Note: Descriptions are shown in the official language in which they were submitted.
CA 02385228 2002-03-19
Ribozyrmes for prevention of restenosis
The present invention relates to arteriosclerosis therapy
and in particular to prevention of restenosis of blood vessels
after stenoses that have been treated using angioplastic tech-
niques.
Coronary heart disease and other arteriosclerotic disor-
ders are the leading cause for morbidity and mortality in the
industrialized Western world. On average, every other patient
suffers from a cardiovascular disorder as a result of arterio-
sclerotic vascular changes.
Besides surgery, in most cases bypass surgery, angioplas-
tic techniques in which the vascular constriction or occlusion,
i.e. stenosis, is removed mechanically, for example, by means
of balloon dilatation (PTCA>, has gained strongly in importance
in recent years. In connection with this treatment, in order to
counteract a renewed vascular occlusion, frequently so-called
stents are implanted into the vessels in order to keep their
lumina open and thus to prevent restenosis.
CA 02385228 2002-03-19
2
Despite the latest catheterization techniques and stent
implants and also pharmacological intervention, however, the
above-mentioned therapeutic measures are substantially limited
by restenosis whose incidence is 30-SDi. An abnormal transient
growth stimulation of vascular smooth muscle cells must be re-
garded as the main cause of this restenosis. The uncontrolled
growth of the vascular smooth muscle cells which results there-
from and their migration into the intimal space and also a re-
duction in death processes then lead to stenosis, due to thz
resulting large number of intimal cells. This large number of
intimal smooth muscle cells is the now accepted key finding in
human restenotic tissues.
For the individual patient, restenosis requires a renewed
traumatizing, possibly surgical intervention with the usual
risks of complication. This alone necessitates novel more ef-
fective therapeutic approaches to prevent restenosis.
Furthermore, the dimension of a reduction in cost- and
manpower-intensive acute and follow-up measures due to such
novel therapeutic approaches is becoming apparent in view of a
worldwide angioplasty frequency of approximately one million
interventions per year. Based on the model character of reste-
nosis for accelerated arteriosclerosis, it is also possible to
use these novel therapeutic approaches for treating by-
pass/shunt arteriosclerosis, transplant arteriopathy and also
clinically particularly early or progressive forms of arterio-
sclerosis by way of a primary prevention with comparable effec-
tiveness. In addition to the intraluminal treatment of defined
target stenoses, percutaneous non-surgical catheterization
techniques also provide the option of selective administration
of protective substances into the infarcted vessel with the aim
of reducing the infarct area or preventing the formation of an-
eurysms.
CA 02385228 2002-03-19
3
In this connection, the present invention proposes the
prevention of restenosis by inhibiting proliferation of the
vascular smooth muscle cells, using therapeutic genes which in~~
duce cell cycle arrest and may be administered immediately af-
ter balloon dilatation.
This therapeutic approach is based on the finding that the
massive local increase in various growth factors after balloon
dilatation, with synthesis of G1-phase cyclins resulting there-
from, causes reentry of the smooth muscle cells into the cell
cycle and thus their uncontrolled proliferation.
At a conference from 23rd to 27th September 1998 in Cold
Spring Harbor, New York, USA, the inventors of the present ap-
plication already suggested in this connection the use of ham-
merhead ribozymes, i.e. catalytic RNA molecules, which are di-
rected against mRNA molecules coding for the cell cycle-
relevant proteins cyclin E or E2F1; Grassi et al. , Pathol Res
Pract 1998, 194/4: 267 (Abstract 214). This approach is based
on the finding that it is possible to achieve efficient inhibi-
tion of proliferation if the production of cell cycle-.relevant
proteins which are involved in a great variety of regulatory
pathways is prevented.
This applies to the proteins cyclin E and E2F1 which are
part of many regulatory pathways, among other things of a regu-
latory feedback mechanism in which cyclin E via interaction
with other products releases the transcription factor E2F1 in
active form. E2F1 in turn activates the transcription of genes
whose products are essential for the S phase and stimulates
both cyclin E transcription and transcription of its own gene.
A combined inactivation of cyclin E and E2F1 is therefore a
particularly efficient method of inducing cell cycle arrest.
CA 02385228 2002-03-19
4
Tha functions and sequences of cyclin E and E2F1 are
described in various publications, for example by Koff et al.,
Cell (1991), volume 66, 1217-1228, Geng et al., Oncogene
(1996>, volume 12, 1173-1180, Helin et al., Cell (1992>, volume
70, 337-350, Ohtani et al., PNAS (1995), volume 92, 12146-50.
