U.S. patent application number 10/178325 was filed with the patent office on 2003-10-23 for antisense modulation of human rho family gene expression.
Invention is credited to Cowsert, Lex M., Roberts, M. Luisa.
Application Number | 20030199467 10/178325 |
Document ID | / |
Family ID | 23529468 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030199467 |
Kind Code |
A1 |
Roberts, M. Luisa ; et
al. |
October 23, 2003 |
Antisense modulation of human Rho family gene expression
Abstract
This invention provides compositions and methods for modulating
expression of members of the human Rho gene family, which encode
low molecular weight GTPases that act as molecular switches in
signal transduction. In preferred embodiments, Rho family members
include RhoA, RhoB, RhoC, RhoG, Rac1 and cdc42. This invention is
also directed to methods for inhibiting hyperproliferation of
cells; these methods can be used diagnostically or therapeutically.
Furthermore, this invention is directed to treatment of conditions
associated with expression of the human Rho family members,
particularly in hyperproliferative disorders.
Inventors: |
Roberts, M. Luisa; (Noank,
CT) ; Cowsert, Lex M.; (Carlsbad, CA) |
Correspondence
Address: |
LICATA & TYRRELL P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
23529468 |
Appl. No.: |
10/178325 |
Filed: |
June 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10178325 |
Jun 21, 2002 |
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09387341 |
Aug 31, 1999 |
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6410323 |
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09387341 |
Aug 31, 1999 |
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09156424 |
Sep 18, 1998 |
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5945290 |
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09387341 |
Aug 31, 1999 |
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09156979 |
Sep 18, 1998 |
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5962672 |
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09387341 |
Aug 31, 1999 |
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09156807 |
Sep 18, 1998 |
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6030786 |
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09387341 |
Aug 31, 1999 |
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09161015 |
Sep 25, 1998 |
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5965370 |
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Current U.S.
Class: |
514/44A ;
536/23.2 |
Current CPC
Class: |
C12N 2310/3521 20130101;
C12N 2310/3525 20130101; C12N 2310/3341 20130101; C12Y 301/05001
20130101; C12N 2310/321 20130101; C12N 2310/341 20130101; C12N
2310/318 20130101; C07H 21/00 20130101; C12N 2310/334 20130101;
C12N 2310/345 20130101; C12N 15/1137 20130101; C12N 2310/346
20130101; C12N 2310/315 20130101; A61K 38/00 20130101; C07B 2200/11
20130101; C12N 2310/3183 20130101; C12N 2310/321 20130101; C12N
2310/322 20130101; C12N 2310/316 20130101; C12N 2310/321 20130101;
C12N 2310/335 20130101 |
Class at
Publication: |
514/44 ;
536/23.2 |
International
Class: |
A61K 048/00; C07H
021/04 |
Claims
What is claimed is:
1. An antisense compound targeted to a nucleic acid molecule
encoding a member of the human Rho family of small GTP binding
proteins, wherein said antisense compound inhibits the expression
of said member of the human Rho family.
2. The antisense compound of claim 1 which is an antisense
oligonucleotide.
3. The antisense compound of claim 2 wherein the oligonucleotide
comprises at least one modified internucleoside linkage.
4. The antisense compound of claim 3 wherein the modified
internucleoside linkage is a phosphorothioate linkage.
5. The antisense compound of claim 2 wherein the oligonucleotide
comprises at least one modified sugar moiety.
6. The antisense compound of claim 5 wherein the modified sugar
moiety is a 2'-O-methoxyethyl sugar moiety.
7. The antisense compound of claim 2 wherein the oligonucleotide
comprises at least one modified nucleobase.
8. The antisense compound of claim 7 wherein the modified
nucleobase is a 5-methylcytosine.
9. The antisense compound of claim 2 wherein the oligonucleotide is
a chimeric oligonucleotide.
10. The antisense compound of claim 1 which is from 8 to 30
nucleobases in length.
11. The antisense compound of claim 1 wherein said member of the
human Rho family of small GTP binding proteins is RhoA, RhoB, RhoC,
or RhoG.
12. The antisense compound of claim 1 wherein said member of the
human Rho family of small GTP binding protein is rac1.
13. The antisense compound of claim 12 wherein the oligonucleotide
comprises SEQ ID NO: 193, 195, 199, 201, 204 or 205.
14. The antisense compound of claim 1 wherein said member of the
human Rho family of small GTP binding protein is cdc42.
15. The antisense compound of claim 14 wherein the oligonucleotide
comprises SEQ ID NO: 219, 220, 221, 222, 223, 224, 225, 226, 227,
228, 229 or 230.
16. A pharmaceutical composition comprising the antisense compound
of claim 1 and a pharmaceutically acceptable carrier or
diluent.
17. The pharmaceutical composition of claim 16 further comprising a
colloidal dispersion system.
18. The pharmaceutical composition of claim 16 wherein the
antisense compound is an antisense oligonucleotide.
19. A method of inhibiting the expression of a member of the human
Rho family of small GTP binding proteins in human cells or tissues
comprising contacting said cells or tissues with the antisense
compound of claim 1 so that expression of said human Rho family
member is inhibited.
20. A method of treating a human having a disease or condition
associated with a member of the human Rho family of small GTP
binding proteins comprising administering to said animal a
therapeutically or prophylactically effective amount of the
antisense compound of claim 1 so that expression of said human Rho
family member is inhibited.
21. The method of claim 20 wherein the disease or condition is a
hyperproliferative condition.
22. The method of claim 21 wherein the hyperproliferative condition
is cancer.
23. The method of claim 20 wherein the disease or condition is
abnormal wound healing or clotting.
24. The method of claim 20 wherein the disease or condition is
ischemia/reperfusion or reoxygenation injury.
25. A compound which inhibits JNK activation by a non-cytokine
activator but does not inhibit JNK activation by a cytokine.
26. The compound of claim 25 wherein the non-cytokine activator is
a stress signal.
27. The compound of claim 26 wherein the stress signal is hydrogen
peroxide or ultraviolet radiation.
28. The compound of claim 25 wherein the cytokine is
IL-1.beta..
29. The compound of claim 25 which is an inhibitor of rhoA.
30. The compound of claim 29 which is an antisense inhibitor of
rhoA.
31. A method of inhibiting JNK activation by a non-cytokine
activator without inhibiting JNK activation by a cytokine
comprising contacting JNK or cells or tissues containing JNK with
an inhibitor of rhoA.
32. The method of claim 31 wherein the non-cytokine activator is a
stress signal.
33. The method of claim 32 wherein the stress signal is hydrogen
peroxide or ultraviolet radiation.
34. The method of claim 31 wherein the cytokine is IL-1.beta..
35. The method of claim 31 wherein the Inhibitor of rhoA is an
antisense oligonucleotide inhibitor of rhoA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. Ser. No. 09/387,341 filed
Aug. 31, 1999 which is a continuation-in-part application of U.S.
Ser. No. 09/156,424 filed Sep. 18, 1998, now issued as U.S. Pat.
No. 5,945,290, and U.S. Ser. No. 09/156,979 filed Sep. 18, 1998,
now issued as U.S. Pat. No. 5,962,672, and U.S. Ser. No. 09/156,807
filed Sep. 18, 1998, now issued as U.S. Pat. No. 6,030,786, and
U.S. Ser. No. 09/161,015, filed on Sep. 25, 1998, now issued as
U.S. Pat. No. 5,965,370.
FIELD OF THE INVENTION
[0002] This invention relates to compositions and methods for
modulating expression of members of the human Rho gene family,
which encode low molecular weight GTPases that act as molecular
switches in signal transduction. This invention is also directed to
methods for inhibiting hyperproliferation of cells; these methods
can be used diagnostically or therapeutically. Furthermore, this
invention is directed to treatment of conditions associated with
expression of the human Rho family member genes.
BACKGROUND OF THE INVENTION
[0003] The Rho family of genes are a sub-family of low molecular
weight GTPases and are related to each other based on sequence
homology and function (Vojtek, A. B., and Cooper, J. A., Cell 1995,
82, 527-529). Other sub-families include Ras, Rab, Arf, and Ran. As
GTPases, these proteins bind and hydrolyze GTP. In an active state,
they bind to GTP and transduce signals of other proteins in signal
transduction pathways. In their inactive state, they are bound to
GDP. Members of the Rho family are typically involved in regulation
of the actin cytoskeleton. Members of the Rho family include RhoA,
RhoB, RhoC, RhoD, RhoE, RhoG, Rac1, Rac2, Rac3 and Cdc42.
[0004] Each class appears to have a unique function in actin
reorganization. Rho has been shown to be essential for the
formation of stress fibers and focal adhesions (Ridley, A. J. and
Hall, A., Cell 1992, 70, 389-399). Focal adhesions are an area of
the cell where integrin receptors cluster and extracellular matrix
proteins such as fibronectin and collagen are bound. Stress fibers
attach at these focal adhesions within a cell. Rac has been shown
to be essential for the formation of membrane ruffles, which
results from the formation of large vesicles within the cell
(Ridley, A. J., et al., Cell 1992, 70, 401-410). Cdc42 (also known
as cdc42Hs and G25K) regulates the formation of filopodia, short
bundles of actin filaments that protrude from a cell (Nobes, C. D.
and Hall, A., Cell 1995, 81, 53-62). Such activities on cell
morphology may play an important role in cell motility,
cytokinesis, and endocytosis.
[0005] Additional functions for the Rho family have begun to be
elucidated. Rac and Rho have been found to promote cadherin-based
cell-cell adhesion (Takaishi, K., et al., J. Cell Biol. 1997, 139,
1047-1059). Rac1 and Cdc42 play a critical role in the c-jun
amino-terminal kinase (JNK)/stress-activated protein kinase (SAPK)
signaling pathway, thereby, potentially having an important role in
gene transcription (Coso, O. A. et al., Cell 1995, 81, 1137-1146).
