U.S. patent application number 10/192926 was filed with the patent office on 2003-06-05 for oligoribonucleotide derivatives for specific inhibition of gene expression.
Invention is credited to Gunkel, Niki, Huber, Jochen, Neumann, Sandra, Uhlmann, Eugen.
Application Number | 20030105052 10/192926 |
Document ID | / |
Family ID | 7691545 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030105052 |
Kind Code |
A1 |
Uhlmann, Eugen ; et
al. |
June 5, 2003 |
Oligoribonucleotide derivatives for specific inhibition of gene
expression
Abstract
The present invention relates to oligoribonucleotide derivatives
which have a 2'5'-linked oligoribonucleotide residue without a
5'-phosphate residue on the 3' end and to the use thereof for
specific inhibition of gene expression.
Inventors: |
Uhlmann, Eugen; (Glashutten,
DE) ; Huber, Jochen; (Maxdorf, DE) ; Gunkel,
Niki; (Heidelberg, DE) ; Neumann, Sandra;
(Offenbach, DE) |
Correspondence
Address: |
ROSS J. OEHLER
AVENTIS PHARMACEUTICALS INC.
ROUTE 202-206
MAIL CODE: D303A
BRIDGEWATER
NJ
08807
US
|
Family ID: |
7691545 |
Appl. No.: |
10/192926 |
Filed: |
July 11, 2002 |
Current U.S.
Class: |
514/44A ;
435/375; 435/6.16; 536/23.2 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/321 20130101; A61P 11/06 20180101; C12N 2310/315
20130101; C12N 2310/3521 20130101; C12N 2310/3183 20130101; A61K
38/00 20130101; A61P 3/00 20180101; A61P 3/10 20180101; A61P 29/00
20180101; A61P 31/00 20180101; A61P 31/12 20180101; C12N 2310/321
20130101; C12N 2310/31 20130101; A61P 9/10 20180101; C07K 2319/00
20130101; A61P 35/00 20180101; C12N 2310/319 20130101 |
Class at
Publication: |
514/44 ; 435/6;
435/375; 536/23.2 |
International
Class: |
A61K 048/00; C12Q
001/68; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 2001 |
DE |
101 33915.1 |
Claims
Patent claims:
1. An oligonucleotide of the formula I5'-(N).sub.x--(Z).sub.n
Formula Iwhere N is naturally or not naturally occurring
nucleotides which are at least partly complementary to a target
RNA, x is independently 10 to 100, n is 2 to 20, Z is naturally or
not naturally occurring nucleotides which are linked via a 2'5'
internucleoside bond. with the proviso that its homologous target
RNA has one of the following sequence patterns.
5'-(U).sub.v--(N).sub.z--(U).sub.w 5'-(U).sub.v--(N).sub.z--UX
5'-UX--(N).sub.z--UX and 5'-(U).sub.v--(N).sub.z, where v is
independently 2 to 20, where w is independently 2 to 20, z is
independently 15 to 25, U is uridine, N is adenosine (A), guanosine
(G), cytidine (C) or U and X is A, G or C, preferably A and to its
physiologically tolerated salts.
2. The oligonucleotide of the formula I as claimed in claim 1,
wherein x is 15 to 45.
3. The oligonucleotide of the formula I as claimed in claim 2,
wherein x is 16 to 25.
4. The oligonucleotide of the formula I as claimed in one or more
of claims 1 to 3, wherein n is 2 to 10.
5. The oligonucleotide of the formula I as claimed in claim 4,
wherein n is 3 to 6.
6. The oligonucleotide of the formula I as claimed in one or more
of claims 1 to 5, wherein N is a ribonucleotide.
7. The oligonucleotide of the formula I as claimed in one or more
of claims 1 to 6, wherein v is 2 to 10.
8. The oligonucleotide of the formula I as claimed in claim 7,
wherein v is 3 to 6.
9. The oligonucleotide of the formula I as claimed in one or more
of claims 6 to 8, wherein w is 2 to 10.
10. The oligonucleotide of the formula I as claimed in claim 9,
wherein w is 3 to 6.
11. The oligonucleotide of the formula I as claimed in one or more
of claims 1 to 10, wherein z is 16 to 23.
12. The oligonucleotide of the formula I as claimed in claim 11,
wherein z is 19 to 21.
13. The oligonucleotide of the formula I as claimed in one or more
of claims 1 to 12, wherein Z is adenosine or 3'-deoxyadenosine.
14. The oligonucleotide of the formula I as claimed in one or more
of claims 1 to 13, in which one or more natural phosphodiester
bonds have been replaced by unnatural internucleotide bonds which
stabilize against nuclease degradation.
15. The oligonucleotide of the formula I as claimed in one or more
of claims 1 to 14, in which one or more natural phosphodiester
bonds have been replaced by phosphorothioate bonds.
16. The oligonucleotide of the formula I as claimed in one or more
of claims 1 to 15, in which a plurality of natural phosphodiester
bonds have been replaced by phosphorothioate bonds, with said
modifications being located on the ends and on internal pyrimidine
nucleotides.
17. A method for inhibiting gene expression of a target gene in a
cell with the aid of one or more oligonucleotides as claimed in one
or more of claims 1 to 16, wherein first an oligonucleotide
complementary to an appropriate target gene is prepared, said
oligonucleotide is introduced into a cell, said cell is incubated
and inhibition of the gene expression of the target gene is then
determined by comparative measurements of the amount of the
corresponding mRNA or corresponding gene product in a control
cell.
18. The method as claimed in claim 17 for inhibiting gene
expression of a target gene in a cell in which 2'5'-oligoadenylate
synthase is underexpressed in comparison with a control cell or is
defective.
19. A pharmaceutical comprising an oligonucleotide as claimed in
one or more of claims 1 to 18 and also additives and/or carriers
and, where appropriate, excipients for preparing or formulating a
pharmaceutical.
20. The use of a pharmaceutical as claimed in claim 19 in tumor
therapy.
21. The use of a pharmaceutical as claimed in claim 19 in the
therapy or prevention of infectious diseases.
22. The use of a pharmaceutical as claimed in claim 19 in the
therapy or prevention of viral diseases.
23. The use of a pharmaceutical as claimed in claim 19 in the
therapy of inflammations or asthma.
24. The use of a pharmaceutical as claimed in claim 19 in the
therapy of cardiovascular or metabolic disorders.
25. The use of an oligonucleotide as claimed in one or more of
claims 1 to 16 for identifying or validating novel therapeutic
target genes.
26. The use of an oligonucleotide as claimed in one or more of
claims 1 to 16 for identifying or validating novel target genes in
crop protection research.
27. A method for preparing an oligonucleotide as claimed in one or
more of claims 1 to 16, wherein the oligonucleotides are first
prepared in solution or on the solid phase by successive coupling
or coupling in blocks and are, after the preparation, isolated and
purified.
28. A method for preparing a pharmaceutical, wherein an
oligonucleotide derivative as claimed in claim 27 is prepared and,
where appropriate, admixed with further additives and/or carriers
and, where appropriate, excipients.
Description
[0001] The present invention relates to novel oligoribonucleotide
derivatives which have a 2'5'-linked oligoribonucleotide residue
without a 5'-phosphate residue on the 3' end and to the use thereof
for specific inhibition of gene expression.
[0002] The inhibition of gene expression with the aid of synthetic
nucleic acids is becoming increasingly important. Typical
representatives of these synthetic nucleic acids (oligonucleotides)
are antisense oligonucleotides, ribozymes, DNA enzymes and external
guide sequences (EGS). "Antisense oligonucleotides" are short
single-stranded nucleic acid derivatives which bind via
Watson-Crick base pairing to a complementary messenger ribonucleic
acid (mRNA) whose translation into the corresponding protein is to
be inhibited. In most cases antisense oligonucleotides exhibit
their action according to a mechanism which is supported by
cellular ribonuclease H (RNase H); numerous studies have shown
evidence for this. RNase H which is present in all cells recognizes
a double strand of DNA and RNA and cuts the mRNA complementary to
said oligonucleotide via hydrolysis of one or in most cases more
phosphodiester bonds. The way in which the oligonucleotides have to
be modified in order for activation of RNase H to take place is
known and is described, for example, in Uhlmann (2000) Curr. Opin.
