ribozyme RNA molecules need to be present in the cell than antisense RNA
molecules for an efficieiZt'knock-down' of the target mRNA.
The PSZ 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 jo accessibility of the mRNA target sites for the ribozyme, (ii) the strength of the ribozyme-target RNA binding, and (iii) the stability of the ribozyme. (i) To select the most accessible target sites of the PS2 mRNA, the most probable mRNA secondar,~ structures were calculated using the "enfold" software (described supra in Example 1; Zuker et al., 1989) (Fig.
lb). By that, three GUX triplets (GUU232, GUC30g, and GUC1173, numbering of nucleotides according to EMBL
Data Bank, Accession No. L43964) were identified in open loop regions of the PS2 mRNA that should be accessible to ribozymes in vivo (Figs. Ib, lc). Gne of these ribozymes was targeted to the coding region of PS2, whereas two ribozymes were directed to the 5' untranslated region of s the mRNA (Fig. lc). For these three target sites, synthetic PS2-specific ribozymes (rz 232, 308, 1173) were designed for in vitro cleavage reactions. (ii) It is known that the length of the substrate binding domain of a hammerhead ribozyme effc;cts both its specificity and its turnover number. However, strong interactions between a ribozyrne and the substrate RNA
can prevent rapid dissociation of the ribozyme following cleavage of the target that could significantly reduce the catalytic activity of the ribozyme. The optimum length of the substrate binding domain has been reported to be in the order of 12-16 nucleotides. In order to carefully select the most efficient ribozyme for cell culture experiments we designed ribozymes targeted to the same site in the PS2 mRNA, but with flanking regions of various lengths (Fig. lc). As a control for the specificity of the ribozyme reaction, we used 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. Any effect on the PS2 protein level observed with the 'antisense-ribozyrr~e' is related to an antisense effect rather than a ribozyme effect. As a further control, we used a ribozyme with a randomized substrate binding sequence to evaluat non-specific effects. (iii) To increase ribozyme stability in cell culture experiments, the synthetic ribozymes have been chemically ri~odified (described supra in Example I ).
Selection of the most effective synthetic ribozyme in vitro To study the cleavage activity of the ribozymes and to compare their efficiencies we started to ~s optimize the ifi vitro ribozyme cleavage reaction {see Example 1). The magnesium dependence of the ribozyme reaction was determined (final concentration of 20 mM MgCl2 data not shown) and , was in concordance with previous observations that hammerhead ribozymes have an absolute requirement for divalent metal ions, preferentially Mg2+ or Mn2+, in order to fulfil their cleavage activity {Uhlenbeck, 1987).
30 In vitro cleavage reactions for the selection of the most efficient ribozyme were earned out using different partial sequences of the PS2 mRNA as substrates (Fig. lc), that were short I6-17 b in size, synthetic oligoribonucIeotides. Each RNA substrate containing the GL1C
(rz308, rz1I73) 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). In standard in vitro cleavage reactions, we could detect the expected 5', end-labeled cleavage products of the RNA substrates (GUC 1173, GLJC232~ GW308) (Fig. 2a).
This was true for all active ribozymes (rz232, rz308, rz1173}. The; synthetic ribozymes with the longer s substrate binding domains ranging up to 15-16 b exhibited a higher in vitro cleavage activity than those with shorter flanking arms (Fig. 2a). When the [32P]-labeled RNA
substrates were incubated with the corresponding 'antisense ribozymes', no cleavage product was detected (Fig.
2a}. In addition, incubation of the RNA substrates without ribozyme did not give rise to a cleavage product (Fig. 2a, lanes "-"). We further analyzed the ribozymeaarget ratios required for efficient in vitro RNA cleavage for the three selected rittozymes rz1173/13.3, rz232/15.1 and rz308/15. As shown in figure 2b, both, rz1173/13.3 and ra308/15, cleaved the target PS2 RNA
substrate at a molar ratio of ribozyme to target of 1:1 under standard in vitro cleavage conditions, though with a lower efficiency than at a greater molar excess of the ribozyme.