RhoA, Rac1 and Cdc42 also regulate transcription through
JNK-independent pathways by binding to either serum response factor
(SRF; Hill, C. S., et al., Cell 1995, 81, 1159-1170) or NF-.kappa.B
(Perona, R., et al., Genes and Develop. 1997, 11, 463-475).
[0006] Members of the Rac subfamily have also been found to
regulate oxygen radical production. Both Rac1 (Sundaresan, M., et
al., Biochem. J. 1996, 318, 379-382) and Rac2 (Knaus, U. G., et
al., Science 1991, 254, 1512-1515) are involved in this
process.
[0007] Members of the Rho family are thought to be involved in
various disease processes, including cancer. Rho, Rac and Cdc42 all
play a role in Ras transformation. Rac was found to essential for
transformation by Ras, but not RafCAAX, a modified Raf kinase with
a localization signal from K-ras (Qiu, R.-G., et al., Nature 1995
374, 457-459). Rho is not essential for Ras transformation, but
acts cooperatively in transformation by Ras and RafCAAX (Qiu,
R.-G., et al., Proc. Natl. Acad. Sci. USA 1995, 92, 11781-11785).
Cdc42 was also found to be essential for Ras transformation, but
its role is distinct from that of Rac (Qiu, R.-G., et al., Mol.
Cell Biol. 1997, 17, 3449-3458). In addition to transformation,
members of the Rho family may also play a role in invasion and
metastasis. Michiels, F. et al. (Nature 1995, 375, 338-340)
demonstrated that T-lymphoma cells that constitutively expressed
Rac1 became invasive. Yoshioka, K. et al. (J. Biol. Chem. 1998,
273, 5146-5154) found that cells stably transfected with RhoA were
also invasive. The RhoB gene has been classified as an
immediate-early gene, which means that its transcription is rapidly
activated upon exposure to certain growth factors or mitogens. The
factors shown to activate RhoB transcription include epidermal
growth factor (EGF), platelet-derived growth factor (PDGF),
genotoxic stress from UV light, alkylating xenobiotics and the
retroviral oncogene v-fps. Each of these stimuli triggers DNA
synthesis in cultures of high cell density (Engel et al., J. Biol.
Chem., 1998, 273, 9921-9926). The response of RhoB to these factors
implies a role for RhoB in wound repair and tissue regeneration
upon growth factor stimulation and tumorigenesis upon mitogen
stimulation.
[0008] The involvement of Rho family proteins in ras-mediated
transformation and tumor cell invasion suggests that they could be
novel targets for cancer treatment (Ridley, A. J., Int. J. Biochem.
Cell Biol. 1997, 29, 1225-1229). In particular, overexpression of
the RhoC gene has been associated with pancreatic cancer. Suwa, H.
et al. (Br. J. Cancer, 1998, 77, 147-152) looked for a role of
RhoA, RhoB and RhoC genes in ductal adenocarcinoma of the pancreas.
They found that expression levels of RhoC were higher in tumors
than in normal tissue and that metastatic tumors expressed RhoC at
higher levels than primary tumors. Rho C expression is also
elevated in a megakaryocytic leukemia cell line, CMK. Takada et
al., Exp. Hematol., 1996, 24, 524-530. Manifestations of altered
RhoB regulation also appear in disease states, including the
development of cancer. Cellular transformation and acquisition of
the metastatic phenotype are the two main changes normal cells
undergo during the progression to cancer. Expression of
constitutively activated forms of RhoB have been shown to cause
tumorigenic transformation of NIH 3T3 and Rat1 rodent fibroblasts
(Khosravi-Far et al., Adv. Cancer Res., 1998, 72, 57-107). RhoB has
also been shown to be overexpressed in human breast cancer tissues
(Zalcman et al., Oncogene, 1995, 10, 1935-1945). RhoA is also
believed to be involved in the development of cancer. Cellular
transformation and acquisition of the metastatic phenotype are the
two main changes normal cells undergo during the progression to
cancer. Recent studies demonstrate that RhoA-regulated pathways can
induce both changes in cells. Injecting cells transformed with rhoA
genes directly into the bloodstream of mice produced metastasis, or
tumor growth, in distant organs (del Peso et al., Oncogene, 1997,
15, 3047-3057).
[0009] It has also been suggested that inhibition of Rac genes may
be useful for preventing reoxygenation injury as it occurs when
ischemic cells undergo reperfusion (Kim, K.-S., et al., J. Clin.
Invest. 1998, 101, 1821-1826). With reoxygenation, reactive oxygen
species are presented to the cell, greatly augmenting cell death.
Kim, K.-S., et al. showed that adenoviral-mediated transfer of a
dominant negative Rac1 could inhibit the formation of reactive
oxygen species and protect cells against
hypoxia/reoxygenation-induced cell death. They suggest that
inhibition of rac1 would be useful, clinically, in treatment in
cases where there is the possibility of reperfusion injury.
[0010] Manifestations of altered RhoA regulation also appear in
both injury and disease states. It has been proposed that acute
central nervous system trauma may contribute to the development of
Alzheimer's disease. Findings that show a high concentration of
thrombin, a serine-protease in the blood clotting cascade,
localized to the plaques of Alzheimer's disease brains support this
claim. An excess of thrombin has been shown to stimulate Rho A
activity with a concomitant increase in apoptosis (programmed cell
death) (Donovan et al., J. Neurosci., 1997, 17, 5316-5326). These
studies also imply a role for RhoA in wound repair and clotting
disorders.
[0011] Although members of the Rho family have been implicated in
various disease processes including cancer and reoxygenation
injury, no effective therapy specifically targeting these proteins
is available. Antisense oligonucleotides have been used to study
the role of some Rho family members in various physiological
processes. Dorseuil, O., et al. (J. Biol. Chem. 1992, 267,
20540-20542) used an 16-mer antisense oligonucleotide targeted to
the start site of both Rac1 and Rac2 and demonstrated a
dose-dependent reduction in superoxide production in whole cells.
Brenner, B., et al. (Biochem. Biophys. Res. Commun. 1997, 231,
802-807) used a similar oligonucleotide (a 15-mer targeted to the
start site) and showed that inhibition of Rac2 protein expression
prevented L-selectin-induced actin polymerization. An 45-mer
antisense oligonucleotide targeted to the 3'-UTR has also been used
as a probe for rac1 (Didsbury, J., et al., J. Biol. Chem. 1989,
264, 16378-16382).
[0012] Thus, there remains an unmet need for compositions and
methods targeting expression of Rho family members, and disease
processes associated there-with.
SUMMARY OF THE INVENTION
[0013] The present invention provides oligonucleotides which are
targeted to nucleic acids encoding members of the human Rho gene
family and are capable of modulating Rho family members expression.
The present invention also provides chimeric oligonucleotides
targeted to nucleic acids encoding human Rho family members. The
oligonucleotides of the invention are believed to be useful both
diagnostically and therapeutically, and are believed to be
particularly useful in the methods of the present invention.
[0014] The present invention also comprises methods of modulating
the expression of human Rho family members using the
oligonucleotides of the invention. Methods of inhibiting Rho family
members expression are provided; these methods are believed to be
useful both therapeutically and diagnostically. These methods are
also useful as tools, for example, for detecting and determining
the role of Rho family member expression in various cell functions
and physiological processes and conditions and for diagnosing
conditions associated with expression of Rho family members.
[0015] The present invention also comprises methods for diagnosing
and treating cancer and preventing reoxygenation injury. These
methods are believed to be useful, for example, in diagnosing Rho
family member-associated disease progression. These methods employ
the oligonucleotides of the invention. These methods are believed
to be useful both therapeutically, including prophylactically, and
as clinical research and diagnostic tools.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Members of the Rho family of GTPases are essential for
transformation by Ras and play a role in tumor cell invasion. In
addition, the Rac subfamily is a regulator of oxygen radical
formation. As such, they represent attractive targets for
antineoplastic therapy and preventative agents for radical
deoxygenation. In particular, modulation of the expression of RhoC
may be useful for the treatment of pancreatic carcinomas and
modulation of Rac1 may be useful for preventing
ischemia/reperfusion injury.
[0017] Antisense oligonucleotides targeting members of the Rho
family represent a novel therapeutic approach.
[0018] The present invention employs antisense compounds,
particularly oligonucleotides, for use in modulating the function
of nucleic acid molecules encoding Rho family members, ultimately
modulating the amount of a Rho family member produced. This is
accomplished by providing oligonucleotides which specifically
hybridize with nucleic acids, preferably mRNA, encoding a Rho
family member.
[0019] This relationship between an antisense compound such as an
oligonucleotide and its complementary nucleic acid target, to which
it hybridizes, is commonly referred to as "antisense". "Targeting"
an oligonucleotide to a chosen nucleic acid target, in the context
of this invention, is a multistep process. The process usually
begins with identifying a nucleic acid sequence whose function is
to be modulated. This may be, as examples, a cellular gene (or mRNA
made from the gene) whose expression is associated with a
particular disease state, or a foreign nucleic acid from an
infectious agent. In the present invention, the targets are nucleic
acids encoding Rho family members; in other words, a gene encoding
a Rho family member, or mRNA expressed from a Rho family member
gene. mRNA which encodes a Rho family member is presently the
preferred target. The targeting process also includes determination
of a site or sites within the nucleic acid sequence for the
antisense interaction to occur such that modulation of gene
expression will result.
[0020] In accordance with this invention, persons of ordinary skill
in the art will understand that messenger RNA includes not only the
information to encode a protein using the three letter genetic
code, but also associated ribonucleotides which form a region known
to such persons as the 5'-untranslated region, the 3'-untranslated
region, the 5' cap region and intron/exon junction ribonucleotides.