Drug Discov. Dev. 3, 203-213. Synthetic ribozymes carry this
nuclease activity in their sequence. The most common type of
ribozyme is the "hammerhead" ribozyme in which the consensus
sequence GAAAC which is derived from naturally occurring ribozymes
forms the RNase part and the flanking sequences form the antisense
oligonucleotide part. DNA enzymes which, however, are not derived
from naturally occurring ribozyme motifs but have been found by
in-vitro selection, act in a similar way. EGS are synthetic RNA
analogs which activate the cellular RNase P and bind via
appropriate flanking sequences to the target mRNA and induce a
specific mRNA degradation.
[0003] A common problem of the inhibition of gene expression with
the aid of synthetic oligonucleotides is that it is always
necessary to assay a relatively large number of oligonucleotides
against various regions of the target nucleic acid, in order to
identify an efficient sequence. Furthermore, antisense
oligonucleotides often inhibit gene expression only inefficiently
or incompletely. Moreover, sequence-unspecific side effects were
observed, which may be caused by the fact that even relatively
short part sequences of about five bases in length activate RNase
H. This is shown, for example, by "Woolf et al. (1992). Proc. Natl.
Acad. Sci. U.S.A. 89, 7305-7309)". However, there are also side
effects which are caused by interaction of the antisense
oligonucleotides with proteins.
[0004] Recently, the use of double-stranded RNA for inhibiting gene
expression has been described. Double-stranded RNA (dsRNA) is a
signal for particular cells and organisms to induce a
sequence-specific degradation of mRNA according to a process which
is known as RNA interference (RNAi). The RNAi phenomenon was
observed in a number of different organisms such as, for example,
C. elegans, flies, fungi, plants and mouse embryos. RNAi is
believed to be very similar or identical to post-transcriptional
gene silencing (PTGS) found in plants. A simple injection of dsRNA
of more than 500 base pairs (bp) in length, whose sense-strand
sequence is identical to the target mRNA to be inhibited, can
specifically inhibit expression of a target gene having the
corresponding DNA sequence. This does not impair the expression of
nonhomologous genes and the base sequence of the target gene is not
altered. RNAi is a post-transcriptional process in which the dsRNA
is first cleaved into relatively small fragments which are then
probably used for sequence-specific degradation of the target
mRNA.
[0005] Previously, the gene expression was efficiently inhibited
mainly by using dsRNA of more than 100 bp in length. This
relatively long dsRNA is accessible only via in-vitro or in-vivo
transcription from the corresponding DNA via suitable transcription
systems. Another limitation of RNAi with long dsRNA is the fact
that only particular organisms such as C. elegans, zebra fish,
plants, particular types of fungi, Drosophila, oocytes and embryos
of mice allow sequence-specific inhibition by dsRNA, while most
animal cells when treated with dsRNA cause apoptosis. Long dsRNA
still inhibits gene expression when the sequence homology is from
70 to 90%. For this reason, it is possible in the case of gene
families with high sequence homology for misinterpretations of the
phenotype to occur by simultaneous inhibition of the expression of
a plurality of not completely homologous genes.
[0006] The treatment of cells with dsRNA, for example with dsRNA
viruses, generally leads to an apoptotic process or to the
sequence-unspecific degradation of the mRNA due to induction of a
2'5'-oligoadenylate-synthas- e activity. The infected cell
synthesizes in response to the viral dsRNA trimeric or tetrameric
adenylate (2'5'-A) with the unusual
2'5'-phosphodiester-internucleoside bond. 2'5'-A is phosphorylated
by cellular kinases on its 5' end and then activates a nuclease
called RNase L. 2'5'-A may also be chemically synthesized and be
introduced into the cell (Torrence et al. (1994) Curr. Med. Chem 1,
176-191). However, synthetic 2'5'-A activates RNase L only if it
has been converted to the 5'-phosphate or 5'-triphosphate form.
RNase L activated by 5'-p-2'5'-A (p is phosphate, diphosphate or
triphosphate) then degrades the entire RNA of the cell in a
sequence-unspecific manner. In addition, it was shown that it is
possible to inhibit gene expression sequence-specifically with the
aid of antisense oligonucleotide conjugates with a 5'-p-2'5'-A
residue. For this purpose, however, it is essential that the 5' end
of the 2'5'-A residue is not linked to the oligonucleotide but is
present as phosphate, thiophosphate or triphosphate (Torrence et
al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1300-4).
Furthermore, the target RNA-recognizing oligonucleotide part
(antisense part) must be in single-stranded form. For the reasons
mentioned above, oligonucleotides having on their 3' ends 2'5'-A
residues which consequently have no free 5'-phosphate or
triphosphate function have not been described previously as
inhibitors of gene expression. The inhibition of gene expression by
the single-stranded, 5'-phosphorylated 5'-p-2'5'-A antisense
oligonucleotide conjugates is a variation of the antisense
principle and is therefore also subject to the limitations of the
antisense-oligonucleotide approach. In this connection, the 2'5'-A
residue is attached to the 5' end of the oligonucleotide via a
spacer (linker) so that the 2' or 3' end of the 2'5'-A residue is
present in bound form. The RNA-binding portion preferably comprises
DNA (FIGS. 4 to 6 in Torence, Curr. Opin. Mol. Ther. (1999) 1,
307).
[0007] Recently, oligonucleotides have been used increasingly as
tools for studying the function of new genes (functional genomics).
The use of antisense oligonucleotides and ribozymes for
sequence-specific inhibition of gene expression of new genes coding
for proteins with unknown function is made more difficult by the
fact that generally a large variety of oligonucleotides of
different sequences have to be assayed, and this is a disadvantage
in particular for a high-throughput process.
[0008] It is therefore an object of the present invention to
provide novel chemically modified oligonucleotides with
significantly improved inhibition of gene expression, which
circumvent the abovementioned limitations of the conventional
methods and agents. In particular, gene expression was intended to
be inhibited in an RNA interference-like process.
[0009] According to the invention, this object is achieved by novel
oligonucleotide derivatives which have a 2'5'-linked
oligonucleotide residue on the 3' end, which carries no phosphate,
thiophosphate or triphosphate group. The sequence of the novel
oligonucleotide derivatives is complementary to the RNA sequence
whose translation is to be inhibited.
[0010] The invention accordingly provides oligonucleotide
derivatives of the formula I,
5'-(N).sub.x--(Z).sub.n Formula I
[0011] where
[0012] N is naturally or not naturally occurring nucleotides,
preferably ribonucleotides, which are at least partly complementary
to a target RNA,
[0013] x is independently 10 to 100, preferably 15 to 45 and
particularly preferably 16 to 25,
[0014] n is 2 to 20, preferably 3 to 10, particularly preferably 3
to 6,
[0015] Z is naturally or not naturally occurring nucleotides which
are linked via a 2'5'internucleoside bond,
[0016] with the proviso that its homologous target RNA has the
following sequence patterns:
[0017] 5'-(U).sub.v--(N').sub.z--(U)w
[0018] 5'-(U).sub.v--(N').sub.z--UX
[0019] 5'-UX--(N').sub.z--UX and
[0020] 5'-(U).sub.v--(N').sub.z
[0021] where
[0022] v and w independently of one another are 2 to 20, preferably
2 to 10, particularly preferably 2 to 6 and
[0023] z is 15 to 25, preferably 16 to 23 and particularly
preferably 19 to 21 and
[0024] U is uridine, N is adenosine (A), guanosine (G), cytidine
(C) or U, and X is A, G or C, preferably A. In a preferred
embodiment, N may be a ribonucleotide.
[0025] If the gene whose expression is to be inhibited contains,
for example, the following DNA sequence
[0026] 5'-TTTTGMGCGAAGGTTGTGGATCTG (Seq ID No. 1)
[0027] or the following RNA sequence
[0028] 5'-UUUUGMGCGAAGGUUGUGGAUCUG (Seq ID No. 2)
[0029] then the target RNA has the following sequence pattern
[0030] 5'-(U).sub.v--(N).sub.z--UX, where v is 4, z is 19 and X is
G.