Inactivation of the said proteins can efficiently prevent pro-
gression of vascular smooth muscle cells through the cell cycle
and transition from the G1 into the S phase of the cell cyclo
does not take place.
Various techniques are available for inactivating genes
and/or products thereof, but the use of catalytic RNA mole-
cules, so-called ribozymes, however has the advantage that on
the one hand said ribozymes break down the mRNA molecules cod-
ing for the proteins cyclin E and E2F1 because of their cata-
lytic activity in traps and on the other hand a selective ef-
fect is achieved because of their sequence-specific interac-
tion.
The catalytic RNA molecules used within the scope of the
invention are so-called hammerhead ribozymes, as described, for
example, in the review by Symons: Small Catalytic RNAs, Annu
Rev Biochem (1992), volume 61, 641-671. Due to their ability to
bind complementary RNA molecules via base pairing and to de-
stroy them by site-specific cleavage, hammerhead ribozymes are
suitable candidates for preparation of therapeutics.
In principle, hammerhead ribozymes are constructed such
that they have two sequence sections, the so-called binding
arms, which are responsible for specific binding to the target
RNA, and a more or less conserved catalytically acting sequence
section which is arranged between the two binding arms. This
catalytic sequence section contains two conserved regions at
the 3' and 5' ends, and another sequence section which is at
CA 02385228 2002-03-19
least partly double-stranded and can be, at least theoreti-
cally, of any length and sequence is present between these two
regions. Binding to the target RNA results in the formation of
the typical hammerhead as shown in Fig. 1. An arrow indicates
the cleavage site and boxes highlight the conserved regions.
As already mentioned, the specificity of the hammerhead
ribozymes is determined by the sequences of the binding arms
which in the region of the target triplet bind to the RNA mole-
cule to be cut. The determination of binding arms for ribozymes
directed against target RNA thus requires firstly identifica-
tion of single-stranded regions in the target RNA molecule and
also selection of suitable cleavage site triplets with subse-
quent determination of the binding arm sequences. In particular
in the case of complicated RNA molecules with relatively long
strands, such as, for example, the mRNA for cyclin E and E2F1,
the binding arm sequences cannot simply be determined on the
basis of the possibly known sequence of the RNA molecule, since
the secondary structure thereof and also the single-stranded
regions freely accessible to the ribozymes cannot readily be
derived from the sequence. Although there are computer models
which may be used for determining the secondary structure of
long RNA molecules, the inventors of the present application
have found that this method cannot successfully be applied for
determining suitable cleavage sites in mRNA molecules for cy-
clin E and E2F1.
The inventors of the present invention have therefore em-
ployed the RNase H technique described in more detail in the
embodiments, in order to determine suitable binding sites for
hammerhead ribozymes. After the possible target sites had been
determined experimentally, possible binding arm sequences had
to be derived therefrom and the corresponding ribozymes had to
be prepared. In this connection, it emerged that the length of
the binding arms is crucial for the efficacy of the catalytic
CA 02385228 2002-03-19
6
activity of the ribozymes so that again only an experimental
determination of efficient ribozymes was possible.
The sequences listed in the attached sequence listing, SEQ
ID No. 1 and SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, SEQ
ID No. 5 and SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, SEQ
ID No. 9 and SEQ ID No~ 10 and SEa ID No. 11 and SEQ ID No. 12
represent binding arm pairs for enzymes against cyclin E mRNA,
and the sequences SEQ ID No. 13 and SEQ ID No. 14, SEQ ID No.
15 and SEQ ID No. 16 and SEQ ID No. 17 and SEQ ID No. 18 repre-
sent binding arm pairs for ribozymes against E2F1 RNA. Ribo-
zymes with these binding arms have a particularly good cata-
lytic effect against the said mRNA molecules, as the inventors
of this application were able to show experimentally.
Instead of the sequences indicated, the binding arms may
also have sequences which bind to sequences binding to the se-
quences mentioned in each case.
In this connection it is preferred, if the catalytic third
sequence section has at its 3' end the sequence SEQ ID No. 19
and at its 5' end the sequence SEQ ID No. 20, this third se-
quence section preferentially comprising the sequence SEQ ID
No. 21.