Thus, oligonucleotides may be formulated in accordance with this
invention which are targeted wholly or in part to these associated
ribonucleotides as well as to the informational ribonucleotides.
The oligonucleotide may therefore be specifically hybridizable with
a transcription initiation site region, a translation initiation
codon region, a 5' cap region, an intron/exon junction, coding
sequences, a translation termination codon region or sequences in
the 5'- or 3'-untranslated region. Since, as is known in the art,
the translation initiation codon is typically 5'-AUG (in
transcribed mRNA molecules; 5'-ATG in the corresponding DNA
molecule), the translation initiation codon is also referred to as
the "AUG codon," the "start codon" or the "AUG start codon." A
minority of genes have a translation initiation codon having the
RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and
5'-CUG have been shown to function in vivo. Thus, the terms
"translation initiation codon" and "start codon" can encompass many
codon sequences, even though the initiator amino acid in each
instance is typically methionine (in eukaryotes) or
formylmethionine (prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA molecule transcribed from a gene encoding a
Rho family member, regardless of the sequence(s) of such codons. It
is also known in the art that a translation termination codon (or
"stop codon") of a gene may have one of three sequences, i.e.,
5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are
5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start codon
region," "AUG region" and "translation initiation codon region"
refer to a portion of such an mRNA or gene that encompasses from
about 25 to about 50 contiguous nucleotides in either direction
(i.e., 5' or 3') from a translation initiation codon. This region
is a preferred target region. Similarly, the terms "stop codon
region" and "translation termination codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation termination codon. This region is a
preferred target region. The open reading frame (ORF) or "coding
region," which is known in the art to refer to the region between
the translation initiation codon and the translation termination
codon, is also a region which may be targeted effectively. Other
preferred target regions include the 5' untranslated region
(5'UTR), known in the art to refer to the portion of an mRNA in the
5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA or corresponding nucleotides on the
gene and the 3' untranslated region (3'UTR), known in the art to
refer to the portion of an mRNA in the 3' direction from the
translation termination codon, and thus including nucleotides
between the translation termination codon and 3' end of an mRNA or
corresponding nucleotides on the gene. The 5' cap of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap. The
5' cap region may also be a preferred target region.
[0021] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a pre-mRNA transcript to yield one or more
mature mRNA. The remaining (and therefore translated) regions are
known as "exons" and are spliced together to form a continuous mRNA
sequence. mRNA splice sites, i.e., exon-exon or intron-exon
junctions, may also be preferred target regions, and are
particularly useful in situations where aberrant splicing is
implicated in disease, or where an overproduction of a particular
mRNA splice product is implicated in disease. Aberrant fusion
junctions due to rearrangements or deletions are also preferred
targets. Targeting particular exons in alternatively spliced mRNAs
may also be preferred. It has also been found that introns can also
be effective, and therefore preferred, target regions for antisense
compounds targeted, for example, to DNA or pre-mRNA.
[0022] Once the target site or sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired modulation.
[0023] "Hybridization", in the context of this invention, means
hydrogen bonding, also known as Watson-Crick base pairing, between
complementary bases, usually on opposite nucleic acid strands or
two regions of a nucleic acid strand. Guanine and cytosine are
examples of complementary bases which are known to form three
hydrogen bonds between them. Adenine and thymine are examples of
complementary bases which form two hydrogen bonds between them.
[0024] "Specifically hybridizable" and "complementary" are terms
which are used to indicate a sufficient degree of complementarity
such that stable and specific binding occurs between the DNA or RNA
target and the oligonucleotide.
[0025] It is understood that an oligonucleotide need not be 100%
complementary to its target nucleic acid sequence to be
specifically hybridizable. An oligonucleotide is specifically
hybridizable when binding of the oligonucleotide to the target
interferes with the normal function of the target molecule to cause
a loss of utility, and there is a sufficient degree of
complementarity to avoid non-specific binding of the
oligonucleotide to non-target sequences under conditions in which
specific binding is desired, i.e., under physiological conditions
in the case of in vivo assays or therapeutic treatment or, in the
case of in vitro assays, under conditions in which the assays are
conducted.
[0026] Hybridization of antisense oligonucleotides with mRNA
interferes with one or more of the normal functions of mRNA. The
functions of mRNA to be interfered with include all vital functions
such as, for example, translocation of the RNA to the site of
protein translation, translation of protein from the RNA, splicing
of the RNA to yield one or more mRNA species, and catalytic
activity which may be engaged in by the RNA. Binding of specific
protein(s) to the RNA may also be interfered with by antisense
oligonucleotide hybridization to the RNA.
[0027] The overall effect of interference with mRNA function is
modulation of expression of a Rho family member. In the context of
this invention "modulation" means either inhibition or stimulation;
i.e., either a decrease or increase in expression. This modulation
can be measured in ways which are routine in the art, for example
by Northern blot assay of mRNA expression, or reverse transcriptase
PCR, as taught in the examples of the instant application or by
Western blot or ELISA assay of protein expression, or by an
immunoprecipitation assay of protein expression. Effects on cell
proliferation or tumor cell growth can also be measured, as taught
in the examples of the instant application. Inhibition is presently
preferred.
[0028] The oligonucleotides of this invention can be used in
diagnostics, therapeutics, prophylaxis, and as research reagents
and in kits. Since the oligonucleotides of this invention hybridize
to nucleic acids encoding a Rho family member, sandwich,
colorimetric and other assays can easily be constructed to exploit
this fact. Provision of means for detecting hybridization of
oligonucleotide with a Rho family member gene or mRNA can routinely
be accomplished. Such provision may include enzyme conjugation,
radiolabelling or any other suitable detection systems. Kits for
detecting the presence or absence of a Rho family member may also
be prepared.
[0029] The present invention is also suitable for diagnosing
abnormal proliferative states in tissue or other samples from
patients suspected of having a hyperproliferative disease such as
cancer. The ability of the oligonucleotides of the present
invention to inhibit cell proliferation may be employed to diagnose
such states. A number of assays may be formulated employing the
present invention, which assays will commonly comprise contacting a
tissue sample with an oligonucleotide of the invention under
conditions selected to permit detection and, usually, quantitation
of such inhibition. In the context of this invention, to "contact"
tissues or cells with an oligonucleotide or oligonucleotides means
to add the oligonucleotide(s), usually in a liquid carrier, to a
cell suspension or tissue sample, either in vitro or ex vivo, or to
administer the oligonucleotide(s) to cells or tissues within an
animal. Similarly, the present invention can be used to distinguish
a Rho family member-associated tumor from tumors having other
etiologies, or those associated with one rho family member from
another, in order that an efficacious treatment regimen can be
designed.
[0030] The oligonucleotides of this invention may also be used for
research purposes. Thus, the specific hybridization exhibited by
the oligonucleotides may be used for assays, purifications,
cellular product preparations and in other methodologies which may
be appreciated by persons of ordinary skill in the art.
[0031] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid or
deoxyribonucleic acid. This term includes oligonucleotides composed
of naturally-occurring nucleobases, sugars and covalent intersugar
(backbone) linkages as well as oligonucleotides having
non-naturally-occurring portions which function similarly. Such
modified or substituted oligonucleotides are often preferred over
native forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced binding to target and increased
stability in the presence of nucleases.
[0032] The antisense compounds in accordance with this invention
preferably comprise from about 5 to about 50 nucleobases.
Particularly preferred are antisense oligonucleotides comprising
from about 8 to about 30 nucleobases (i.e. from about 8 to about 30
linked nucleosides). As is known in the art, a nucleoside is a
base-sugar combination. The base portion of the nucleoside is
normally a heterocyclic base. The two most common classes of such
heterocyclic bases are the purines and the pyrimidines. Nucleotides
are nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. In turn the respective ends of this
linear polymeric structure can be further joined to form a circular
structure, however, open linear structures are generally preferred.
Within the oligonucleotide structure, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
[0033] Specific examples of preferred antisense compounds useful in
this invention include oligonucleotides containing modified
backbones or non-natural internucleoside linkages. As defined in
this specification, oligonucleotides having modified backbones
include those that retain a phosphorus atom in the backbone and
those that do not have a phosphorus atom in the backbone. For the
purposes of this specification, and as sometimes referenced in the
art, modified oligonucleotides that do not have a phosphorus atom
in their internucleoside backbone can also be considered to be
oligonucleosides.
[0034] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
borano-phosphates having normal 3'-5' linkages, 2'-5' linked
analogs of these, and those having inverted polarity wherein the
adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or
2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are
also included.
[0035] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. No. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361 and 5,625,050.
[0036] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
[0037] Representative U.S. patents that teach the preparation of
the above oligonucleosides include, but are not limited to, U.S.
Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312; 5,633,360; 5,677,437; and 5,677,439.
[0038] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative U.S. patents that teach the
preparation of PNA compounds include, but are not limited to, U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of
PNA compounds can be found in Nielsen et al. (Science, 1991, 254,
1497-1500).
[0039] Most preferred embodiments of the invention are
oligonucleotides with phosphorothioate backbones and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0040] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O--, S--, or N-alkyl,
O-alkyl-O-alkyl, O--, S--, or N-alkenyl, or O--, S--or N-alkynyl,
wherein the alkyl, alkenyl and alkynyl may be substituted or
unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10
alkenyl and alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.n(CH.sub.3)].sub.2, where n and
m are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy
(2'--O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'--O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in U.S. patent application Ser. No. 09/016,520, filed
on Jan. 30, 1998, which is commonly owned with the instant
application and the contents of which are herein incorporated by
reference.