[0031] Furthermore, preference is given to oligonucleotides of the
formula I in which one or more phosphodiester bonds have been
replaced, for example by phosphorothioate bonds or
N3',P5'-phosphoramidate bonds. Particular preference is given to
oligonucleotides of the formula I in which one or more
phosphodiester bonds have been replaced by phosphorothioate
residues. The phosphorothioate residues are preferably introduced
on the 3' ends, the 5' ends and on the internal pyrimidine
nucleotides C and U, in particular if several pyrimidine
nucleotides succeed one another in the sequence.
[0032] A particular embodiment of the invention comprises the use
of a mixture of two or more oligonucleotide derivatives in
accordance with formula 1 for inhibiting gene expression. The
oligonucleotide derivatives in this case may be directed against
different regions of an RNA or against the RNA of different
genes.
[0033] The single-stranded oligonucleotides of the fomula I were
originally employed as control oligonucleotides for RNAi
experiments using short dsRNA. Thus, owing to the single-stranded
character, inhibition of gene expression was not expected.
Surprisingly, however, particular single-stranded oligonucleotides
inhibited gene expression, too, in particular when sufficiently
stable toward nucleases. Another surprise was that the
oligonucleotides of the formula I in which the 2'5'-linked
oligoadenylate residue has no free 5'-phosphate, 5'-thiophosphate
or 5'-triphosphate residue inhibited gene expression in a
sequence-specific manner. It also came as a complete surprise that
in this case the 2'5'-linked oligoadenylate residue can be bound to
the 3'5'-linked RNA directly via the 5' function. It has been a
valid dogma up until now that the 2'5'-linked oligoadenylate
residue must have a free phosphate, thiophosphate or triphosphate
residue on the 5' end in order to inhibit gene expression.
Moreover, a 2'5' oligoadenylate-mediated inhibition had previously
always been asscociated with an unspecific, i.e.
sequence-independent, effect (Bass, Nature (2001) 411, 428). It is
therefore obvious that the oligonucleotides of the formula I not
only deviate in their structure from the oligonucleotide conjugates
described by Torrence (Curr. Opin. Mol. Ther. (1999) 1, 307) but
also exhibit a much better inhibitory action which consequently is
based on a different mechanism.
[0034] Surprisingly, the oligonucleotides of the invention also had
an inhibitory sequence-specific effect on human primary cells. As
far as we know, the inhibition of gene expression by
oligonucleotides having 2'5'-linked nucleotides in human primary
cells has not been observed previously.
[0035] The inventive oligonucleotides of the formula I may also be
used for inhibiting gene expression in cells which express only a
small amount of, a defective or no 2'5'-oligoadenylate
synthase.
[0036] It is furthermore also possible to use the oligonucleotides
of the formula I for treating patients having a deficiency or
defect in 2'5'-oligoadenylate synthase. Patients with CFS (chronic
fatigue syndrome), for example, may also be treated.
[0037] The sequences of the oligonucleotides of the formula I which
are used for inhibiting the gene expression of particular targets
are selected on the basis of the corresponding gene sequences. The
sequences of said genes are obtained by sequencing or from gene
databases. An example which may be illustrated here is the
inhibition of luciferase (firefly) by double-stranded nucleic
acids. The accession number for this gene is U47298. The coding
region of firefly luciferase comprises 1 653 nucleotides. The
following four regions may be selected, inter alia, as target
sequences for the inhibition by double-stranded nucleic acids.
1 (Seq ID No.3) gcttttacagatgcacatatcgaggtggacatcacttacg 121
---------+---------+---------+---------+ 160
cgaaaatgtctacgtgtatagctccacctgtagtgaatgc (Seq ID No.4)
ccgcgaacgacatttataatgaacgtgaattgctcaacag 311
---------+---------+---------+---------+ 350
ggcgcttgctgtaaatattacttgcacttaacgagttgtc (Seq ID No.5)
gcggtcggtaaagttgttccattttttgaagcgaaggttg 1081
---------+---------+---------+---------+ 1120
cgccagccatttcaacaaggtaaaaaacttcgcttccaac (Seq ID No.6)
attttttgaagcgaaggttgtggatctggataccgggaaa 1101
---------+---------+---------+---------+ 1140
taaaaaacttcgcttccaacacctagacctatggcccttt
[0038] The corresponding RNA for these regions then has the
following sequence.
2 (Seq ID No.7) GCUUUUACAGAUGCACAUAUCGAGGUGGACAUCACUUACG (Seq ID
No.8) CCGCGAACGACAUUUAUAAUGAACGUGAAUUGCUC- AACAG (Seq ID No.9)
GCGGUCGGUAAAGUUGUUCCAUUUUUUG- AAGCGAAGGUUG (Seq ID No.10)
AUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAA
[0039] The inventive complementary oligonucleotides of the formula
I derived therefrom have the following sequences and are
characterized in that two or more nucleotides (indicated here by
lower-case letters) are linked via a 2'5'-internucleoside bond.
Preference is given to 2'5'-linked adenylate residues.
3 3' aaaaAUGUCUACGUGUAUAGCUCCAC Seq ID No.11 3'
aaaaAUAUUACUUGCACUUAACGAG Seq ID No.12 3' aaaaCCAUUUCAACAAGGUAAAAAA
Seq ID No.13 3' aaaaaaCUUCGCUUCCAACACCUAGAC Seq ID No.14
[0040] In order to improve metabolic stability, it is also possible
to modify the oligonucleotides, for example as phosphorothioates
(asterisks). Stabilization by phosphorothioates is preferably
carried out on the ends and internal pyrimidine nucleotides.
[0041] 3' a*a*a a-C*U*U*C G C*U*U C*C A A*C A C*C*U A G A*C
[0042] The specificity of the inhibition of luciferase expression
can be checked on the basis of control oligonucleotides which are
not completely complementary to the target RNA and have, for
example, 4 base mismatches.
[0043] 3' a*a*a a C*U*U*C U*C U*U*C A A C*C A*C*C G A*G A*C Seq ID
No. 15
[0044] An example of the structure of oligonucleotides of the
formula I is given below: 1
[0045] where
[0046] B is a naturally or not naturally occurring nucleobase,
[0047] U, V and W independently of one another are O, S, NH or
CH.sub.2, preferably O or S,
[0048] R.sup.1 is independently of one another OH, SH, CH.sub.3 or
BH.sub.3 , preferably OH or SH, or physiologically tolerated salts
thereof,
[0049] R.sup.2 is independently of one another OH, H, O--C.sub.1 to
C.sub.12-alkyl , preferably OH (ribonucleotide), where C.sub.1 to
C.sub.12-alkyl preferably is CH.sub.3 or
CH.sub.3--O--CH.sub.2CH.sub.2,
[0050] R.sup.3 is independently of one another OH, H, O--C.sub.1 to
C.sub.12-Alkyl , preferably OH or H, where C.sub.1 to
C.sub.12-alkyl preferably is CH.sub.3 or
CH.sub.3--O--CH.sub.2CH.sub.2,
[0051] x is independently 10 to 100, preferably 15 to 45, and
particularly preferably 16 to 25,
[0052] n is 2 to 20, preferably 3 to 10, particularly preferably 3
to 6,
[0053] A is adenine or an adenine derivative, for example
8-bromoadenine, 8-methyladenine, or hypoxanthine.
[0054] In order to test the inhibition of gene expression using the
oligonucleotides of the invention in animal cells, in particular in
human primary cells, these are directed, for example, against a
human gene or the corresponding RNA thereof and assayed in human
cells (HUVEC, human umbilical vein endothelial cells). For this,
Edg-1 DNA (accession number M31210) from the gene database, for
example, may be transcribed into the corresponding messenger RNA
and the following two regions (175 and 725) could be selected for
synthesizing appropriate oligonucleotides.