The attached sequence listing contains the sequences of
tested ribozymes against cyclin E as SEQ ID No. 22 to SEQ ID
No. 27 and of those against E2F1 as SEG! ID No. 28 - SEQ ID No.
30. Table 1 in the detailed description indicates the kinetic_
constants for these ribozymes which are among the best con-
stants described for hammerhead ribozymes.
Although it is in principle possible to introduce the
catalytic RNA molecules into the target cells using an appro-
CA 02385228 2002-03-19
priate target cell-specific transport system, it is more advan-
tageous to use DNA molecules for this, which comprise a se-
quence section coding for the above-mentioned catalytic RNA
molecules. This DNA sequence section may be part of a vector
plasmid or may be used for generating a vector, preferably a;~
adenoviral vector for application in gene therapy.
Compared with other methods, introduction of the foreign
DNA by means of recombinant adenoviruses in particular has the
advantage that adenoviruses infect target cells with high effi-
ciency and there is no risk of insertion mutagenesis.
The applicability in principle of somatic gene therapy for
cardiovascular disorders by means of replication-incompetent
adenoviral constructs was described, for example, by Barr et
al., J. Cell Biochem (1993), volume 17D, 192.
In general, the therapeutic effect can thus be achieved
either via selective transfer systems or via cell-specific gena
expression, and a selective and efficient expression via tis-
sue-specific expression of the therapeutic genes is achieved by
a promoter which is expressed exclusively in smooth muscle
cells and has a high expression rate.
In this connection the present invention also relates to a
therapeutic composition comprising the novel catalytic RNA
molecule or the novel DNA molecule or the novel vector plasmid,
respectively.
In order to prepare specific vectors or vector plasmids,
the present invention further relates to a kit containing the
novel DNA molecule which has at least one sequence section cod-
ing for the novel RNA molecule.
CA 02385228 2002-03-19
8
In general, the present invention relates to the use of
the novel catalytically acting RNA molecule, the novel DNA
molecule and/or the novel vector plasmid for prevention of re-
stenosis by means of inhibiting proliferation of vascular
smooth muscle cells.
It is understood that the features mentioned above and
still to be illustrated below can be used not only in the com-
bination indicated in each case but also in other combinations
or on their own, without going beyond the scope of the present
invention.
Further features and advantages of the present invention
arise from the following description of preferred embodiments.
The examples below are illustrated on the basis of the at-
tached drawing in which:
Fig. 1 shows the schematic structure of hammerhead ri-
bozymes; and
Fig. 2 shows a table containing the measured kinetic
constants of the tested hammerhead ribozymes.
Structure of hammerhead ribozyrmes
Fig. 1 shows the basic structure of a hammerhead ribozyme,
as can be found, for example, in the publication by Symons men-
tioned at the outset.
CA 02385228 2002-03-19
9
Fig~ 1 shows a ribozyme hybridized with a substrate (tar-
get mRNA), whereby two double-stranded regions are formed be-
tween the substrate and the ribozyme, between which regions the
cleavage site indicated by an arrow can be found on the sub-
strate side. The ribozyme side comprises the catalytic region
III located between the two sequence sections I and II which
are denoted ~binding arm", which region III comprises conserved
regions and variable regions, the conserved regions being high-
lighted by boxes.
On the substrate side, a box likewise highlights the tar-
get triplet NUH. It should be noted that N denotes any of the
four nucleotides, H denotes any of the nucleotides A, U and C,
Y denotes a pyrimidine and R a purine.
The catalytic sequence section of the ribozyme comprises
at its 3' end the sequence SEQ ID No. 19 and at its 5' end the
sequence SEQ ID No. 20 of the attached sequence listing. SEQ ID
No. 21 of the attached sequence listing shows an example of them
catalytic sequence section III and also indicates the double-
stranded region and the loop region from the third sequence
section III by way of example with defined nucleotides.
The specificity of the ribozyme of Fig. 1 for the target
mRNA results from the specific sequence of binding arms I and
II which must be selected such that they flank a target triplet
NUH on the substrate, as indicated.
Example 2 describes how to determine the binding arm se-
quences.
,~,xamQle 2
CA 02385228 2002-03-19
RNase H mapoina of the ribozYme-accessible cleavage sites of
cvclin E and E2F1
The cleavage sites of cyclin E and E2F1, which are acces-
sible for hammerhead ribozymes, cannot be determined immedi-
ately on the basis of the known mRNA sequences for cyclin E
<Koff et al., loc. cit., Geng et al., loc. cit.) and E2F1 (He-
lin et al., loc. cit.)~or on the basis of mathematical folding
models of these mRNAs so that experimental methods had to be
applied.