[0041] Other preferred modifications include 2'-methoxy
(2'--O--CH.sub.3), 2'-aminopropoxy
(2'--OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'--F).
Similar modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative U.S. patents that teach the
preparation of such modified sugars structures include, but are not
limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,0531
5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920.
[0042] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C or m5c), 5-hydroxymethyl cytosine,
xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl
derivatives of adenine and guanine, 2-propyl and other alkyl
derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and
cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further
nucleobases include those disclosed in U.S. Pat. No. 3,687,808,
those disclosed in the Concise Encyclopedia Of Polymer Science And
Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley
& Sons, those disclosed by Englisch et al. (Angewandte Chemie,
International Edition 1991, 30, 613-722), and those disclosed by
Sanghvi, Y. S., Chapter 15, Antisense Research and Applications
1993, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press.
Certain of these nucleobases are particularly useful for increasing
the binding affinity of the oligomeric compounds of the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and O-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and
Lebleu, B., eds., Antisense Research and Applications 1993, CRC
Press, Boca Raton, pages 276-278) and are presently preferred base
substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0043] Representative U.S. patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include, but are not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.:
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941.
[0044] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA
1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Lett. 1994, 4, 1053-1059), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10,
1111-1118; Kabanov et al., FEBS Lett. 1990, 259, 327-330;
Svinarchuk et al., Biochimie 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res. 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett. 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al. , J.
Pharmacol. Exp. Ther. 1996, 277, 923-937).
[0045] Representative U.S. patents that teach the preparation of
such oligonucleotide conjugates include, but are not limited to,
U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;
5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;
5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;
4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;
4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;
5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;
5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;
5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;
5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928
and 5,688,941.
[0046] The present invention also includes oligonucleotides which
are chimeric oligonucleotides. "Chimeric" oligonucleotides or
"chimeras," in the context of this invention, are oligonucleotides
which contain two or more chemically distinct regions, each made up
of at least one nucleotide. These oligonucleotides typically
contain at least one region wherein the oligonucleotide is modified
so as to confer upon the oligonucleotide increased resistance to
nuclease degradation, increased cellular uptake, and/or increased
binding affinity for the target nucleic acid. An additional region
of the oligonucleotide may serve as a substrate for enzymes capable
of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H
is a cellular endonuclease which cleaves the RNA strand of an
RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the
efficiency of antisense inhibition of gene expression. Cleavage of
the RNA target can be routinely detected by gel electrophoresis
and, if necessary, associated nucleic acid hybridization techniques
known in the art. This RNAse H-mediated cleavage of the RNA target
is distinct from the use of ribozymes to cleave nucleic acids.
Ribozymes are not comprehended by the present invention.
[0047] Examples of chimeric oligonucleotides include but are not
limited to "gapmers," in which three distinct regions are present,
normally with a central region flanked by two regions which are
chemically equivalent to each other but distinct from the gap. A
preferred example of a gapmer is an oligonucleotide in which a
central portion (the "gap") of the oligonucleotide serves as a
substrate for RNase H and is preferably composed of
2'-deoxynucleotides, while the flanking portions (the 5' and 3'
"wings") are modified to have greater affinity for the target RNA
molecule but are unable to support nuclease activity (e.g.,
2'-fluoro- or 2'-O-methoxyethyl-substituted). Other chimeras
include "wingmers," also known in the art as "hemimers," that is,
oligonucleotides with two distinct regions. In a preferred example
of a wingmer, the 5' portion of the oligonucleotide serves as a
substrate for RNase H and is preferably composed of
2'-deoxynucleotides, whereas the 3' portion is modified in such a
fashion so as to have greater affinity for the target RNA molecule
but is unable to support nuclease activity (e.g., 2'-fluoro- or
2'-O-methoxyethyl-substituted), or vice-versa. In one embodiment,
the oligonucleotides of the present invention contain a
2'-O-methoxyethyl (2'--O--CH.sub.2CH.sub.2OCH.sub.3) modification
on the sugar moiety of at least one nucleotide. This modification
has been shown to increase both affinity of the oligonucleotide for
its target and nuclease resistance of the oligonucleotide.
According to the invention, one, a plurality, or all of the
nucleotide subunits of the oligonucleotides of the invention may
bear a 2'-O-methoxyethyl (--O--CH.sub.2CH.sub.2OCH.sub.3)
modification. Oligonucleotides comprising a plurality of nucleotide
subunits having a 2'-O-methoxyethyl modification can have such a
modification on any of the nucleotide subunits within the
oligonucleotide, and may be chimeric oligonucleotides. Aside from
or in addition to 2'-O-methoxyethyl modifications, oligonucleotides
containing other modifications which enhance antisense efficacy,
potency or target affinity are also preferred. Chimeric
oligonucleotides comprising one or more such modifications are
presently preferred.
[0048] The oligonucleotides used in accordance with this invention
may be conveniently and routinely made through the well-known
technique of solid phase synthesis. Equipment for such synthesis is
sold by several vendors including Applied Biosystems. Any other
means for such synthesis may also be employed; the actual synthesis
of the oligonucleotides is well within the talents of the
routineer. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and 2'-alkoxy or
2'-alkoxyalkoxy derivatives, including 2'-O-methoxyethyl
oligonucleotides (Martin, P., Helv. Chim. Acta 1995, 78, 486-504).
It is also well known to use similar techniques and commercially
available modified amidites and controlled-pore glass (CPG)
products such as biotin, fluorescein, acridine or psoralen-modified
amidites and/or CPG (available from Glen Research, Sterling, Va.)
to synthesize fluorescently labeled, biotinylated or other
conjugated oligonucleotides.
[0049] The antisense compounds of the present invention include
bioequivalent compounds, including pharmaceutically acceptable
salts and prodrugs. This is intended to encompass any
pharmaceutically acceptable salts, esters, or salts of such esters,
or any other compound which, upon administration to an animal
including a human, is capable of providing (directly or indirectly)
the biologically active metabolite or residue thereof. Accordingly,
for example, the disclosure is also drawn to pharmaceutically
acceptable salts of the nucleic acids of the invention and prodrugs
of such nucleic acids. APharmaceutically acceptable salts@ are
physiologically and pharmaceutically acceptable salts of the
nucleic acids of the invention: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto (see, for example, Berge et
al., "Pharmaceutical Salts," J. of Pharma. Sci. 1977, 66,
1-19).
[0050] For oligonucleotides, examples of pharmaceutically
acceptable salts include but are not limited to (a) salts formed
with cations such as sodium, potassium, ammonium, magnesium,
calcium, polyamines such as spermine and spermidine, etc.; (b) acid
addition salts formed with inorganic acids, for example
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; (c) salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like;
and (d) salts formed from elemental anions such as chlorine,
bromine, and iodine.
[0051] The oligonucleotides of the invention may additionally or
alternatively be prepared to be delivered in a Aprodrug@ form. The
term Aprodrug@ indicates a therapeutic agent that is prepared in an
inactive form that is converted to an active form (i.e., drug)
within the body or cells thereof by the action of endogenous
enzymes or other chemicals and/or conditions. In particular,
prodrug versions of the oligonucleotides of the invention are
prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives
according to the methods disclosed in WO 93/24510 to Gosselin et
al., published Dec. 9, 1993.
[0052] For therapeutic or prophylactic treatment, oligonucleotides
are administered in accordance with this invention. Oligonucleotide
compounds of the invention may be formulated in a pharmaceutical
composition, which may include pharmaceutically acceptable
carriers, thickeners, diluents, buffers, preservatives, surface
active agents, neutral or cationic lipids, lipid complexes,
liposomes, penetration enhancers, carrier compounds and other
pharmaceutically acceptable carriers or excipients and the like in
addition to the oligonucleotide. Such compositions and formulations
are comprehended by the present invention.
[0053] Pharmaceutical compositions comprising the oligonucleotides
of the present invention may include penetration enhancers in order
to enhance the alimentary delivery of the oligonucleotides.
Penetration enhancers may be classified as belonging to one of five
broad categories, i.e., fatty acids, bile salts, chelating agents,
surfactants and non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems 1991, 8, 91-192; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7,
1-33). One or more penetration enhancers from one or more of these
broad categories may be included.
[0054] Various fatty acids and their derivatives which act as
penetration enhancers include, for example, oleic acid, lauric
acid, capric acid, myristic acid, palmitic acid, stearic acid,
linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate,
monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic
acid, arachidonic acid, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono-
and di-glycerides and physiologically acceptable salts thereof
(i.e., oleate, laurate, caprate, myristate, palmitate, stearate,
linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems 1991, page 92; Muranishi, Critical Reviews in
Therapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J.
Pharm. Pharmacol. 1992 44, 651-654).
[0055] The physiological roles of bile include the facilitation of
dispersion and absorption of lipids and fat-soluble vitamins
(Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological
Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill,
New York, N.Y., 1996, pages 934-935). Various natural bile salts,
and their synthetic derivatives, act as penetration enhancers.
Thus, the term "bile salt" includes any of the naturally occurring
components of bile as well as any of their synthetic
derivatives.
[0056] Complex formulations comprising one or more penetration
enhancers may be used. For example, bile salts may be used in
combination with fatty acids to make complex formulations.
[0057] Chelating agents include, but are not limited to, disodium
ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,
sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl
derivatives of collagen, laureth-9 and N-amino acyl derivatives of
beta-diketones (enamines)[Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical
Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et
al., J. Control Rel. 1990, 14, 43-51). Chelating agents have the
added advantage of also serving as DNase inhibitors.
[0058] Surfactants include, for example, sodium lauryl sulfate,
polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems
1991, page 92); and perfluorochemical emulsions, such as FC-43
(Takahashi et al., J. Pharm. Phamacol. 1988, 40, 252-257).