[0055] Edg-1 RNA:
4 "175" GACCUCGGUGGUGUUCAUUCUCAUCUGCUGCU (Seq ID No.16)
UUAUCAUCCUGGAGAACAUCUUUGUCUU "725" AUUUCCAAGGCCAGCCGCAGCUCUGAGAAUGU
(Seq ID No.17) GGCGCUGCUCAAGACCGUAAUUAUCGUC
[0056] Examples of the possible structure of the corresponding
oligonucleotides are disclosed below:
5 3'-aaaaUAGUAGGACCUCUUGUAGAAA; Seq ID No.18
3'-aaaaGGUUCCGGUCGGCGUCGAGAC; Seq ID No.19 Mismatch control
3'-aaaaGGUGCCUGUCUGCGGCGACAC; Seq ID No.20
[0057] The mismatch control differs in 5 nucleotides (underlined as
mismatch) from the edg-1 RNA.
[0058] Furthermore, the following oligonucleotides directed against
edg-1 were prepared, which have improved nuclease stability and
increased inhibitory activity and are derived from the above edg-1
sequences.
6 3'-a*a*a a U*A G*U A G G A C*C*U C*U*U G*U*A G A A *A 3'-a*a*a a
G G U*U*C*C G G*U*C G G*C G*U*C G A G A *C 3'-a*a*a a G G U*G C*C*U
G*U*C*U G*C G G*C G A*C A *C
[0059] The inventive nucleic acid derivatives of formula I are
synthesized from oligonucleotides. For example, an oligonucleotide
may be synthesized completely from the nucleotides adenosine
phosphate, guanosine phosphate, inosine phosphate, cytidine
phosphate, uridine phosphate and thymidine phosphate. Preference is
given to oligonucleotides which are synthesized from
ribonucleotides, the "oligoribonucleotides". In other embodiments
of the present invention, an oligonucleotide may contain, where
appropriate, one or more modifications, for example chemical
modifications. An oligonucleotide may have a plurality of identical
and/or different modifications.
[0060] The 2'5'-linked residue may contain, for example, adenosine,
3'-deoxyadenosine (cordycepin), inosine, 8-bromoadenosine,
8-methyladenosine and other 8-substituted adenosine derivatives.
The ribose residue may also be derivatized as 3'-O-methyladenosine.
The internucleoside bonds in the 2'5'-linked portion are preferably
phosphodiester and phosphorothioate bonds. Common derivatives of
2'5'-adenylate, their synthesis and activation of RNase L are
described in the literature (Player et al. (1998) Pharmacol. Ther.
78, 55).
[0061] Examples of chemical modifications are known to the skilled
worker and are described, for example, in E. Uhlmann and A. Peyman,
Chemical Reviews 90 (1990) 543 and "Protocols for Oligonucleotides
and Analogs" Synthesis and Properties & Synthesis and
Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA
1993, J. Hunziker and C. Leumann `Nucleic Acid Analogs: Synthesis
and Properties` in Modern Synthetic Methods (Ed. Beat Ernst and C.
Leumann) Verlag Helvetica Chimica Acata, Basle, p. 331-417, R P
lyer et al. Curr Opin Mol Therap (1999) 1:344-358; S. Verma and F.
Eckstein, Annu Rev Biochem (1998) 67:99-134; J W Engels and E.
Uhlmann : Chemistry of oligonucleotides. In: Pharmaceutical aspects
of oligonucleotides. Couvreur P, Malvy C (Eds), Taylor &
Francis, London, (2000): 35-78.
[0062] The chemical modification of an oligonucleotide may include,
for example,
[0063] a) replacing completely or partially the phosphoric diester
bridges with, for example, phosphorothioate, phosphorodithioate,
NR.sup.1R.sup.1' phosphoramidate, boranophosphate,
(C.sub.1-C.sub.21)--O-alkyl phosphate,
[(C.sub.6-C.sub.12)aryl-(C.sub.1-C.sub.21)--O-alkyl]phosphate,
(C.sub.1-C.sub.8)alkyl phosphonate and/or (C.sub.6-C.sub.12)aryl
phosphonate bridges, where
[0064] R.sup.1 and R.sup.1' independently of one another are
hydrogen, (C.sub.1-C.sub.18)alkyl, (C.sub.6-C.sub.20)aryl,
(C.sub.6-C.sub.14)aryl-(- C.sub.1-C.sub.8)alkyl, preferably
hydrogen, (C.sub.1-C.sub.8)alkyl and/or methoxyethyl, particularly
preferably hydrogen, (C.sub.1-C.sub.4)alkyl and/or methoxyethyl,
or
[0065] R.sup.1 and R.sup.1', together with the nitrogen atom to
which they are bound, form a 5-6-membered heterocycle which may
additionally contain another heteroatom selected from the group
consisting of O, S, N;
[0066] b) replacing completely or partially the 3'- and/or
5'-phosphoric diester bridges with "dephospho" bridges (described,
for example, in Uhlmann, E. and Peyman, A. in "Methods in Molecular
Biology", Vol. 20, "Protocols for Oligonucleotides and Analogs", S.
Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, 355ff), for
example with formacetal, 3'-thioformacetal, methylhydroxylamine,
oxime, methylenedimethylhydrazo, dimethylenesulfone and/or silyl
groups;
[0067] c) replacing partially the sugar phosphate backbone, for
example with "morpholino" oligomers (described, for example, in E.
P. Stirchak et al., Nucleic Acids Res. 17 (1989) 6129 and in J.
Summerton and D. Weller, Antisense and Nucleic Acid Drug Dev. 7
(1997) 187-195) and/or with polyamide nucleic acids ("PNAs")
(described, for example, in P. E. Nielsen et al, Bioconj. Chem. 5
(1994) 3) and/or phosphomonoester nucleic acids ("PHONAs")
(described, for example, in Peyman et al., Angew. Chem. Int. Ed.
Engl. 35 (1996) 2632-2638);
[0068] d) replacing partially the .beta.-D-ribose units with, for
example, .beta.-D-2'-deoxyribose, .alpha.-D-2'-deoxyribose,
L-2'-deoxyribose, 2'-F-2'-deoxyribose,
2'-F-2'-deoxyarabinofuranose, 2'-O--(C.sub.1-C.sub.6)alkylribose,
2'-O--(C.sub.2-C.sub.6)alkenylribose,
2'-[O--(C.sub.1-C.sub.6)alkyl-O--(C.sub.1-C.sub.6)alkyl]ribose,
2'-NH.sub.2-2'-deoxyribose, .beta.-D-xylofuranose,
.beta.-D-arabinofuranose, .alpha.-arabinofuranose,
2,4-dideoxy-.beta.-D-erythrohexopyranose, conformationally
restricted sugar analogs such as LNA (Locked nucleic acids; Singh
et al., Chem. Commun. 4 (1998) 455; Singh et al. Chem. Commun. 12
(1998) 1247) and carbocyclic (described, for example, in Froehler,
J.Am.Chem.Soc. 114 (1992) 8320) and/or open-chain sugar analogs
(described, for example, in Vandendriessche et al., Tetrahedron 49
(1993) 7223) and/or bicyclo sugar analogs (described, for example,
in M. Tarkov et al., Helv. Chim. Acta 76 (1993) 481). The
2'-modified oligonucleotide analogs are described in detail in
Manoharan, Biochim. Biophys. Acta (1999) 117 and conformationally
restricted oligonucleotide analogs in Herdewijn, Biochim. Biopyhs.
Acta (1999) 167;
[0069] e) modifying and, respectively, completely or partially
replacing the natural nucleoside bases with, for example,
5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil,
pseudoisocytosine, dihydrouracil, 5-(C.sub.1-C.sub.6)alkyluracil,
5-(C.sub.2-C.sub.6)-alkenyluracil,
5-(C.sub.2-C.sub.6)alkynyluracil, 5-(C.sub.1-C.sub.6)alkylcytosine,
5-(C.sub.2-C.sub.6)alkenyl-cytosine,
5-(C.sub.2-C.sub.6)alkynylcytosine, 5-fluorouracil,
5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil,
5-bromocytosine or 7-deaza-7-substituted purines.
[0070] Heterocyclic base modifications are described, for example,
in Herdewijn, Antisense & Nucl. Acid Drug Dev. (2000) 297.