In contrast, ribozyme rz232/I5. I cleavage products were only detectable, when the ribozyme was in a 50 fold ~s molar excess over the target RNA (Fig. 2b).
Since ribozyme rzl 173 very effectively cleaved the PS2 enF~NA in the coding region, we selected this ribozyme for the PS2 'knock-down' im cultured cells and investigated the optimal length of its substrate binding domain in more detail (described supra in Example 1).
Whereas rz1173/13.3 (substrate binding domain: 13 bases and 3 bases forming wobble base pairs with the target :o mRNA) produced significant amounts of the expected cleavage product within lh at a molar ribozymeaarget ratio of 1:1, much higher molar ratios were required when the binding domain was shortened to 9 bases (Fig. 3a). Time course studies with ribozyme rz1173/13.3 revealed that ribozyme-mediated target RNA cleavage could be detected already after 5 min of incubation (Fig.
3b}. After 6 h the RNA substrate was almost completely degraded (Fig. 3b).
~s In vitro transcribed ribozyme rz1173/13.3 also cleaves longer substrate RNAs To ensure that ribozyme rzi 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 3o rz1173113.3auto (Fig. S). The autocataiytic ribozyme should be able to splice itself out of the initial transcript to generate ribozyme rzl 173 with a defined 3' end. This approach was reported to keep the ribozyme in the nucleus, because of an inhibition of the transport into the cytoplasm.
where it can directly act on newly transcribed substrate; RNA (Liu and Carmichael, 1994).
WO 00/03004 PCT/EP99/04$04 Calculations with the "mfold" software (described supra in Example 1 ) predicted the same secondary structure for the 367 b in vitro transcribed substrate RNA as for the same sequence stretch in the context of the full length PS2 mRNA. Incubation of the substrate RNA (PS2.NcoI
mRNA fragment) with increasing amounts of biosynthetic ribozyme rz 1173113.3 resulted in a s cleavage of the target RNA into the predicted fragments of 259 and 108 bases (Fig. 5). The cleavage of the longer RNA substrate was not as efficient as the cleavage of the shorter synthetic oligoribonucleotides (compare to Figs. 2 and 3).
'Knock-dmvn' of endogenous PS2 in a ribozyrne expressing HeLa cell line For the PS2 'knock-down' in cells we applied the inducible "Tet-off' expression system. The efficacy of ribozyme rzl 173/13.3 in cells was first tested in transient transfection experiments.
HtTA cells transiently transfected with the plasmid pUHDlO-3/PS2-rz1173.13.3auto (Fig. 4b) resulted in an about 30% reduction of PS2 mRNA (data not shown). We then produced over 49 clonal cell lines and analyzed their PS2 mRNA level in R2~lase protection assays (RPA) using the ~32p~_labeled antisense PS2.NcoI mRNA fragment as probe. The PS2 mRNA levels of a number of cell clones are shown in figure 6a. The PS2 RNA contents of these clones were analyzed after complete induction of ribozyme expression by omission of doxycycline for 3 days. The time course of the induction of ribozyme expression after the system was shut off with either I pg or 2 ng doxycycline/ml was experimentally determined in detail {data not shown). We selected clone zo 40 for further analyses and demonstrated the 'knock-down' of PS2 also on the protein level (Fig.
6b}. Expression of ribozyme rzl 173/13.3 resulted in an almost complete extinction of the PS2 protein 2 days after omission of doxycycline. Although cells were continously cultivated under doxycycline-free conditions, the PS2 protein, surprisingly, returned to basal level within 2 weeks (Fig. 6b)_ Therefore, further functional analyses in PSZ k.d cells were performed 2 days after ~s doxycycline omission.