The accessible cleavage sites were therefore determined by
applying the method of RNase H mapping, as described by Za-
rinkar et al., Nature Struct Biol (1996) volume 3, 432 and
Lapham et al., RNA (1996>, volume 2, 289. To this end, com-
pletely randomized nonameric oligodeoxynucleotides were incu-
bated with the 5' end-radiolabelled target RNA, with complemen-
tary base pairing taking place only in open RNA structures,
i.e. single-stranded "loops", but not in closed regions, the
so-called stems. Based on the ability of RNase H to cleave suc~~
RNA/DNA hybrids and depending on the RNase H cleavage sites, a
mixture of RNA fragments is obtained whose lengths reflect
those positions of the open regions, which are in principle ac-
cessible for ribozymes.
The length of the RNase H fragments was then determined
electrophoretically, and it was then possible via the use of
sequence-specific oligodeoxynucleotides to confirm possible ri-
bozyme cleavage sites.
For cyclin E, RNase H mapping was carried out using class
III and class I transcripts which differ from one another only
in the absence of two exons in the class I transcript (exons I
and II>.
CA 02385228 2002-03-19
11
For E2F1 transcripts, too, two RNAs of different lengths
were used, but the truncated transcript has no physiological
importance.
The open RNA regions determined experimentally for cyclin
E and E2F1 were compared with various computer-assisted folding
analyses, and it was found that it was not possible to find an
optimal agreement between experimentally mapped cleavage sites
and the predicted structure.
In knowledge of the target triplet NUH mentioned in Exam-
ple 1, various binding arm I and II sequences were determined
for the open RNA regions, which were then used to prepare and
test hammerhead ribozymes, as is described in Example 3 below.
It was found in these experiments that the length of the
binding arms is one of the deciding factors for the efficiency
of the catalytic effect of the ribozymes synthesized in this
way, and it was furthermore found that the ribozymes generated
were not able to recognize and cleave any possible cleavage
site.
The experimentally confirmed binding arm pairs I and II
are represented in the attached sequence listing as sequences
SEQ ID No. 1 and 2, SEQ ID No. 3 and 4, SEQ ID No. 5 and 6, SEA
ID No. 7 and 8, SEQ ID No. 8 and 9, SEQ ID No. 9 and 1D, SEQ ID
No. 11 and 12, SEQ ID No. 13 and 14, SEQ ID No. 15 and 16 and
SEQ ID No. 17 and 18, the sequence having the odd SEQ ID in
each case denoting the binding arm at the 5' end and the se-
quence having the even SEQ ID denoting the binding arm at the
3' end of the ribozyme.
ExamQle 3
CA 02385228 2002-03-19
12
Determination of the activityr of the hammerhead ribozymes
On the basis of binding arms SEQ ID No. 1 to 18 and the
catalytically acting sequence SEQ ID No. 21, nine hammerhead
ribozymes were prepared whose sequences are listed in the at-
tached sequence listing under SEQ ID No. 22 to SEQ ID No. 30.
The activity was determined by incubating transcribed RNAs
for E2F1, class I cyclin E and class III cyclin E with the
relevant ribozyme in the presence of 32P-UTP, and determining
the kinetic constant Kcat/Km tin 104 M-1 min-1>. The in vitro
activity of the hammerhead ribozymes having the sequences SEQ
ID No. 22-30 is shown in Table 1 which is contained in Fig. 2.
Example 4
Construction of reRlication-incompetent adenoviruses
DNA molecules coding for the catalytic RNA molecules of
Examples 2 and 3 are cloned into an adenoviral vector, the re-
combinant adenoviruses having been made replication incompetent
by mutation or deletion.
SEQUENCE LISTING
<110> Eberhard-Karls-University TUbingen University Clinic
CA 02385228 2002-03-19
13
<120> Ribozymes for prevention of restenosis
<130> 5402p178
<140>
<141>
<160> 30
<170> PatentIn Ver. 2.1
<210> 1
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding arm for
ribozyme against cyclin E
<400> 1
ggaucagg 8
<210> 2
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding arm for
ribozyme against cyclin E
<400> 2
CA 02385228 2002-03-19
14
agcagggg
<210> 3
<211> 12
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding arm for
ribozyme against cyclin E
<400> 3
guggggauca 9g 12
<210> 4
<211> 12
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding arm for
ribozyme against cyclin E
<400> 4
agcagggguc ug 1':.'