[0071] The chemical modification of the oligonucleotide furthermore
comprises conjugating an oligonucleotide with one or more molecules
which influence advantageously the properties (e.g. nuclease
stability, affinity for target sequence, pharmacokinetics) of said
oligonucleotide and/or, during hybridization of the modified
oligonucleotide to the target sequence, attack said target sequence
with binding and/or crosslinking (oligonucleotide conjugates).
Examples thereof are conjugates with polylysine, with intercalators
such as pyrene, acridine, phenazine, phenanthridine, with
fluorescent compounds such as fluorescein, with crosslinkers such
as psoralen, azidoproflavin, with lipophilic molecules such as
(C.sub.12-C.sub.20)alkyl, with lipids such as
1,2-dihexadecyl-rac-glycerol, with steroids such as cholesterol or
testosterone, with vitamins such as vitamin E, with poly- or
oligoethylene glycol, with (C.sub.12-C.sub.18)alkyl phosphate
diesters and/or with
--O--CH.sub.2--CH(OH)--O--(C.sub.12-C.sub.18)alkyl. Such molecules
may be conjugated at the 5' and/or 3' end and/or within the
sequence, for example at a nucleobase. Examples of oligonucleotide
conjugates known to the skilled worker are described in Manoharan
(2001) Conjugated Oligonucleotides in Antisense technology. In:
Crooke (Editor) Antisense Technology. Marcel Dekker, New York.
[0072] A specific embodiment of the chemical modification relates
to conjugation of the oligonucleotide a) with lipophilic molecules,
for example (C.sub.12-C.sub.20)alkyl, b) with steroids such as
cholesterol and/or testosterone, c) with poly- and/or oligoethylene
glycol, d) with vitamin E, e) with intercalators such as pyrene, f)
with (C.sub.14-C.sub.18)alkyl phosphate diesters and/or g) with
--O--CH.sub.2--CH(OH)--O--(C.sub.12-C.sub.16)alkyl.
[0073] Another specific embodiment of the chemical modification
relates to derivatization of the oligonucleotide, as described in
HMR 99/L045, as aryl ester conjugate, for example as FDA conjugate,
which derivatization benefits the cellular uptake of said
oligonucleotides.
[0074] Methods for preparing said oligonucleotide derivatives are
known to the skilled worker and described, for example, in Uhlmann,
E. & Peyman, A., Chem. Rev. 90 (1990) 543 and/or M. Manoharan
in "Antisense Research and Applications", Crooke and Lebleu, Eds.,
CRC Press, Boca Raton, 1993, chapter 17, p. 303ff. and/or EP-A 0
552 766.
[0075] In further specific embodiments of the present invention,
the oligonucleotide may have on its 5' end a 5'-5' inversion. This
type of chemical modification is known to the skilled worker and
described, for example, in M. Koga et al., J. Org. Chem. 56 (1991)
3757. Moreover, the 5' end is a preferred position for conjugating
the oligonucleotide with one or more molecules which have a
beneficial effect on the properties (for example stability against
nucleases, cellular uptake, affinity for the target sequence,
pharmacokinetics) of the oligonucleotide.
[0076] The invention further provides methods for preparing the
oligonucleotides. The oligonucleotides described may be prepared
with the aid of various known chemical methods, as described, for
example, in Eckstein, F. (1991) "Oligonucleotides and Analogues, A
Practical Approach", IRL Press, Oxford. The oligonucleotides may
also be prepared by methods which, where appropriate, contain one
or more enzymic steps.
[0077] The invention furthermore provides the use of the
oligonucleotides for modulating and for completely or partially
inhibiting the expression of particular target genes, for example
for completely or partially inhibiting translation. The invention
furthermore relates to the use of said oligonucleotides for
modulating and for completely or partially inhibiting expression in
cells which have only a small amount of, a defective or no
2'5'-oligoadenylate synthase.
[0078] The invention furthermore provides the use of said
oligonucleotides as pharmaceuticals or to the use of said
oligonucleotides for the production of pharmaceuticals. In
particular, it is possible to use said oligonucleotides in
pharmaceuticals which are suitable for the prevention and/or
treatment of diseases which accompany the expression or
overexpression of particular genes.
[0079] The invention further provides the use of said
oligonucleotides or of pharmaceuticals containing said
oligonucleotides for the treatment of diseases in which specific
genes are the cause or are involved, due to overexpression.
[0080] The pharmaceuticals of the present invention may be used,
for example, for the treatment of disorders caused by viruses, for
example by CMV, HIV, HSV-1, HSV-2, hepatitis B, hepatitis C
viruses, or papillomaviruses. Pharmaceuticals of the present
invention are particularly suitable for the treatment of RNA
viruses such as, for example, polio viruses, VSV or Influenza
virus, in particular also of double-stranded RNA viruses such as
reoviruses, for example.
[0081] The pharmaceuticals of the present invention are also
suitable, for example, for cancer treatment. In this case it is
possible, for example, to use oligonucleotide sequences which are
directed against targets responsible for the development or growth
of cancers. Examples of such targets are:
[0082] 1) nuclear oncoproteins such as, for example, c-myc, N-myc,
c-myb, c-fos, c-fos/jun, PCNA, p120,
[0083] 2) cytoplasmic/membrane-associated oncoproteins such as, for
example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1,
c-mos, c-src, c-abl, c-ets,
[0084] 3) cellular receptors such as, for example, EGF receptor,
Her-2, c-erbA, VEGF receptor (KDR-1), retinoid receptors, protein
kinase regulatory subunit, c-fms, Tie-2, c-raf-1 kinase, PKC-alpha,
protein kinase A (R1 alpha),
[0085] 4) cytokines, growth factors, extracellular matrix such as,
for example, CSF-1, IL-6, IL-1 a, IL-1b, IL-2, IL-4, IL-6, IL-8,
bFGF, VEGF, myeloblastin, fibronectin,
[0086] 5) inhibitors of tumor suppressor genes such as, for
example, MDM-2.
[0087] The pharmaceuticals of the present invention are further
suitable, for example, for the treatment of disorders which are
influenced by integrins or cell-cell adhesion receptors, for
example by VLA-4, VLA-2, ICAM, VCAM or ELAM.
[0088] The pharmaceuticals of the present invention are also
suitable, for example, for preventing restenosis. In this
connection, it is possible to use, for example, oligonucleotide
sequences which are directed against targets responsible for
proliferation or migration. Examples of such targets are:
[0089] 1) nuclear transactivator proteins and cyclins such as, for
example, c-myc, c-myb, c-fos, c-fos/jun, cyclins and cdc2
kinase,
[0090] 2) mitogens or growth factors such as, for example, PDGF,
bFGF, VEGF, EGF, HB-EGF and TGF-.beta.
[0091] 3) cellular receptors such as, for example, bFGF receptor,
EGF receptor and PDGF receptor.
[0092] The invention further relates to oligonucleotides for the
treatment of asthma, with expression of the adenosine-A1 receptor,
adenosine-A3 receptor, Bradikinin receptor or of IL-13 being
inhibited with the aid of suitable oligonucleotides.
[0093] The invention also relates to oligonucleotides, for example,
for the treatment of cardiovascular diseases, with, for example,
expression of the .beta.1-adrenergic receptor or of a protein from
the EDG family such as, for example, Edg-1 being inhibited.
[0094] The invention also relates to oligonucleotides, for example,
for the treatment of diabetes, with expression of PTP-1 B being
inhibited, for example.
[0095] The pharmaceuticals may be used, for example, in the form of
pharmaceutical preparations which may be administered orally, for
example in the form of tablets, coated tablets, hard or soft
gelatin capsules, solutions, emulsions or suspensions. They may
also be administered rectally, for example in the form of
suppositories, or parenterally, for example in the form of
injection solutions. Pharmaceutical preparations may be produced by
processing said compounds in therapeutically inert organic and
inorganic carriers. Examples of such carriers for tablets, coated
tablets and hard gelatin capsules are lactose, corn starch or
derivatives thereof, talc and stearic acid or salts thereof.