Apoptosis sensitivity is decreased in PS2 'knock-down' cells and increased in cells overexpressing wildtype or mutant PS2 It has been reported in the literature that wildtype and mutant PS2 have a proapoptotic potential, 3o when overexpressed in various cell lines (Deng et al., 1996; Vito et al., 1996; Wolozin et al., 1996; 3anicki et aL, 1997). Furthermore, cells expressing mutant forms of PS2 show an increased sensitivity to apoptotic stimuli compared to wildtype PS2 expressing cells (Wolozin et al., 1996;
Janicki et al., 1997). To address the question whether PS2 is actively involved in ~apoptosis, we studied the sensitivity of PS2 k.d. HeLa cells to stauros~porine compared HeLa cells inducibly overexpressing wildtype and 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, AIamar Blue reduction and LDH release was measured.
Apoptosis was induced by increasing concentrations of sta.urosporine. Cells were incubated with an ethidiumbromide/ acridine orange mixture that staine;s living cells green.
Apoptotic cells showed a characteristic chromatin condensation, nuclear fragmentation and the generation of ~o apoptotic bodies, and their chromatin was stained orange upon ethidiumbromide intercalation {Fig. 7b). In the absence of an apoptotic stimulus, 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). At very low concentrations of staurosporine (1 pM), apoptotic cells appeared in the PS2 wt and PS2 mut cultures, with a higher frequency of apoptotic cells in the PS2 mut cell line (Fig. 7b). No apoptotic cells were observed at the same staurosporine concentration in the PS2 k.d. cell line I;Fig. 7b). Higher concentrations of staurosporine yielded a comparable number of apoptotic cells in the PS2 wt and PS2 mut cultures (Fig. 7b). In the ribozyme-mediated PS2 k.d. cells only the :highest concentration of staurosporine ( 1 nM) resulted in the occurence of apoptotis. Thus, 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.
Interestingly, the PS2 'knock-down' resulted iri a significant reduction of apo~ptosis sensitivity.
Similar data were obtained when cells were stained with Hoechst 33258 to visualize apoptotic cells (data not shown).
.-s Since visualization of apoptotic cells with ethidiumbromide/ acridine orange staining is not a quantitative method, we applied a cell death detection ELISA that determines mono- and oligonucleosomes in the cytoplasmic fraction of cell lysate;s (Fig. 8a). In addition, cell viability was assessed using an Alamar Blue reduction assays (Fig. 8b). To distinguish between apoptosis and necrosis, the release of LDH was measured (Fig. 8c). As shown in figure 8a, PSZ wt and PS2 _. mut cells exhibited a more pronounced response to subtoxic concentrations of staurosporine (1-100 pM) than HeLa cells expressing endogenous PS2 levels. At higher concentrations of staurosporine (>1nM), secondary necrosis set in, and the. difference between these cell lines became blurred. A lower degree of apoptosis (Fig. 8a) together with the increased LDH release (Fig. 8c) clearly indicated that at staurosporine concentrations >10 nM, necrosis instead of apoptosis was the prevailing mode of cell injury.
The ELISA results reflected the marked resistance of PS:? k.d. cells to apoptosis stimulation by 1 pM - 1 nM staurosporine, compared to cells expressing normal levels of PS2 (Fig. 8a). In this concentration range, staurosporine had no significant effect on cell viability (Fig. 8b).
The PS2 expression level does not affect the kinetics of caspase 3. activation and PARP
cleavage To establish whether PS2 'knock-down' or overexpression changes the kinetics of processes characteristic of the execution phase of apoptosis, we studied the time-course of caspase 3 activation and poly(ADP)ribose polymerase (PAR.P) cleavage following the induction of apoptosis with 1 ~.M staurosporine. It is known that the final step in the cascade of protease activation during apoptosis is the activation of caspase 3 that in turn leads to the cleavage of specific proteins that either are actively involved in the apoptosis or just 'innocent bystanders' ~.s (Martin and Green, 1995; Alnemri et al., 1996; Chinnaiyan and Dixit, 1996). Kim et al. (Science 1997, 277: 373-376) and Loetscher et al. (199'7} reported that preseniilin 1 and 2 are both cleaved during apoptosis by a protease that belongs to the caspase 3 protease family.