<210> 5
<211> 8
<212> RNA
<213> Artificial sequence
<220>
CA 02385228 2002-03-19
<223> Description of artificial sequence: 5' binding arm fo~
ribozyme against cyclin E
<40D> 5
ucaucagc
<210> 6
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding arm for
ribozyme against cyclin E
<400> 6
acgccccu
<210> 7
<211> 12
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding arm for
ribozyme against cyclin E
<400> 7
aucuucauca gc 12
<210> 8
<211> 12
<212> RNA
CA 02385228 2002-03-19
16
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding arm for
ribozyme against cyclin E
<400> 8
acgccccuga ag 12
<210> 9
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding arm for
ribozyme against cyclin E
<400> 9
cucggcau 8
<210> 10
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding arm for
ribozyme against cyclin E
<400> 10
auggggcu 8
CA 02385228 2002-03-19
17
<210> 11
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding arm for
ribozyme against cyclin E
<400> 11
ucugcaca 8
<210> 12
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding arm for
ribozyme against cyclin E
<400> 12
acugcauu 8
<210> 13
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding arm for
ribozyme against E2F1
CA 02385228 2002-03-19
18
<400> 13
cguccgcc 'S
<210> 14
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding arm for
ribozyme against E2F1
<400> 14
acgcccac 8
<210> 15
<211> 12
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding arm for
ribozyme against E2F1
<400> 15
aagccguccg cc 12
<210> 16
<211> 12
<212> RNA
<213> Artificial sequence
<220>
CA 02385228 2002-03-19
19
<223> Description of artificial sequence: 3' binding arm for
ribozyme against E2F1
<400> 16
acgcccacug u9 12
<210> 17
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding arm for
ribozyme against E2F1
<400> 17
ugaguaga 8
<210> 18
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding arm for
ribozyme against E2F1
<400> 18
acuggcug 8
<210> 19
<211> 4
<212> RNA
CA 02385228 2002-03-19
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' end of the cata-
lytic region of a ribozyme
<400> 19
Ygaa
<210> 20
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' end of the cata-
lytic region of a ribozyme
<400> 20
cugangar 8
<210> 21
<211> 22
<212> RNA
<213> Artificial sequence
<22D>
<223> Description of artificial sequence: catalytic region of
a ribozyme
<400> 21
cugaugaguc cgugaggacg as 22
CA 02385228 2002-03-19
21
<210> 22
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme against
cyclin E
<400> 22
ggaucaggcu gaugaguccg ugaggacgaa agcagggg 38
<210> 23
<211> 46
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme against
cyclin E
<400> 23
guggggauca ggcugaugag uccgugagga cgaaagcagg ggucug 46
<210> 24
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme agains'
cyclin E
CA 02385228 2002-03-19
22
<400> 24
ucaucagccu gaugaguccg ugaggacgaa acgccccu 36
<210> 25
<211> 46
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme against
cyclin E
<400> 25
aucuucauca gccugaugag uccgugagga cgaaacgccc cugaag 46
<210> 26
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme against
cyclin E
<400> 26
cucggcaucu gaugaguccg ugaggacgaa auggggcu 38
<210> 27
<211> 38
<212> RNA
<213> Artificial sequence
<220>
CA 02385228 2002-03-19
23
<223> Description of artificial sequence: ribozyme against
cyclin E
<400> 27
ucugcacacu gaugaguccg ugaggacgaa acugcauu 38
<210> 28
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme against
E2F1
<400> 28
cguccgcccu gaugaguccg ugaggacgaa acgcccac 38
<210> 29
<211> 46
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme against
E2F1
<400> 29
aagccguccg cccugaugag uccgugagga cgaaacgccc acugug 4~
<210> 30
<211> 38
<212> RNA
CA 02385228 2002-03-19
24
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme against
E2F1
<400> 30
ugaguagacu gaugaguccg ugaggacgaa acuggcug 38
CA 02385228 2002-03-19
SEQUENCE LISTING
<110> Eberhard-Karls-Universitat Tubingen University
Clinic
<120> Ribozymes used for Restenosis Prevention
<130> 8052-18 LAB
<160> 30
<170> PatentIn Ver. 2.0
<210> 1
<211> 8
<212> RNA
<213> Artificial sequence
<220> ,
<223> Description of artificial sequence: 5' binding
arm for ribozyme against cyclin E