Carriers suitable for the preparation of solutions are water,
polyols, sucrose, invert sugar and glucose. Carriers suitable for
injection solutions are water, alcohols, polyols, glycerol and
vegetable oils. Carriers suitable for suppositories are vegetable
and hardened oils, waxes, fats and semisolid polyols. The
pharmaceutical preparations may also contain preservatives,
solvents, stabilizers, wetting agents, emulsifiers, sweeteners,
colorants, flavorings, salts for modifying the osmotic pressure,
buffers, coating agents, antioxidants and, where appropriate, other
therapeutically active substances.
[0096] Preferred administration forms are topical administrations,
local administrations such as, for example, with the aid of a
catheter or by inhalation, injections or infusions, and oral
administration. For injection, the oligonucleotide derivatives are
formulated in a liquid solution, preferably in a physiologically
acceptable buffer such as, for example, Hank's solution or Ringer's
solution. However, the oligonucleotides may also be formulated in
solid form and be dissolved or suspended prior to use. The dosages
preferred for systematic administration are from approx. 0.01 mg/kg
to approx. 50 mg/kg body weight and day.
[0097] The invention furthermore relates to pharmaceutical
preparations which contain oligonucleotides and/or physiologically
tolerated salts thereof in addition to pharmaceutically suitable
carriers and/or additives.
[0098] The oligonucleotides and/or physiologically tolerated salts
thereof may be administered to animals, preferably to mammals, and
in particular to humans as pharmaceuticals on their own, in
mixtures with one another or in the form of pharmaceutical
preparations which permit topical, percutaneous, parenteral or
enteral application and which contain as active ingredient an
active dose of at least one oligonucleotide in addition to common
pharmaceutically suitable carriers and additives. The preparations
normally contain about from 0.1 to 90% by weight of the
therapeutically active compound. For the treatment of skin
disorders such as, for example, psoriasis or vitiligo, a topical
application, for example in the form of ointments, lotions or
tinctures, emulsions, or suspensions is preferred.
[0099] The pharmaceutical preparations are produced in a manner
known per se (e.g. Remingtons Pharmaceutical Sciences, Mack Publ.
Co., Easton, Pa.), with pharmaceutically inert inorganic and/or
organic carriers being used. For the production of pills, tablets,
coated tablets and hard gelatin capsules, lactose, corn starch
and/or derivatives thereof, talc, stearic acid and/or salts
thereof, etc. may be used, for example. Examples of carriers for
soft gelatin capsules and/or suppositories are fats, waxes,
semisolid and liquid polyols, natural and/or hardened oils, etc.
Examples of carriers suitable for the preparation of solutions
and/or syrups are water, sucrose, invert sugar, glucose, polyols,
etc. Carriers suitable for the preparation of injection solutions
are water, alcohols, glycerol, polyols, vegetable oils, etc.
Carriers suitable for microcapsules, implants and/or rods are mixed
polymers of glycolic acid and lactic acid. Liposome formulations
which are known to the skilled worker (N. Weiner, Drug Develop Ind
Pharm 15 (1989) 1523; "Liposome Dermatics, Springer Verlag 1992),
for example HVJ liposomes (Hayashi, Gene Therapy 3 (1996) 878), are
also suitable. Dermal administration may also be carried out, for
example, with the aid of ionophoretic methods and/or with the aid
of electroporation. In addition, it is possible to use lipofectins
and other carrier systems, for example those which are used in gene
therapy. Particularly suitable systems are those which can be used
to introduce oligonucleotides into eukaryotic cells with great
efficiency.
[0100] In addition to the active substances and the carriers, a
pharmaceutical preparation may also contain additives such as, for
example, fillers, extenders, disintegrants, binding agents,
lubricants, wetting agents, stabilizers, emulsifiers,
preservatives, sweeteners, colorants, flavorings or aromatizers,
thickening agents, diluents, buffer substances, furthermore
solvents and/or solubilizers and/or agents for achieving a depot
effect, and also salts for modifying the osmotic pressure, coating
agents and/or antioxidants. They may also contain two or more
different oligonucleotides and/or their physiologically tolerated
salts and furthermore, in addition to at least one oligonucleotide,
one or more other therapeutically active substances.
[0101] The dose may vary within wide limits and, in each individual
case, has to be adjusted to the individual circumstances.
EXAMPLES
[0102] 1. Synthesis of the Oligonucleotides of the Formula 1
[0103] a) 3' aaaaaaCUUCGCUUCCAACACCUAGAC (The bases indicated by
lower-case letters have a 2'5'-internucleoside bond).
[0104] The syntheses were carried out in an ABI 394 DNA or Expedite
synthesizer (Applied Biosystems, Weiterstadt, Germany). The
synthesis cycles recommended by the manufacturer were used but for
the ribonucleoside-2'-O-phosphoramidites the condensation step was
doubled (with a coupling time of in each case 400 s) and the length
of the iodine oxidation step was increased to 30 s. The solid phase
used was a 1000 .ANG. controlled pore glass (CPG) support which had
5'-O-dimethoxytrityl-N-6-benzoyladenosine (NSS-6101-10A, Chemgenes,
Waltham, Mass.) bound via the 2' or 3' position of the sugar. After
removing the 5'-O-dimethoxytrityl group by cleavage with
trichloroacetic acid, the 2'5'-linked oligonucleotide part was
synthesized by five condensations with
5'-O-dimethoxytrityl-N-6-benzoyl-3'-O-tertbutyldimethy-
lsilyladenosine-2'-O-phosphoramidite (ANP-5681, Chemgenes). This
was followed by synthesizing the 3'5'-linked oligonucleotide part
by repeated condensation with the corresponding
5'-O-dimethoxytrityl-2'-O-tertbutyldi-
methylsilyladenosine-3'-O-phosphoramidites (ANP-5671 to ANP-5680,
Chemgenes). The CPG support was incubated with 750 .mu.l of conc.
ammonia/ethanol (3:1, v:v) with shaking at 30.degree. C. for 24
hours in order to remove the oligomer from the support and to
deprotect the phosphate and amino protective groups. The
supernatant was separated from the support which was then washed
twice more with 150 .mu.l of conc. ammonia/ethanol (3:1, v:v). The
combined supernatants were concentrated under reduced pressure and
the residue was incubated with shaking in 1200 .mu.l of
triethylamine.times.3HF (very toxic) at 30.degree. C. for 24 hours
in order to remove the silyl protective groups. This is followed by
adding 700 .mu.l of n-butanol, cooling the mixture on dry ice for
30 minutes and centrifugation. The pellet was washed with butanol
two more times. In addition, a sodium chloride precipitation was
then carried out. 112 OD (260) of the crude product which shows
only one main band in gelelectrophoresis were obtained. The product
was further characterized by means of HPLC and electrospray mass
spectrometry (negative mode) (calc. 8527.2, found 8527.5).
[0105] b) 3' a*a*a a-C*U*U*C G C*U*U C*C A A*C A C*C*U A G A*C
[0106] The synthesis was carried out analogously to that of example
1a), with the 2'5'-linked oligonucleotide part being synthesized by
three condensations with
5'-O-dimethoxytrityl-N-6-benzoyl-3'-O-tertbutyldimethy-
lsilyladenosine-2'-O-phosphoramidite (ANP-5681, Chemgenes). The
phosphorothioate residue was introduced by using the Beaucage
reagent (RN-1535, Chemgenes, Waltham, Mass.) rather than the iodine
solution in the particular oxidation step. 128 OD (260) of the
crude product which shows only one main band in gelelectrophoresis
were obtained. The product was further characterized by means of
HPLC and electrospray mass spectrometry (negative mode) (calc.
8061.6, found 8062.8).
[0107] 2. Inhibition of Luciferase Expression in SL-3 Cells
[0108] In order to test for biological activity, the following
oligonucleotides as described in example 1 were prepared and tested
for inhibition of luciferase activity.