The caspase 3, or CPP32, is activated by cleavage into two proteolytic fragments (I7 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 alaoptosis could be detected between PSZ
wt, PS2 mut and PS2 k.d. cetls (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 199?, 277: 373-376}. Therefore, PARP is quite often used as marker for apoptosis :f (example shown in Fig. l0a). We then analyzed the kinetiics 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).
Normal proteolytic, not alternative PSZ fragments seemed to be directly involved in apoptosis 3o The 'knock-down' of endogenous PS2 resulted in a marked inhibition of apoptosis i8 h after the induction with subtoxic staurosporine concentrations (Figs. 7 and 8}. On the other hand, no difference could be observed between the ribozyme-mediated PS2 k.d. cell line and control cells regarding the kinetics of caspase 3 activation and PARP' cleavage using 1 uM
staurosporine in time-course experiments (Figs. 9 and 10). The analysis of CTF generation in the course of cell death revealed that the PS2 k.d. reduced both, the normal proteolytic PS2 fragments and also the alternative PSZ fragments (Fig, l la). The generation of CTF16 occured at earlier time points in the PS2 mut than in the PS2 wt cultures, pointing to an earlier onset of apoptosis caused by the N141V mutation in PS2 (compare to Fig. 7b). The presence or absence of the PS2 alternative fragments can account for the difference in the sensitivity to an apoptotic stimulus (Fig. 1lb), arguing for a direct involvement of the alternative fragments in the execution of apoptosis.
Another implication of our findings would be that the 'knock-down' of endogenous normal PS2 CTF22 renders cells less vulnerable to apoptotic stimuli suggesting normal CTF22 as active to mediator of cell death. In order to address the question whether normal or alternative PS2 ..
fragments are actively involved in programmed cell death, we analyzed fragment formation at subtoxic staurosporine concentrations (Fig. 12). Surprisingly, at low concentrations of the apoptotic stimulus, at which cells clearly underwent apoptosis without loss of cell viability and at which PS2 'knock-down' exerted a strong inhibitory effect on apoptosis (see Fig. 8), no is alternative CTF16 generation could be observed. This finding speaks against an active role of the alternative PS2 fragments in the apoptotic cascade and rather suggests that the normal endoproteolytic cleavage products are active mediators o:P programmed cell death.
References Alnemri, ES, Livingston, DJ, Nicholson, DW, Salvesen, G, Thornberry, NA, Wong;
WW, and Yuan, JY ( 1996). Human ICE/CED-3 protease nomenclature. Cell 87, 171.
=s Beaton et al. (1991), in: Eckstein, F. (ed.) Oligonucleotidles and analogues - A practical approach - Oxford, JRL Press, 109-135.
Been MD (1994). Cis- and traps-acting ribozymes from a human pathogen, hepatitis delta virus.
Trends Biochem Sci 19, 251-256.
Boado RJ, Tsukamoto H, Pardridge WM (1998). Drug delivery of antisense molecules to the brain for treatment of Alzheimer's disease and cerebral AmS. J Pharm Sci 87:
1308-1315.
Borchelt DR, Thinakaran G, Eckman CB, Lee MK, Davenport F, Ratovitsky T, Prada CM, Kim G, Seekins S, Yager D, Slunt HH, Wang R, Seeger M, Levey Al:, Gandy SE, Copeland NG, Jenkins NA, Price DL, Younkin SG (1996). Familial Alzheimer's disease-linked presenilin 1 variants elevate ABetal-42/1-40 ratio in vitro and in vivo. Neuron 17, 1005-1013.
Borchelt DR, Ratovitski T, Van Lare J, Lee MK, Gonzales V, Jenkins NA, Copeland NG, Price DL, Sisodia SS (1997}. Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron 19, 939-945.
Chartier-Harlin, MC, Crawford, F, Houlden, H; Warren, A, Hughes, D, Fidani, L, Goate, A, Rossor, M, Rogues, P, Hardy, J, and MulIan, M (1991). Early-onset Alzheimer's disease caused by mutations at codon 717 of the beta amyloid precursor protein gene. Nature 353, 844-846.