[0059] Non-surfactants include, for example, unsaturated cyclic
ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems 1991,
page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol. 1987, 39, 621-626).
[0060] As used herein, "carrier compound" refers to a nucleic acid,
or analog thereof, which is inert (i.e., does not possess
biological activity per se) but is recognized as a nucleic acid by
in vivo processes that reduce the bioavailability of a nucleic acid
having biological activity by, for example, degrading the
biologically active nucleic acid or promoting its removal from
circulation. The coadministration of a nucleic acid and a carrier
compound, typically with an excess of the latter substance, can
result in a substantial reduction of the amount of nucleic acid
recovered in the liver, kidney or other extracirculatory
reservoirs, presumably due to competition between the carrier
compound and the nucleic acid for a common receptor.
[0061] In contrast to a carrier compound, a "pharmaceutically
acceptable carrier" (excipient) is a pharmaceutically acceptable
solvent, suspending agent or any other pharmacologically inert
vehicle for delivering one or more nucleic acids to an animal. The
pharmaceutically acceptable carrier may be liquid or solid and is
selected with the planned manner of administration in mind so as to
provide for the desired bulk, consistency, etc., when combined with
a nucleic acid and the other components of a given pharmaceutical
composition. Typical pharmaceutically acceptable carriers include,
but are not limited to, binding agents (e.g., pregelatinized maize
starch, polyvinyl-pyrrolidone or hydroxypropyl methylcellulose,
etc.); fillers (e.g., lactose and other sugars, microcrystalline
cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose,
polyacrylates or calcium hydrogen phosphate, etc.); lubricants
(e.g., magnesium stearate, talc, silica, colloidal silicon dioxide,
stearic acid, metallic stearates, hydrogenated vegetable oils, corn
starch, polyethylene glycols, sodium benzoate, sodium acetate,
etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.);
or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained
release oral delivery systems and/or enteric coatings for orally
administered dosage forms are described in U.S. Pat. No. 4,704,295;
4,556,552; 4,309,406; and 4,309,404.
[0062] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional
compatible pharmaceutically-active materials such as, e.g.,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the composition of present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the invention.
[0063] Regardless of the method by which the oligonucleotides of
the invention are introduced into a patient, colloidal dispersion
systems may be used as delivery vehicles to enhance the in vivo
stability of the oligonucleotides and/or to target the
oligonucleotides to a particular organ, tissue or cell type.
Colloidal dispersion systems include, but are not limited to,
macromolecule complexes, nanocapsules, microspheres, beads and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, liposomes and lipid:oligonucleotide complexes of
uncharacterized structure. A preferred colloidal dispersion system
is a plurality of liposomes. Liposomes are microscopic spheres
having an aqueous core surrounded by one or more outer layers made
up of lipids arranged in a bilayer configuration (see, generally,
Chonn et al., Current Op. Biotech. 1995, 6, 698-708).
[0064] The pharmaceutical compositions of the present invention may
be administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration may be topical (including ophthalmic, vaginal,
rectal, intranasal, epidermal, and transdermal), oral or
parenteral. Parenteral administration includes intravenous drip,
subcutaneous, intraperitoneal or intramuscular injection, pulmonary
administration, e.g., by inhalation or insufflation, or
intracranial, e.g., intrathecal or intraventricular,
administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration.
[0065] Formulations for topical administration may include
transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders. Conventional
pharmaceutical carriers, aqueous, powder or oily bases, thickeners
and the like may be necessary or desirable. Coated condoms, gloves
and the like may also be useful.
[0066] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets or tablets. Thickeners, flavoring agents,
diluents, emulsifiers, dispersing aids or binders may be
desirable.
[0067] Compositions for parenteral administration may include
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives. In some cases it may be more
effective to treat a patient with an oligonucleotide of the
invention in conjunction with other traditional therapeutic
modalities in order to increase the efficacy of a treatment
regimen. In the context of the invention, the term "treatment
regimen" is meant to encompass therapeutic, palliative and
prophylactic modalities. For example, a patient may be treated with
conventional chemotherapeutic agents, particularly those used for
tumor and cancer treatment. Examples of such chemotherapeutic
agents include but are not limited to daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,
bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphor- amide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide, trimetrexate,
teniposide, cisplatin and diethylstilbestrol (DES). See, generally,
The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp.
1206-1228, Berkow et al., eds., Rahway, N.J. Preferred are
chemotherapeutic agents which are direct or indirect inhibitors of
a Rho family member. These include MTX, Tomudex and fluorinated
pyrimidines such as 5-FU and 5-FUdR. When used with the compounds
of the invention, such chemotherapeutic agents may be used
individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,
5-FU and oligonucleotide for a period of time followed by MTX and
oligonucleotide), or in combination with one or more other such
chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or
5-FU, radiotherapy and oligonucleotide).
[0068] The formulation of therapeutic compositions and their
subsequent administration is believed to be within the skill of
those in the art. Dosing is dependent on severity and
responsiveness of the disease state to be treated, with the course
of treatment lasting from several days to several months, or until
a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides,
and can generally be estimated based on EC.sub.50s found to be
effective in vitro and in in vivo animal models. In general, dosage
is from 0.01 .mu.g to 100 g per kg of body weight, and may be given
once or more daily, weekly, monthly or yearly, or even once every 2
to 20 years. Persons of ordinary skill in the art can easily
estimate repetition rates for dosing based on measured residence
times and concentrations of the drug in bodily fluids or tissues.
Following successful treatment, it may be desirable to have the
patient undergo maintenance therapy to prevent the recurrence of
the disease state, wherein the oligonucleotide is administered in
maintenance doses, ranging from 0.01 .mu.g to 100 g per kg of body
weight, once or more daily, to once every 20 years.
[0069] Thus, in the context of this invention, by "therapeutically
effective amount" is meant the amount of the compound which is
required to have a therapeutic effect on the treated individual.
This amount, which will be apparent to the skilled artisan, will
depend upon the age and weight of the individual, the type of
disease to be treated, perhaps even the gender of the individual,
and other factors which are routinely taken into consideration when
designing a drug treatment. A therapeutic effect is assessed in the
individual by measuring the effect of the compound on the disease
state in the animal. For example, if the disease to be treated is
cancer, therapeutic effects are assessed by measuring the rate of
growth or the size of the tumor, or by measuring the production of
compounds such as cytokines, production of which is an indication
of the progress or regression of the tumor.
[0070] The following examples illustrate the present invention and
are not intended to limit the same.
EXAMPLES
Example 1
Synthesis of Oligonucleotides
[0071] Unmodified oligodeoxynucleotides are synthesized on an
automated DNA synthesizer (Applied Biosystems model 380B) using
standard phosphoramidite chemistry with oxidation by iodine.
.beta.-cyanoethyldiisopropyl-phosphoramidites are purchased from
Applied Biosystems (Foster City, Calif.). For phosphorothioate
oligonucleotides, the standard oxidation bottle was replaced by a
0.2 M solution of .sup.3H-1,2-benzodithiole-3-one 1,1-dioxide in
acetonitrile for the stepwise thiation of the phosphite linkages.
The thiation cycle wait step was increased to 68 seconds and was
followed by the capping step.
[0072] 2'-methoxy oligonucleotides were synthesized using
2'-methoxy.beta.-cyanoethyldiisopropyl-phosphoramidites (Chemgenes,
Needham, Mass.) and the standard cycle for unmodified
oligonucleotides, except the wait step after pulse delivery of
tetrazole and base was increased to 360 seconds. Other 2'-alkoxy
oligonucleotides were synthesized by a modification of this method,
using appropriate 2'-modified amidites such as those available from
Glen Research, Inc., Sterling, Va.
[0073] 2'-fluoro oligonucleotides were synthesized as described in
Kawasaki et al. (J. Med. Chem. 1993, 36, 831-841). Briefly, the
protected nucleoside N.sup.6-benzoyl-2'-deoxy-2'-fluoroadenosine
was synthesized utilizing commercially available
9-.beta.-D-arabinofuranosyladenine as starting material and by
modifying literature procedures whereby the 2'-.alpha.-fluoro atom
is introduced by a S.sub.N2-displacement of a 2'-.beta.-O-trifyl
group. Thus N.sup.6-benzoyl-9-.beta.-D-arabinofuranosy- ladenine
was selectively protected in moderate yield as the
3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the
THP and N.sup.6-benzoyl groups was accomplished using standard
methodologies and standard methods were used to obtain the
5'-dimethoxytrityl-(DMT) and 5'-DMT-3'-phosphoramidite
intermediates.
[0074] The synthesis of 2'-deoxy-2'-fluoroguanosine was
accomplished using tetraisopropyldisiloxanyl (TPDS) protected
9-.beta.-D-arabinofuranosylgua- nine as starting material, and
conversion to the intermediate
diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS
group was followed by protection of the hydroxyl group with THP to
give diisobutyryl di-THP protected arabinofuranosylguanine.
Selective O-deacylation and triflation was followed by treatment of
the crude product with fluoride, then deprotection of the THP
groups. Standard methodologies were used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidi- tes.
[0075] Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by
the modification of a known procedure in which
2,2'-anhydro-1-.beta.-D-arabin- ofuranosyluracil was treated with
70% hydrogen fluoride-pyridine. Standard procedures were used to
obtain the 5'-DMT and 5'-DMT-3' phosphoramidites.
[0076] 2'-deoxy-2'-fluorocytidine was synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N.sup.4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures
were used to obtain the 5'-DMT and 5'-DMT-3' phosphoramidites.
[0077] 2'-(2-methoxyethyl)-modified amidites are synthesized
according to Martin, P. (Helv. Chim. Acta 1995, 78, 486-506). For
ease of synthesis, the last nucleotide was a deoxynucleotide.