[0109] a) 3' aaaaaaCUUCGCUUCCAACACCUAGAC
[0110] b) 3' a*a*a a-C*U*U*C G C*U*U C*C A A*C A C*C*U A G A*C
[0111] Transfection: on the day before the experiment,
2.times.10.sup.6 cells/ml were plated out into 6-well plates. The
oligonucleotides were taken up in 100 .mu.l of SF 900II SFM (SF-900
serum-free insect medium II; Gibco BRL 10902-096). For
transfection, 10 .mu.l of lipofectin (1 mg/ml; Gibco BRL) were
mixed with 100 .mu.l of SF 900II SFM and incubated at room
temperature for 15 min. This was followed by pipetting together the
lipofectin mix and the nucleic acid and incubating at room
temperature for 15-45 min. In the meantime, the cells were washed
with 3 ml of serum-free medium and 800 .mu.l of SF 900II SFM and
the nucleic acid/lipofectin mixture were successively added to the
cells, followed by incubation at 25 degrees overnight. On the next
day, 1 ml of medium and serum (Gibco BRL 10122-166; final
concentration 2%) is added.
[0112] Dual-luciferase reporter (DLR; Promega E 1960) assay
system:
[0113]
(hftp://www.promega.com/catalog/CatalogProducts.asp?catalog%5Fname=-
Promega%5FProducts&category%5Fname=Dual%2DLuciferase+Reporter+Assay+System-
&description%5Ftext=Dual%2DLuciferase%3Csup%3E%26reg%3B%3C%2Fsup%3E+Report-
er+Assay+System)
[0114] The Promega DLR assay allows the sequential determination of
the firefly luciferase and Renilla luciferase activities having
different nucleic acid sequences from a single sample. The
oligonucleotides according to the formula I, which were to be
measured, were directed against firefly luciferase. Thus, only
firefly luciferase activity but not Renilla luciferase activity
should be inhibited. Thus, apart from the inhibitory action, the
specificity may also be tested for.
[0115] The passive lysis of the cells in the well plates was
carried out by first removing the medium and washing the cells with
PBS (phosphate-buffered saline (Gibco BRL 14200-067). The medium
was completely removed by suction and then the PLB (passive lysis
buffer, diluted 1:5 with water; 500 .mu.l of PLB (1.times.) to be
introduced into one well of a 6-well plate) was added thereto. This
was followed by a 15-minute incubation with shaking at room
temperature.
[0116] The luciferase assay reagent II (LAR II) was prepared by
resuspending the luciferase assay substrate (LAS) in 10 ml of
luciferase assay buffer II (LAB II). The Stop & Glo reagent was
prepared by adding 200 .mu.l of the Stop & Glo substrate
(solution) into the bottle containing dry Stop & Glo substrate
and mixing the solution for 10 seconds using a vortexer. In order
to produce a 1.times.Stop & Glo solution, 20 .mu.l of the
50.times.Stop & Glo substrate and 1 ml of the Stop & Glo
buffer are combined. This is sufficient for 10 assays.
[0117] DLR-assay: 100 .mu.l of LAR II were introduced together with
20 .mu.l of cell lysate into a well and mixed by pipetting up and
down for 2-3 seconds. After luminometric measurement of firefly
luciferase activity, 100 .mu.l of Stop & Glo reagent were
added, the solution was mixed and then the Renilla-luciferase
activity was determined. The luminescence was determined using the
Fluoroskan Ascent FL luminometer (Thermo Labsystems, Frankfurt,
Germany).
7 Oligonucleotide % Inhibition* a) 3' aaaaaaCUUCGCUUCCAACACCUAGAC
43 (RNA in antisense orientation, with 2'5' A) b) 3' a*a*a
a-C*U*U*C G C*U*U C*C A A*C A C*C*U A G A*C 43 (RNA in antisense
orientation, with 2'5' A) c) 3' aaaaTTTTTTACCTTGTTGAAATGG 12 (not
complementary to target RNA; sense orientation) d) 3' a*a*a
a-C*U*U*C G C*U*U C*C A A*C A C*C*U A G A*C 7 (antisense
orientation, underlined 2'-O-methyl) 5'-G A A G*C G A A G G*U*U G*U
G G A U*C*U*G-teg 0 (Seq ID No.20; sense orientation, without 2'5'
A, teg: triethylene glycol phosphate) 3'-teg-G*C*T*T C*C*A A*C
A*C*C*T A G A*C*C*T*A 0 (Seq ID No.21; antisense orientation, DNA,
underlined 2'-O-methyl) 1100 bp dsRNA 94 without dsRNA 0 %
Inhibition of firefly-luciferase activity ( )
[0118] The firefly luciferase-complementary oligonucleotide a)
inhibited firefly-luciferase activity to a substantially greater
extent than the non-complementary oligonucleotide c). The
stabilization of the oligonucleotide by phosphorothioate residues
(oligonucleotide b) at particular positions on the oligomer
resulted in a markedly improved action. When the entire 3'5'-linked
complementary sequence was derivatized as 2'-O-methyl derivative,
virtually no activity was detectable (oligonucleotide d).
[0119] 3. Inhibition of the Edg-1 Expression in Human Primary
Umbilical Cells (HUVEC)
[0120] In order to test the oligonucleotides of the invention for
inhibition of gene expression in human primary cells, said
oligonucleotides were also directed against a human gene or the
corresponding RNA and tested on human cells (HUVEC, human umbilical
vein endothelial cells).
[0121] The appropriate oligonucleotides were synthesized. The first
two sequences are complementary to edg-1 RNA, while the third
oligonucleotide has base mismatches.
8 #2: 5' A U*C A U*C*C*U G G A G A A*C A*U C* U*U*U-teg #3:
3'-a*a*a a U*A G*U A G G A C*C*U C*U*U G*U*A G A A*A #5: 5' C*C*A A
G G*C*C A G*C*C G*C A G C*U* C*U*G-teg #6: 3'-a*a*a a G G U*U*C*C G
G*U*C G G*C G*U*C G A G A*C #7: 5' C*C*A C*G G A C*A G A C*G C*C*G
C*U* G*U*G-teg #8: 3'-a*a*a a C G U*G C*C*U G*U*C*U G*C G G*C G A
*C A*C
[0122] The control oligonucleotides used were the complementary
sequences (sense orientation) without 2'5'-oligoadenylate,
[0123] where * is phosphorothioate; a*a*a a is a 2'5'-linked
adenylate (partially modified with *) and teg is triethylene glycol
phosphate.
[0124] The oligoribonucleotide analogs which had been modified with
phosphothioate at particular positions were used in human primary
cells as follows, in order to inhibit gene expression of Edg-1 in
human cells (HUVEC, human umbilical vein endothelial cells).
[0125] Cells (HUVECS) and detection of cellular uptake.
Transfection: 24 h prior to the actual transfection, primary HUVECs
(2nd passage, isolated according to Jaffe et al., 1973, J.
Clin.Invest 52, pp.2745), were plated out at a density of
2.5.times.10.sup.5 cells/well in 6-well plates coated with
collagen-I from rats (Biocoat, #354400, Becton Dickinson). For
transfection, 6 .mu.l of lipofectin (1 mg/ml; Gibco BRL, #
18292-011) were mixed with 200 .mu.l of serum-free Opti-MEM 1
medium (Gibco BRL, 31985-047) and incubated at room temperature for
15 minutes. In a parallel reaction, 10 .mu.M (.fwdarw.final
concentration 0.1 .mu.m) or 100 .mu.m (.fwdarw.final concentration
1 .mu.m) of an oligonucleotide solution (in PBS, pH 7.4) was
diluted in a ratio of 1:10 with serum-free Opti-MEM 1 medium and
mixed with the same volume of preincubated lipofectin solution.
After incubation at room temperature for 15 minutes, the volume of
said mixture was increased to 2 ml with serum-free Opti-MEM 1
medium and the cell lawn was washed once with PBS and then
incubated with said mixture at 37.degree. C., 5% CO.sub.2 and 95%
humidity for 4 hours. Subsequently, the cell lawn was washed again
with PBS and then overlaid with serum-containing EGM medium
(CellSystems, # CC-3024+EGM supplements # CC-3124) and incubated
for a further 24 or 48 h. In the case of uptake studies using
fluorescently labeled oligonucleotides, the cells were incubated
for 4 hours, then fixed with 5% paraformaldehyde (in PBS, pH 7.4)
and directly photographed in an inverted fluorescence microscope
(Zeiss Axiovert 135M) with its 200-fold magnification using a
cooled CCD camera (ORCA-1, Bfi optilas) and excitation through an
FITC filter (excitation: 490 nm, emission: 510 nm) and processed
via AQM2000 software (Kinetic Imaging). Western blot analysis: the
cells were lysed by washing the cell lawn once with PBS and then
overlaying it with 200 .mu.l/well 2.times.Laemmli buffer (Bio-Rad
#161-0737). After incubation at room temperature for five minutes,
the cell lysate was collected using a cell scraper (Becton
Dickinson, #3085) and, prior to discontinuous 12% SDS
polyacrylamide gel electrophoresis (SDS-PAGE, Laemmli et al., 1970,
Bio-Rad-Criterion-System #345-0014), heated at 95.degree. C. for 5
minutes and 45 .mu.l of this solution were applied to each slot.