Chinnaiyan, AM, and Dixit, VM (1996). The cell-death machine. Curr Biol 6, SSS-562.
Deng GM, Pike CJ, Cotman CW ( 1996). .Alzheimer-associated presenilin-2 confers increased sensitivity to apoptosis in PC12 cells. FEBS Lett 397, SO-S4.
Duff K, Eckman C, Zehr C, Yu X, Prada CM, Perez-Tur J, Hutton M, Buee L, Harigaya Y, Yager D, Morgan D, Gordon MN, Holcomb L, RefoIo L, Zenk B, Hardy J, Younkin S
(1996).
Increased amyloid-Beta42{43) in brains of mice expressing ;mutant presenilin 1. Nature 383, 710-713.
Eckstein F (1985). Nucleoside phosphorothioates. Annu Rev Biochem 54, 367-402.
ZS
Fraley R and Papahadjopoulos D ( i 981 ). New generation liposomes - The engineering of an efficient vehicle for intracellular delivery of nucleic acids. Trends Biochem Sci 6, 77-80.
Goate, A, Chartier-Harlin, MC, Mullan, M, Brown, J, CrawjPord, F, Fidani, L, Giuffra, L, Haynes, 3o A, living, N, James, L, et al. (1991). Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature 349, i'04-706.
WO 00/03004 PCT/EP99l04804 Gossen M, and Bujard H ( 1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U;SA 89, 5547-5551.
Hampel A, Tritz R, Hicks M, Cruz P (1990). 'Hairpin' catalytic RNA model:
evidence for helices s and sequence requirement for substrate RNA. Nucleic Acids Res 18, 299-304.
HaseIofl', J, and Gerlach, WL (1988). Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334, 585-591.
fo Hendriks, L, van Duijn, CM, Cras,'P, Cruts, M, Van :Elul, W, van Harskamp, F, Warren, A, Mclnnis, MG, Antonarakis, SE, Martin, JJ, et al. (T992). Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the beta-amyloid precursor protein gene.
Nature Genet 1, 218-221.
~s Janicki S, Monteiro MJ (1997). Increased apoptosis arising from increased expression of the Alzheimer's disease-associated presenilin-2 mutation (N141I). J Cell Biol I39, 485-495.
Jarrett, JT, Berger, ET, and Lansbury, PT (1993). The. carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Zo Alzheimer's disease. Biochemistry 32, 4693-4697.
Jarvis TC, Wincott FE, Alby LJ, McSwiggen JA, Beigelman L, Gustofson J, DiRenzo A, Levy K, Arthur M, Matulic-Adamic J, Karpeisky A, Gonzalez C, Woolf TM, Usman N, and Stinchcomb DT (1996) Optimizing the cell efl~cacy of synthetic ribozymes. J Biol Chem 27I, 29107-29112.
as Joyce (1992). In: Murray J.A.H. (Ed.,) Antisense RNA and DNA, Wiley-Liss, New York 353-3 72.
Kim TW, Pettingell WH, Jung YK, Kovacs DM, Tar~zi RE (/997). Alternative cleavage of jo Alzheimer-associated presenilins during apoptosis by a caspase-3 family protease. Science 277, 373-376.
Kim TW, Pettingell WH, Hallmark OG, Moir RD, Wasco W, and Tanzi RE (1997).
Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells. J Biol Chem 272, 11006-11 O 10.
s Koizumi M, Hayase Y, Iwai S, Kamiya H, moue H, Ohtsuka E { 1989). Design of 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. (E,d.), Antisense RNA and DNA, Wiley-Liss, New York, 373-38I.
ra Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima J, PettingeIl WH, Yu CE, Jondro PD, Schmidt SD, Wang K, Crowley AC, Fu Y, Guenette SY, Cialas D, Nemens E, Wijsman EM, Bird TD, Scheilenberg GD, Tanzi RE (1995). Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science 269, 973-977.