2'--O--CH.sub.2CH.sub.2OCH.sub- .3-- cytosines may be 5-methyl
cytosines.
[0078] Synthesis of 5-Methyl Cytosine Monomers
[0079]
2,2'-Anhydro[1-(.beta.-D-arabinofuranosyl)-5-methyluridine]:
[0080] 5-Methyluridine (ribosylthymine, commercially available
through Yamasa, Choshi, Japan) (72.0 g, 0.279 M),
diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g,
0.024 M) were added to DMF (300 mL). The mixture was heated to
reflux, with stirring, allowing the evolved carbon dioxide gas to
be released in a controlled manner. After 1 hour, the slightly
darkened solution was concentrated under reduced pressure. The
resulting syrup was poured into diethylether (2.5 L), with
stirring. The product formed a gum. The ether was decanted and the
residue was dissolved in a minimum amount of methanol (ca. 400 mL).
The solution was poured into fresh ether (2.5 L) to yield a stiff
gum. The ether was decanted and the gum was dried in a vacuum oven
(60.degree. C. at 1 mm Hg for 24 h) to give a solid which was
crushed to a light tan powder (57 g, 85% crude yield). The material
was used as is for further reactions.
[0081] 2'-O-Methoxyethyl-5-methyluridine:
[0082] 2,2'-Anhydro-5-methyluridine (195 g, 0.81 M),
tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol
(1.2 L) were added to a 2 L stainless steel pressure vessel and
placed in a pre-heated oil bath at 160.degree. C. After heating for
48 hours at 155-160.degree. C., the vessel was opened and the
solution evaporated to dryness and triturated with MeOH (200 mL).
The residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 mL) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2Cl.sub.2/acetone/MeOH (20:5:3) containing 0.5% Et.sub.3NH.
The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL) and adsorbed
onto silica (150 g) prior to loading onto the column. The product
was eluted with the packing solvent to give 160 g (63%) of
product.
[0083] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:
[0084] 2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was
co-evaporated with pyridine (250 mL) and the dried residue
dissolved in pyridine (1.3 L). A first aliquot of dimethoxy-trityl
chloride (94.3 g, 0.278 M) was added and the mixture stirred at
room temperature for one hour. A second aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the reaction stirred for
an additional one hour. Methanol (170 mL) was then added to stop
the reaction. HPLC showed the presence of approximately 70%
product. The solvent was evaporated and triturated with CH.sub.3CN
(200 mL). The residue was dissolved in CHCl.sub.3 (1.5 L) and
extracted with 2.times.500 mL of saturated NaHCO.sub.3 and
2.times.500 mL of saturated NaCl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered and evaporated. 275 g of residue was
obtained. The residue was purified on a 3.5 kg silica gel column,
packed and eluted with EtOAc/-Hexane/Acetone (5:5:1) containing
0.5% Et.sub.3NH. The pure fractions were evaporated to give 164 g
of product. Approximately 20 g additional was obtained from the
impure fractions to give a total yield of 183 g (57%).
[0085]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:
[0086] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106
g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from
562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38
mL, 0.258 M) were combined and stirred at room temperature for 24
hours. The reaction was monitored by tlc by first quenching the tlc
sample with the addition of MeOH. Upon completion of the reaction,
as judged by tlc, MeOH (50 mL) was added and the mixture evaporated
at 35.degree. C. The residue was dissolved in CHCl.sub.3 (800 mL)
and extracted with 2.times.200 mL of saturated sodium bicarbonate
and 2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approx. 90% product). The residue was purified on a 3.5 kg silica
gel column and eluted using EtOAc/Hexane(4:1). Pure product
fractions were evaporated to yield 96 g (84%).
[0087]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triaz-
oleuridine:
[0088] A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methox-
yethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in
CH.sub.3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M)
was added to a solution of triazole (90 g, 1.3 M) in CH.sub.3CN (1
L), cooled to -5.degree. C. and stirred for 0.5 h using an overhead
stirrer. POCl.sub.3 was added dropwise, over a 30 minute period, to
the stirred solution maintained at 0-10.degree. C., and the
resulting mixture stirred for an additional 2 hours. The first
solution was added dropwise, over a 45 minute period, to the later
solution. The resulting reaction mixture was stored overnight in a
cold room. Salts were filtered from the reaction mixture and the
solution was evaporated. The residue was dissolved in EtOAc (1 L)
and the insoluble solids were removed by filtration. The filtrate
was washed with 1.times.300 mL of NaHCO.sub.3 and 2.times.300 mL of
saturated NaCl, dried over sodium sulfate and evaporated. The
residue was triturated with EtOAc to give the title compound.
[0089] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine:
[0090] A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5--
methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and
NH.sub.4OH (30 mL) was stirred at room temperature for 2 hours. The
dioxane solution was evaporated and the residue azeotroped with
MeOH (2.times.200 mL). The residue was dissolved in MeOH (300 mL)
and transferred to a 2 liter stainless steel pressure vessel. MeOH
(400 mL) saturated with NH.sub.3 gas was added and the vessel
heated to 100.degree. C. for 2 hours (tlc showed complete
conversion). The vessel contents were evaporated to dryness and the
residue was dissolved in EtOAc (500 mL) and washed once with
saturated NaCl (200 mL). The organics were dried over sodium
sulfate and the solvent was evaporated to give 85 g (95%) of the
title compound.
[0091]
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcyti-
dine:
[0092] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85
g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride
(37.2 g, 0.165 M) was added with stirring. After stirring for 3
hours, tlc showed the reaction to be approximately 95% complete.
The solvent was evaporated and the residue azeotroped with MEOH
(200 mL). The residue was dissolved in CHCl.sub.3 (700 mL) and
extracted with saturated NaHCO.sub.3 (2.times.300 mL) and saturated
NaCl (2.times.300 mL), dried over MgSO.sub.4 and evaporated to give
a residue (96 g). The residue was chromatographed on a 1.5 kg
silica column using EtOAc/Hexane (1:1) containing 0.5% Et.sub.3NH
as the eluting solvent. The pure product fractions were evaporated
to give 90 g (90%) of the title compound.
[0093]
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcyti-
dine-3'-amidite:
[0094]
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcyti-
dine (74 g, 0.10 M) was dissolved in CH.sub.2Cl.sub.2 (1 L)
Tetrazole diisopropylamine (7.1 g) and
2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were
added with stirring, under a nitrogen atmosphere. The resulting
mixture was stirred for 20 hours at room temperature (tlc showed
the reaction to be 95% complete). The reaction mixture was
extracted with saturated NaHCO.sub.3 (1.times.300 mL) and saturated
NaCl (3.times.300 mL). The aqueous washes were back-extracted with
CH.sub.2Cl.sub.2 (300 mL), and the extracts were combined, dried
over MgSO.sub.4 and concentrated. The residue obtained was
chromatographed on a 1.5 kg silica column using
EtOAc.backslash.Hexane (3:1) as the eluting solvent. The pure
fractions were combined to give 90.6 g (87%) of the title
compound.
[0095] 5-methyl-2'-deoxycytidine (5-me-C) containing
oligonucleotides were synthesized according to published methods
(Sanghvi et al., Nucl. Acids Res. 1993, 21, 3197-3203) using
commercially available phosphoramidites (Glen Research, Sterling
Va. or ChemGenes, Needham Mass.).
[0096] Oligonucleotides having methylene (methylimino) (MMI)
backbones are synthesized according to U.S. Pat. No. 5,378,825,
which is coassigned to the assignee of the present invention and is
incorporated herein in its entirety. For ease of synthesis, various
nucleoside dimers containing MMI linkages were synthesized and
incorporated into oligonucleotides. Other nitrogen-containing
backbones are synthesized according to WO 92/20823 which is also
coassigned to the assignee of the present invention and
incorporated herein in its entirety.
[0097] Oligonucleotides having amide backbones are synthesized
according to De Mesmaeker et al. (Acc. Chem. Res. 1995, 28,
366-374). The amide moiety is readily accessible by simple and
well-known synthetic methods and is compatible with the conditions
required for solid phase synthesis of oligonucleotides.
[0098] Oligonucleotides with morpholino backbones are synthesized
according to U.S. Pat. No. 5,034,506 (Summerton and Weller).
[0099] Peptide-nucleic acid (PNA) oligomers are synthesized
according to P. E. Nielsen et al. (Science 1991, 254,
1497-1500).
[0100] After cleavage from the controlled pore glass column
(Applied Biosystems) and deblocking in concentrated ammonium
hydroxide at 55.degree. C. for 18 hours, the oligonucleotides are
purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes
ethanol. Synthesized oligonucleotides were analyzed by
polyacrylamide gel electrophoresis on denaturing gels and judged to
be at least 85% full length material. The relative amounts of
phosphorothioate and phosphodiester linkages obtained in synthesis
were periodically checked by .sup.31P nuclear magnetic resonance
spectroscopy, and for some studies oligonucleotides were purified
by HPLC, as described by Chiang et al. (J. Biol. Chem. 1991, 266,
18162). Results obtained with HPLC-purified material were similar
to those obtained with non-HPLC purified material.
Example 2
Human RhoA Oligonucleotide Sequences
[0101] Antisense oligonucleotides were designed to target human
RhoA. Target sequence data are from the RhoA cDNA sequence
published by Yeramian, P., et al. (Nucleic Acids Res. 1987, 15,
1869); Genbank accession number X05026, provided herein as SEQ ID
NO: 1. Oligonucleotides were synthesized primarily with
phosphorothioate linkages. Oligonucleotide sequences are shown in
Table 1.