<400> 1
ggaucagg 8
<210> 2
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding
arm for ribozyme against cyclin E
<400> 2
agcagggg 8
<210> 3
<211> 12
CA 02385228 2002-03-19
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding
arm for ribozyme against cyclin E
<400> 3
guggggauca gg 12
<210> 4
<211> 12
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding
arm for ribozyme against cyclin E
<400> 4
agcagggguc ug 12
<210> 5
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding
arm for ribozyme against cyclin E
<400> 5
ucaucagc 8
<210> 6
<211> 8
<212> RNA
<213> Artificial sequence
<220>
CA 02385228 2002-03-19
<223> Description of artificial sequence: 3' binding
arm for ribozyme against cyclin E
<400> 6
acgccccu 8
<210> 7
<211> 12
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding
arm for ribozyme against cyclin E
<400> 7
aucuucauca gc 12
<210> 8
<211> 12
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding
arm for ribozyme against cyclin E
<400> 8
acgccccuga ag 12
<210> 9
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding
arm for ribozyme against cyclin E
<400> 9
CA 02385228 2002-03-19
cucggcau
<210> 10
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding
arm for ribozyme against cyclin E
<400> 10
auggggcu 8
<210> 11
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding
arm for ribozyme against cyclin E
<400> 11
ucugcaca 8
<210> 12
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding
arm for ribozyme against cyclin E
<400> 12
acugcauu 8
<210> 13
<211> 8
CA 02385228 2002-03-19
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding
arm for ribozyme against E2F1
<400> 13
cguccgcc 8
<210> 14
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding
arm for ribozyme against E2F1
<400> 14
acgcccac 8
<210> 15
<211> 12
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding
arm for ribozyme against E2F1
<400> 15
aagccguccg cc 12
<210> 16
<211> 12
<212> RNA
<213> Artificial sequence
<220>
CA 02385228 2002-03-19
<223> Description of artificial sequence: 3' binding
arm for ribozyme against E2F1
<400> 16
acgcccacug ug 12
<210> 17
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' binding
arm for ribozyme against E2F1
<400> 17
ugaguaga 8
<210> 18
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' binding
arm for ribozyme against E2F1
<400> 18
acuggcug 8
<210> 19
<211> 4
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' end of
the catalytic region of a ribozyme
<400> 19
CA 02385228 2002-03-19
Ygaa 4
<210> 20
<211> 8
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' end of
the catalytic region of a ribozyme
<400> 20
cugangar 8
<210> 21
<211> 22
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: catalytic
region of a ribozyme
<400> 21
cugaugaguc cgugaggacg as 22
<210> 22
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme
against cyclin E
<400> 22
ggaucaggcu gaugaguccg ugaggacgaa agcagggg 38
<210> 23
<211> 46
CA 02385228 2002-03-19
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme
against cyclin E
<400> 23
guggggauca ggcugaugag uccgugagga cgaaagcagg ggucug 46
<210> 24
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme
against cyclin E
<400> 24
ucaucagccu gaugaguccg ugaggacgaa acgccccu 38
<210> 25
<211> 46
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme
against cyclin E
<400> 25
aucuucauca gccugaugag uccgugagga cgaaacgccc cugaag 46
<210> 26
<211> 38
<212> RNA
<213> Artificial sequence
<220>
CA 02385228 2002-03-19
<223> Description of artificial sequence: ribozyme
against cyclin E
<400> 26
cucggcaucu gaugaguccg ugaggacgaa auggggcu 38
<210> 27
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme
against cyclin E
<400> 27
ucugcacacu gaugaguccg ugaggacgaa acugcauu 38
<210> 28
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme
against E2F1
<400> 28
cguccgcccu gaugaguccg ugaggacgaa acgcccac 38
<210> 29
<211> 46
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme
against E2F1
<400> 29
CA 02385228 2002-03-19
aagccguccg cccugaugag uccgugagga cgaaacgccc acugug 46
<210> 30
<211> 38
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: ribozyme
against E2F1
<400> 30
ugaguagacu gaugaguccg ugaggacgaa acuggcug 38