IS
Lisziewicz J, Sun D; Smythe J, Lusso P, Lori F, Louie A, Markham P, Rossi J, Reitz M, Gallo RC (1993). Inhibition of human immunodeficiency virus type 1 replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS. Proc Natl Acad Sci USA 90, 8000-8004.
zo Liu Z, and Carmichael GG (1994). Nuclear antisense RNA. Mol Biotechnol 2, 107-118.
Loetscher H, Deuschle U, Brockhaus M, Reinhardt D, Nelboeck P, Mous J, Gruenberg J, Haass C, Jacobsen H (1997). Presenilins are processed by caspase-type proteases. 3 Biol Chem 272, =s 20655-20659.
Mann, DMA, Iwatsubo, T, Cairns, NJ, Lantos, PL, Nochlin, D, Sumi, SM, Bird, TD, Poorkay, P, Hardy, J, Hutton, M, Prihar, G, Crook, R, Rossor, MN, and Haltia, M ( 1996).
Amyloid beta protein (Abeta) deposition in chromosome 14-linked Alzheimer's disease:
predominance of 3o Abeta42(43}. Ann Neurol 40, 149-156.
Mannino RJ, and Gould-Fogerite S ( 1988). Liposome mediated gene transfer.
BioTechniques 6, 682-690.
WO 00/03004 PCT/EP99/04$04 Marcus-Sekura, CJ. (1988). Techniques for using antisense oligodeoxyribonucleotides to study gene expression. Anal. Biochem. 172, 289-295.
Martin, SJ, and Green, DR (1995). Protease activation during apoptosis: death by a thousand cuts. Cell 82, 349-352.
Miller et al. (1991), in: Eckstein, F. (ed.) Oligonucleotides. and analogues -A practical approach - Oxford, JRL Press, 137-154.
Morgan RA, and Anderson WF (1993). Human gene therapy. Annu Rev Biochem 62, 19I-217.
Mullan, M, Crawford, F, Axelman, K, Houlden, H, Lilius, L, Winblad, B, and Lannfelt, L ( 1992).
A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of ~s beta-amyloid. Nature Genet I, 345-347.
Murrell, J, Farlow, M, Ghetti, B, and Benson, MD (1991). A mutation in the amyloid precursor protein associated with the hereditary Alzheimer diseasc; with a guanine to thymine missence change at position 1924 of the APP gene. Science 254, 97.-99.
~o Paolella G, Sproat BS, Lamond AI (1992). Nuclease resistant ribozymes with high catalytic activity. EMBO J lI, 1913-1919 Pieken WA, Olsen DB, Benseler F, Aurup H, Eckstein F (1991). Kinetic characterization of 2f ribonuclease-resistant 2'-modified hammerhead ribozymes. Science 253, 314-317.
Podlisny, MB, Citron, M, Amarante, P, Sherrington, R, Xia, W, Zhang, J, Diehl, T, Levesque, G, Fraser, P, Haass, C, Koo, EH, Seubert, P, St. George-Hyslop, P, Teplow, DB, and Selkoe, DJ
{ 1997). Presenilin proteins undergo heterogenous endoproteolysis between Thr291 and A1a299 3o and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue. Neurobiol Dis 3, 325-337.
Rogaev Ei, Sherrington R, Rogaeva EA, Levesque G, Ike:da M; Liang Y, Chi H, Lin C, Holman K, Tsuda T, Mar L, Sorbi S, Nacmias B, Piacentini S, Amaducci L, Chumakov I, Cohen D, Lannfelt L, Fraser PE, Rommens JM, St George-Hyslop F'H (1995). Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosc>me I related to the Alzheimer's disease s type 3 gene. Nature 376, 775-778.
Rossi JJ (1993). Introductory remarks on the general application of antisense RNAs and ribozymes. Methods 5, I-5.
Ruffner, DE, Stormo, GD, and Uhlenbeck, OC {1990). Sequence requirements of the hammerhead RNA self cleavage reaction. Biochemistry 29;; 10695-10702.