[0102] A549 cells, human lung carcinoma cells (obtained from
American Type Culture Collection) were cultured in Dulbecco's
modified Eagle's medium (DMEM) low glucose, 10% fetal calf serum,
and penicillin (50 units/ml)/streptomycin (50 mg/ml). Cells were
passaged at 90-95% confluency. All culture reagents were obtained
from Life Technologies (GIBCO BRL, Rockville, Md).
[0103] A549 cells were plated at a starting cell number of
approximately 2.times.10.sup.5 cells per well. After twenty-four
hours, at 80-90% confluency, the cells were washed twice with
Opti-Mem (GIBCO BRL) and the oligonucleotide formulated in
LIPOFECTIN (GIBCO BRL), a 1:1 (w/w) liposome formulation of the
cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n-
,n,n-trimethylammonium chloride (DOTMA), and dioleoyl
phosphotidylethanolamine (DOPE) in membrane filtered water, at a
constant ratio of 2.5 mg/ml LIPOFECTIN to 100 nM oligonucleotide,
in Opti-Mem. For an initial screen, the oligonucleotide
concentration was 300 nM. Treatment was for four hours. After
treatment, the media was removed and the cells were further
incubated in DMEM containing 10% FCS, and penicillin/streptomycin
for 24 or 48 hours.
[0104] mRNA was isolated using the MICRO-FASTTRACK kit (Invitrogen,
Carlsbad, Calif.), separated on a 1% agarose gel, transferred to
Hybond-N+ membrane (Amersham, Arlington Heights, Ill.), a
positively charged nylon membrane, and probed. A RhoA probe was
generated using asymmetric PCR, in the presence of a[.sup.32P]-dCTP
(Amersham), with the following primers:
1 Forward: 5'-TGCAAGCACAGCCCTTATG-3' SEQ ID NO. 2 Reverse:
5'-TGTCAAAAGGACCCTGGTG-3' SEQ ID NO. 3
[0105] A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe was
purchased from Clontech (Palo Alto, Calif.), Catalog Number 9805-1.
The probe was labeled by random primer using the Large Fragment of
DNA polymerase (Klenow fragment) (GIBCO BRL) as described in
Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989.
mRNA was quantitated by a PhosphoImager (Molecular Dynamics,
Sunnyvale, Calif.).
2TABLE 1 Nucleotide Sequences of RhoA Oligonucleotides TARGET GENE
SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET NO. (5'
-> 3') NO: ORDINATES.sup.1 REGION 16191 AGTCGCAAACTCGGAGAC 4
0085-0102 5'-UTR 16192 TTGCTCAGGCAACGAATC 5 0142-0159 AUG 16193
CTGAACACTATCACCAAGCATG 6 0214-0235 Coding 16194 CTCATCATTCCGAAGATCC
7 0515-0533 Coding 16195 CCAATCCTGTTTGCCATATCTC 8 0592-0613 Coding
16196 CCATCTTTGGTCTTTGCTGAAC 9 0634-0655 Coding 16197
CCAGAGCAGCTCTCGTAGCCA 10 0676-0696 Coding 16198
TCACAAGACAAGGCAACCAG 11 0721-0740 Stop 16199 AGGCCAGTAATCATACACTA
12 0799-0818 3'-UTR 16200 GTTGGCTTCTAAATACTGCT 13 0871-0890 3'-UTR
16201 GGCTGTTAGAGCAGTGTCAA 14 0937-0956 3'-UTR 16202
AGCGCCTGGTGTGTCAGGTG 15 0971-0990 3'-UTR 16203 TAGTTACAGCCTAATTGACA
16 1051-1073 3'-UTR 16913 GGCACCTGTTGGGTGAGCTG 17 16202 control
16914 ACACTCTTGCTTACCGTACCTT 18 16195 control 16915
TCCCGTAAGTGCGGTATCAA 19 16201 control .sup.1All linkages are
phosphorothioate linkages. .sup.2Co-ordinates from Genbank
Accession No. X05026, locus name "HSRHOB" SEQ ID NO. 1.
[0106] Results are shown in Table 2. Oligonucleotides 16193 (SEQ.
ID NO. 6), 16195 (SEQ ID NO. 8), 16196 (SEQ ID NO. 9), 16197 (SEQ
ID NO. 10), 16198 (SEQ ID NO. 11), 16199 (SEQ ID NO. 12), 16200
(SEQ ID NO. 13), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15)
gave better than 50% inhibition of RhoA expression.
Oligonucleotides 16195 (SEQ ID NO. 8), 16197 (SEQ ID NO. 10), 16199
(SEQ ID NO. 12), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15)
gave better than 75% inhibition of RhoA expression.
3TABLE 2 Activities of Phosphorothioate Oligonucleotides Targeted
to Human RhoA SEQ GENE ISIS ID TARGET % mRNA % mRNA No: NO: REGION
EXPRESSION INHIBITION LIPOFECTIN -- -- 100.0% 0.0% only 16191 4
5'-UTR 66.4% 33.6% 16192 5 AUCG 68.0% 32.0% 16193 6 Coding 31.9%
68.1% 16194 7 Coding 79.9% 20.1% 16195 8 Coding 3.9% 96.1% 16196 9
Coding 31.4% 68.6% 16197 10 Coding 19.2% 81.8% 16198 11 Stop 46.4%
53.6% 16199 12 3'-UTR 22.9% 77.1% 16200 13 3'-UTR 36.9% 63.1% 16201
14 3'-UTR 22.0% 78.0% 16202 15 3'-UTR 14.4% 85.6% 16203 16 3'-UTR
88.0% 12.0%
Example 3
Dose Response and Specificity of Antisense Oligonucleotide Effects
on Human RhoA mRNA Levels in A549 Cells
[0107] Three of the most active oligonucleotides from the initial
screen were chosen for dose response assays. These include
oligonucleotides 16195 (SEQ ID NO. 8), 16201 (SEQ ID NO. 14), and
16202 (SEQ ID NO. 15). A549 cells were grown, treated and processed
as described in Example 2. LIPOFECTIN was added at a ratio of 2.5
mg/ml per 100 nM of oligonucleotide. The control included
LIPOFECTIN at a concentration of 7.5 mg/ml. Results are shown in
Table 3. Each oligonucleotide showed a dose response effect with
maximal inhibition greater than 90%.
[0108] The specificity of these oligonucleotides was investigated
using scrambled controls, i.e. oligonucleotides with the same base
composition and a scrambled sequence. Oligonucleotide 16915 (SEQ ID
NO. 19) is a scrambled control for 16201 (SEQ ID NO. 14) and
oligonucleotide 16913 (SEQ ID NO. 17) is a scrambled control for
16202 (SEQ ID NO. 15). Both antisense oligonucleotides showed a
dose dependent effect on mRNA expression, while scrambled controls
showed much less inhibition which was only seen at higher does.
4TABLE 3 Dose Response of A549 Cells to RhoA Antisense
Oligonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO:
Target Dose Expression Inhibition control -- LIPOFECTIN -- 100.0%
0.0% only 16195 8 Coding 75 nM 72.7% 27.3% 16195 8 " 150 nM 35.0%
65.0% 16195 8 " 300 nM 20.3% 79.7% 16201 14 3'-UTR 75 nM 79.1%
20.9% 16201 14 " 150 nM 35.7% 64.3% 16201 14 " 300 nM 9.5% 90.5%
16202 15 3'-UTR 75 nM 68.7% 31.3% 16202 15 " 150 nM 28.8% 71.2%
16202 15 " 300 nM 6.1% 93.7%
[0109]
5TABLE 4 Specificity of RhoA Antisense Oligonucleotides (ASOs) in
A549 Cells SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose
Expression Inhibition control -- LIPOFECTIN -- 100% 0% only 16201
14 3'-UTR 75 nM 64.4% 35.6% 16201 14 " 150 nM 35.3% 64.7% 16201 14
" 300 nM 5.7% 94.3% 16915 19 control 75 nM 89.9% 10.1% 16915 19 "
150 nM 98.3% 1.7% 16915 19 " 300 nM 84.8% 15.2% 16202 15 3'-UTR 75
nM 39.9% 60.1% 16202 15 " 150 nM 20.2% 79.8% 16202 15 " 300 nM
10.8% 89.2% 16913 17 control 75 nM 97.6% 2.4% 16913 17 " 150 nM
89.8% 10.2% 16913 17 " 300 nM 55.6% 44.4%
Example 4
Design and Testing of Chimeric (Deoxy Gapped) 2'-O-methoxyethyl
RhoA Antisense Oligonucleotides on RhoA Levels in A549 Cells
[0110] Oligonucleotides having SEQ ID NO: 14 were synthesized as a
uniformly phosphorothioate or mixed phosphorothioate/phosphodiester
chimeric oligonucleotides having variable regions of
2'-O-methoxyethyl (2'-MOE) nucleotides and deoxynucleotides. All
2'-MOE cytosines were 5-methyl-cytosines. Additionally, some
oligonucleotides were synthesized with deoxycytosines as
5-methyl-cytosines. Additional oligonucleotides were synthesized,
with similar chemistries, as scrambled controls.