The gel was run in 1.times.Tris/glycine/SDS buffer (Bio-Rad #
161-0732). For the immunoblot, the gel was transferred with the aid
of the Bio-Rad criterion Western blot apparatus (#170-4070) to a
nitrocellulose (NC) membrane (Amersham # RPN 2020D) in
1.times.Tris/glycine buffer (Bio-Rad #161-0732, +10% methanol). The
NC membrane was then saturated at room temperature for 1 hour using
1.times.TBS buffer (Bio-Rad # 170-6435), which contained 5% milk
powder ("Blotto", Bio-Rad #170-6404) and 0.1% Tween 20 (Bio-Rad #
170-6531). After washing the membrane three times in Blotto-free
TBS-Tween (TBST) buffer, the membrane was incubated with the
anti-hEDG-1 primary antibody (polyclonal rabbit serum obtained by
immunization with the EDG-1-specific peptide sequence
CKAHRSSVSDYVNYD, coupled to KLH and affinity-purified against the
abovementioned peptide sequence) in a 1:50 dilution in TBST-Blotto
at 4.degree. C. overnight. After washing three times with TBST, the
secondary antibody (anti-rabbit, alkaline phosphatase-coupled,
Dianova # 111-055-045) was incubated in a 1:2000 dilution in
TBST-Blotto at room temperature for one hour. After another washing
step (see above), the ECF ("enhanced chemifluorescence") detection
reaction (Amersham #RPN5785) was carried out, and the NC membrane
which was covered with clingfilm was incubated with 1 ml of ECF
substrate (Amersham Pharmacia #RPN5785) at room temperature for 5
minutes and then detected using a Fluor-imager 595 scanner
(Amersham Pharmacia). The signal was quantified using the
ImageQuant software (Amersham Pharmacia) and normalized to the
.beta.-tubulin signal which was obtained after destaining (Alpha
Diagnostic Kit # 90100) the NC membrane once and incubating the
.beta.-tubulin-specific primary antibody (affinity-purified rabbit
antibody, Santa Cruz # sc-9104) according to the above-described
method.
9 EDG-1 protein (% of control) Concentration Oligo #2 Oligo #3
Oligo #5 Oligo #6 Oligo #7 Oligo #8 (.mu.M) region "175" region
"175" region "725" region "725" mismath mismath 0 100.0 100.0 100.0
100.0 100.0 100.0 0.01 87.7 51.4 98.6 47.2 89.4 128.3 0.05 100.8
44.2 129.3 35.5 109.7 107.5 0.1 103.0 35.5 109.4 25.1 121.8 103.6
0.5 119.2 40.3 107.2 27.1 95.7 85.6 1.0 104.4 34.0 96.2 22.6 100.1
83.5
[0126] Treatment of the primary HUVEC cells with the chemically
modified single-stranded oligoribonucleotides of the invention led
to a dose-dependent inhibition of edg-1 expression. Only the
oligoribonucleotides #3 and #6 with antisense orientation inhibited
gene expression, while the oligoribonucleotides #2 and #5 with
sense orientation did not inhibit expression. The inhibition proved
to be target gene-specific, since, after treatment with the
edg-1-specific oligoribonucleotides #3 and #6, only the EDG-1
protein levels and not the tubulin level were reduced. The
inhibition proved to be also sequence-specific with regard to the
oligoribonucleotides used, since only the edg-1-homologous
oligoribonucleotides #3 and #6 inhibited edg-1 expression, while
the oligoribonucleotide #8 with antisense orientation, which
differs from the edg-1 sequence by 5 nucleotides, did not inhibit
edg-1 expression.
Sequence CWU 1
1
23 1 25 DNA Artificial Sequence Part of Photinus pyralis Luciferase
1 ttttgaagcg aaggttgtgg atctg 25 2 25 RNA Artificial Sequence Part
of Photinus pyralis Luciferase 2 uuuugaagcg aagguugugg aucug 25 3
80 DNA Artificial Sequence Part of Photinus pyralis Luciferase 3
gcttttacag atgcacatat cgaggtggac atcacttacg cgaaaatgtc tacgtgtata
60 gctccacctg tagtgaatgc 80 4 80 DNA Artificial Sequence Part of
Photinus pyralis Luciferase 4 ccgcgaacga catttataat gaacgtgaat
tgctcaacag ggcgcttgct gtaaatatta 60 cttgcactta acgagttgtc 80 5 80
DNA Artificial Sequence Part of Photinus Pyralis Luciferase 5
gcggtcggta aagttgttcc attttttgaa gcgaaggttg cgccagccat ttcaacaagg
60 taaaaaactt cgcttccaac 80 6 80 DNA Artificial Sequence Part of
Photinus Pyralis Luciferase 6 attttttgaa gcgaaggttg tggatctgga
taccgggaaa taaaaaactt cgcttccaac 60 acctagacct atggcccttt 80 7 40
RNA Artificial Sequence Part of Photinus Pyralis Luciferase 7
gcuuuuacag augcacauau cgagguggac aucacuuacg 40 8 40 RNA Artificial
Sequence Part of Photinus pyralis Luciferase 8 ccgcgaacga
cauuuauaau gaacgugaau ugcucaacag 40 9 40 RNA Artificial Sequence
Part of Photinus pyralis Luciferase 9 gcggucggua aaguuguucc
auuuuuugaa gcgaagguug 40 10 40 RNA Artificial Sequence Part of
Photinus pyralis Luciferase 10 auuuuuugaa gcgaagguug uggaucugga
uaccgggaaa 40 11 26 RNA Artificial Sequence Part of Photinus
pyralis Luciferase 11 caccucgaua ugugcaucug uaaaaa 26 12 25 RNA
Artificial Sequence Part of Photinus pyralis Luciferase 12
gagcaauuca cguucauuau aaaaa 25 13 25 RNA Artificial Sequence Part
of Photinus pyralis Luciferase 13 cagauccaca accuucgcuu caaaa 25 14
25 RNA Artificial Sequence Part of Photinus pyralis Luciferase 14
cagauccaca accuucgcuu caaaa 25 15 25 RNA Artificial Sequence Part
of Photinus Pyralis Luciferase 15 cagagccacc aacuucucuu caaaa 25 16
60 RNA Artificial Sequence Part of human EDG1 16 gaccucggug
guguucauuc ucaucugcug cuuuaucauc cuggagaaca ucuuugucuu 60 17 60 RNA
Artificial Sequence Part of human EDG1 17 auuuccaagg ccagccgcag
cucugagaau guggcgcugc ucaagaccgu aauuaucguc 60 18 25 RNA Artificial
Sequence Part of human EDG1 18 aaagauguuc uccaggauga uaaaa 25 19 25
RNA Artificial Sequence Part of human EDG1 19 cagagcugcg gcuggccuug
gaaaa 25 20 25 RNA Artificial Sequence Part of human EDG1 20
cacagcggcg ucuguccgug gaaaa 25 21 21 RNA Artificial Sequence Part
of Photinus Pyralis Luciferase 21 gaagcgaagg uuguggaucu g 21 22 16
RNA Artificial Sequence Part of Photinus pyralis Luciferase 22
accagaccac aacccg 16 23 15 PRT Artificial Sequence An
EDG-1-specific peptide sequence 23 Cys Lys Ala His Arg Ser Ser Val
Ser Asp Tyr Val Asn Tyr Asp 1 5 10 15
* * * * *