Sambrook et al. (1989). Molecular Cloning: A Labarator,~ Manual, 2°d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
~s Scheuner D, Eckman C, Jensen M, Song X, Citron M, Suzuki N, Bird TD, Hardy J, Hutton M, Kukull W, Larson E, Levy-Lahad E, Viitanen M, Peskind lE, Poorkaj P, Schellenberg G, Tanzi R, Wasco W, Lannfeit L, Selkoe D, Younkin S (1996). Secreted amyloid Beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin I and 2 and APP
2o mutations linked to familial Alzheimer's disease. Nature Me;d 2, 864-870.
Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin J, Bruni AC, Montesi MP, Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A, Sanseau P, St George-Hyslop PH (1995).
Cloning of a a gene bearing missense mutations in early-onset familial Alzheimer's disease.
Nature 375, 7S4-760.
Symons RH (1992}. Small catalytic RNAs. Annu Rev Biochem 61, 641-671.
Thinakaran, G, Borchelt, DF;, Lee, MK, Slunt, HH, Spitzer, L, Kim, G, Ratovitzky, T, 3o Davenport, F, Nordstedt, C, Seeger, M, Hardy, J, Levey, AI, Gandy, SE, Jenkins, NA, Copeland, NG, Price, DL, and Sisodia, SS (1996). Endoproteolysis of presenilin I and accumulation of processed derivates in vivo. Neuron 17, 181-190.
WO 00/03004 . PCT/LP99/04804 Thomsan JB, Tuschl T, Eckstein F (1993). Activity of hammerhead ribozymes containing non-nucleotidic linkers. Nucleic Acids Res 21, 5600-5603.
Vito P, Wolozin B, Ganjei JK, Iwasaki K, Lacana E, and D'.Adamio L (1996}.
Requirement of the s familial Alzheimer's disease gene PS2 for apoptosis. J Bial C:hem 271, 31025-31028.
Weintraub HM (1990}. Antisense RNA and DNA. Scientific American, 262, 34-40.
Wolozin B, Iwasaki K, Vito P, Ganjei JK, Lacana E, Sunderland T, Zhao B, Kusiak JW, Wasco u' W, D'Adamia L (1996). Participation of presenilin 2 in apoptosis: enhanced basal activity conferred by an Alzheimer mutation. Science 274, 1710-1713.
Wu HN, Lee 3Y, Huang HW, Huang YS, Hsueh TG (1993). Mutagenesis analysis of a hepatitis delta virus genomic ribozyme. Nucleic Acids Res 21, 4193-4.199.
is Yamada O, Yu M, Yee JK, Kraus G, Looney D, Wong-Staal F (1994). Intracellular immunization of human T cells with a hairpin ribozyme against human immunodeficiency virus type 1. Gene Ther 1, 3 8-45.
:o Yu M, Ojwang J, Yamada O, Hampel A, Rapapport J, IJooney D, Wong-Staal F
(1993). A
hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1 Proc Natl Acad Sci USA 90, 6340-6344. [published erratum appears in Proc Natl Acad Sci USA
(1993) 90, 8303).
.5 Yuan Y, Altman S (1994}. Selection of guide sequences that direct efficient cleavage of mRNA
by human ribonuclease P: Science 263, 1269-1273.
Zuker M. ( 1989). Computer prediction of RNA structure. Methods Enzymol. 18Q, 262-288.
WO 00/03004 ~ PCT/EP99/04804 SEQUENCE LISTING
<110> Boehringer Ingelheim GmbH
<120> Presenilin 2 specific ribozyme <130> 1-0154-PCT
<140>
<241>
<150> 98112653.5 <151> 1998-07-09 <150> 60/126200 <151> 1999-03-25 <160> 24 <170> PatentIn Ver. 2.1 <210> 1 <211> 38 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozym~e Sequence <400> 1 uucuuuggcu gaugaggccg ugaggccgaa acacagcg 38 <210> 2 <211> 34 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence <400> 2 cuuuggcuga ugaggccgug aggccgaaac acag 34 <210> 3 <211> 30 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence <400> 3 uuggcugaug aggccgugag gccgaaacac 30 <210> 4 <211> 34 <212> RNA
<213> Artificial Sequence <220>
i;
<223> Description of Artificial Sequence: Ribozyme:
Sequence <400> 4 cuuuggcuga ugaggccgug aggccgaaac acaa 34 <210> 5 <211> 38 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme:
Sequence <400> 5 ugguuuuucu gaugaggccg uuaggccgaa acacgucg 38 <210> 6 <211> 34 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme:
Sequence <400> 6 guuuuucuga ugaggccguu aggccgaaac acgu 34 <210> 7 <211> 32 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme:
Sequence <400> 7 uuuucugaug aggccguuag gccgaaacac gu 32 <210> 8 <211> 37 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme:
Sequence <40a> 8 gaaucccgcu gaugaggccg uuaggccgaa accuugg 37 <210> 9 <211> 35 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme~
Sequence <400> 9 gaucccgcug augaggccgu uaggccgaaa ccuug 35 <220> 10 <211> 55 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence <400> 10 gauccgucga cggacucgag uccguccuga ugaguccgug aggacgaaac ggauc 55 <210> 11 <211> 93 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence <400> 11 uucuuuggcu gaugaggccg ugaggccgaa acacagcgga uccgucgacg gacucgaguc 60 cguccugaug aguccgugag gacgaaacgg auc 93 <220> 12 <211> 89 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence <400> 12 cuuuggcuga ugaggccgug aggccgaaac acaggauccg ucgacggacu cgaguccguc 60 cugaugaguc cgugaggacg aaacggauc 89 <210> 13 <211> 85 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence <400> 13 uuggcugaug aggccgugag gccgaaacac gauccgucga cggacucgag uccguccuga 60 ugaguccgug aggacgaaac ggauc 85 <210> 14 <211> 89 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence WO 00/03004 4 FCTlEP99/04804 <400> 14 cuuuggcuga ugaggccgug aggccgaaae acaagauccg cgaguccguc ucgacggacu 60 cugaugaguc cgugaggacg aaacggauc 89 <210> IS
<211> 93 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:
Ribozyme:
Sequence <400> 15 ugguuuuucu gaugaggccg uuaggccgaa acacgucgga gacucgaguc uccguc:gacg 60 cguccugaug aguccgugag gacgaaacgg auc 93 <210> 16 <221> 89 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:
Ribozyme Sequence <400> 16 guuuuucuga ugaggccguu aggccgaaac acgugauccg cgaguccguc ucgacggacu 60 cugaugaguc cgugaggacg aaacggauc 89 <210> 17 <211> 87 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:
Ribozyme Sequence <400> 17 uuuucugaug aggccguuag gccgaaacac gugauccguc aguccguccu gacggacucg 60 gaugaguccg ugaggacgaa acggauc g7 <210> 18 <211> 92 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:
Ribozyme Sequence <400> 18 gaaucccgcu gaugaggccg uuaggccgaa accuugggau acucgagucc ccgucgacgg 60 guccugauga guccgugagg acgaaacgga uc 92 <210> 19 <211> 90 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:
Ribozyme WO 00/03004 ~ PCTlEP99104804 Sequence <400> 19 gaucccgcug augaggccgu uaggccgaaa ccuuggaucc gucgac:ggac ucgaguccgu 60 ccugaugagu ccgugaggac gaaacggauc 90 <210> 20 <211> 33 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence <400> 20 cuuuggcuga uucggccgug aggccgauac aca 33 <210> 21 <211> 37 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Ribozyme Sequence <400> 21 ugguuuuucu gauucggccg uuaggccgau acacguc 37 <210> 22 <211> 17 <212> RNA
<213> Homo Sapiens <400> 22 cgcugugucc caaagaa 17 <210> 23 <211> 17 <212> RNA
<213> Homo sapiens <400> 23 cgacguguua aaaacca 17 <210> 24 <211> 16 <212> RNA
<213> Homo Sapiens <400> 24 ccaagguccg ggauuc