6TABLE 5 Nucleotide Sequences of 16201 Analogues SEQ TARGET GENE
GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5' ->
3').sup.1 NO: CO-ORDINATES.sup.2 REGION 17130
GsGcCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3'-UTR 17131
GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsT- sCsAsA 14 0937-0956 3'-UTR 17132
GsGsCsTsGsTsTsAsGsAsGsCsA- sGsTsGsTsCsAsA 14 0937-0956 3'-UTR 17133
GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3'-UTR 17134
GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956 3'-UTR 17818
GoGoCsTsGsTsTsAsGsAsGsCsAoGoToGoToCoAoA 14 0937-0956 all 5-meC
17819 ToGoCsGsCsTsAsAsGsTsGsCsGoGoToAoToCoAoA 19 16201 control all
5-meC 18550 TsGsCsGsGsTsAsAsCsTsGsCsGsG- sTsAsTsCsAsA 19 16201
control 20459 GsGsCsTsCsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA 14 0937-0956
all 5-meC 21919 GsTsCsGsTsTsAsCsTsCsGsGsAsAsAsTsGsGsAsGsGsC 20
16201 control 21920 AsGsCsTsTsGsTsTsGsAsAsCsGsAsGsTsGsTsCsGsA 21
16201 control 21921 TsGsCsAsGsTsTsCsGsCsAsGsAsGsTsCsTsGsAsA 22
16201 control .sup.1Emboldened residues are 2'-methoxyethoxy
residues (others are 2'-deoxy). All 2'-methoxyethoxy cytidines are
5-methyl-cytidines; where indicated "all 5-meC", 2'-deoxycytidines
are also 5-methyl-cytidines; "s" linkages are phosphorothioate
linkages, "o" linkages are phosphodiester linkages.
.sup.2Co-ordinates from Genbank Accession No. X05026, locus name
"HSRHOB" SEQ ID NO. 1
[0111] Dose response experiments were performed using chimeric
oligonucleotides as discussed in Example 3. Results are shown in
Table 6. The introduction of 2'-MOE nucleotides into the sequence
improved the maximum inhibition from 60%, with a phosphorothioate
oligodeoxynucleotide, to greater than 75%. The exception was the
fully modified 2-MOE oligonucleotide which was less effective than
the oligodeoxynucleotide.
7TABLE 6 Dose Response of A549 Cells to RhoA Antisense Gapmer
Oliqonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO:
Target Dose Expression Inhibition control -- LIPOFECTIN -- 100% 0%
only 16201 14 3'-UTR 75 nM 119.5% -- 16201 14 " 150 nM 54.5% 45.5%
16201 14 " 300 nM 39.5% 60.5% 17130 14 3'-UTR 75 nM 56.2% 43.8%
17130 14 " 150 nM 31.5% 68.5% 17130 14 " 300 nM 14.1% 85.9% 17131
14 3'-UTR 75 nM 55.5% 44.5% 17131 14 " 150 nM 35.4% 64.6% 17131 14
" 300 nM 24.7% 75.3% 17132 14 3'-UTR 75 nM 71.3% 28.7% 17132 14 "
150 nM 31.3% 68.7% 17132 14 " 300 nM 13.1% 86.9% 17133 14 3'-UTR 75
nM 41.7% 58.3% 17133 14 " 150 nM 33.8% 66.2% 17133 14 " 300 nM
14.4% 85.6% 17134 14 3'-UTR 75 nM 76.6% 23.4% 17134 14 " 150 nM
35.9% 64.1% 17134 14 " 300 nM 68.5% 31.5%
Example 5
Time Course of Antisense Oligonucleotide Effects on Human RhoA
Protein Levels in A549 Cells
[0112] Oligonucleotide 17131 was tested by treating for varying
times and measuring the effect of the oligo on RhoA protein levels.
A549 cells were grown and treated with oligonucleotide (300 nM) as
described in Example 2. Cells were harvested at 24, 48 and 72 hours
after treatment. RhoA protein levels were measured by Western
blotting. After oligonucleotide treatment, cells were washed twice
in phosphate-buffered saline (PBS) and lysed in 25 mM Tris-HCl pH
7.5, 1% Triton X-100, 0.2% SDS, 0.5% sodium deoxycholate, 450 mM
NaCl, and 10 mg/ml aprotinin and leupeptin. After 15 minutes on
ice, the samples were centrifuged at maximum speed in a microfuge.
Protein concentration was determined by Bradford reagent (Bio-Rad
Laboratories, Hercules, Calif.). Fifty mg of protein was separated
by SDS-PAGE (15%). Following electrophoresis, proteins were
transferred to IMMOBILON-P membranes (Millipore, Bedford, Mass.)
The membrane was blocked in 5% fish gelatin (Sigma Chemicals, St.
Louis, Mo.) and RhoA specific antibodies were used to visualize the
proteins. After incubation with the appropriate secondary antibody,
proteins were visualized using either LUMIGLO Reagent (New England
Biolabs, Beverly, Mass.) or ECL PLUS (Amersham Pharmacia Biotech,
Piscataway, N.J.) . Inhibition of RhoA protein was observable after
24 hours. After 48 hours, RhoA protein concentration was reduced by
80% using 17131 (SEQ ID NO. 14). Minimal inhibition was seen with
17163 (SEQ ID NO. 190), an oligonucleotide targeted to Rac1.
Results are shown in Table 7.
8TABLE 7 Time course of RhoA Antisense Oligonucleotides (ASOs) in
A549 Cells Time SEQ ID ASO Gene after % protein % protein ISIS #
NO: Target treatment Expression Inhibition control -- LIPOFECTIN --
100% 0% only 17131 14 3'-UTR 24 hr 46.2% 53.8% 17131 14 " 48 hr
16.0% 84.0% 17131 14 " 72 hr 12.4% 87.6% 17163 190 Rac1 control 24
hr 104.1% -- 17163 190 " 48 hr 82.3% 17.7% 17163 190 " 72 hr 95.2%
4.8%
Example 6
Dose Response of Antisense Oligonucleotide Effects on Human RhoA
Protein Levels in A549 Cells
[0113] Oligonucleotide 17131 was tested for a dose response by
using varying concentrations of oligonucleotide and measuring the
effect of the oligonucleotide on RhoA protein levels. A549 cells
were grown and treated with oligonucleotide (concentrations
indicated in Table 8) as described in Example 2. Western blotting
was performed to measure protein levels as described in Example 5.
A dose response effect is seen with 17131 (SEQ ID NO. 14), whereas
the scrambled control 18550 (SEQ ID NO. 19) had no effect on RhoA
protein levels.
9TABLE 8 Dose response of RhoA antisense oligonucleotide on protein
levels in A549 cells SEQ ID ASO Gene % protein % protein ISIS # NO:
Target Dose Expression Inhibition control -- LIPOFECTIN -- 100% 0%
only 17131 14 3'-UTR 75 nM 51% 49% 17131 14 " 150 nM 23% 77% 17131
14 " 300 nM 20% 80% 18550 19 control 75 nM 101% -- 18550 19 " 150
nM 101% -- 18550 19 " 300 nM 104% --
Example 7
Inhibition of JNK Activation by RhoA Antisense Oligonucleotides in
A549 Cells Stimulated with H.sub.2O.sub.2
[0114] Oligonucleotide 17131 (SEQ ID NO. 14) was tested for its
ability to inhibit JNK activation by stimulation with
H.sub.2O.sub.2 or Il-1b. A549 cells were grown as described in
Example 2. Cells were treated with 150 nM of oligonucleotide for
four hours. After treatment, the media was replaced with DMEM, 0.1%
FCS, and the cells were left in culture for 48 hours prior to
stimulation. Stimulation was with either 30 ng/ml IL-1b or 1 mM
H.sub.2O.sub.2 for 30 minutes. After stimulation, the cells were
washed twice in PBS, and lysed in 25 mM Hepes pH 7.7, 0.3 M NaCl,
1.5 mM MgCl.sub.2, 0.1% Triton X-100, 20 mM b-glycerophosphate, 0.1
mM sodium orthovanadate (Na.sub.3VO.sub.4), 0.5 mM PMSF, and 10
mg/ml of aprotinin and leupeptin. After 20 minutes on ice, the
lysates were centrifuged at maximum speed in a microfuge for 20
minutes. The protein concentration in the supernatant was
determined using Bradford reagent (Bio-Rad Laboratories, Hercules,
Calif.). To 150 mg of protein, 25 ml of c-Jun fusion beads (New
England Biolabs, Beverly, Mass.) were added and incubated at
4.degree. C. on a rotating wheel overnight. The samples were then
washed four times in 20 mM Hepes pH 7.7, 50 mM NaCl, 0.1 mM EDTA,
2.5 mM MgCl.sub.2, and 0.05% Triton X-100 (HIBI buffer). The kinase
reaction was run for 20 minutes at 30.degree. C. in 20 mM Hepes pH
7.7, 20 mM MgCl.sub.2, 20 mM b-glycerophosphate, 20 mM
p-nitrophenyl phosphate, 0.1 mM Na.sub.3VO.sub.4, 2 mM DTT, 20 mM
ATP, and 5 mCi of g[.sup.32P]-ATP. The reaction was stopped with
500 ml of ice cold HIBI buffer. The beads were pelleted,
resuspended in PAGE loading buffer, boiled for 5 minutes, and the
products separated on a 12% SDS gel (Novex, La Jolla, Calif.).
Bands were quantitated using a PhosphorImager.
[0115] Results are shown in Table 9. Oligonucleotide 17131 (SEQ ID
NO. 14) showed moderate but specific inhibition of
H.sub.2O.sub.2-induced JNK activation.
10TABLE 9 Inhibition of JNK activation by RhoA antisense
oligonucleotides SEQ ID ASO Gene % inhibition % inhibition ISIS #
NO: Target Dose Il-1b induced H.sub.2O.sub.2 induced control 13
LIPOFECTIN -- 0% 0% only 17131 14 3'-UTR 150 nM -- 37.6% 18550 19
control 150 nM 2.2% 5.8%
Example 8
Synthesis of Additional RhoA Sequences
[0116] Additional oligonucleotides were synthesized in 96 well
plate format via solid phase P(III) phosphoramidite chemistry on an
automated synthesizer capable of assembling 96 sequences
simultaneously in a standard 96 well format. Phosphodiester
internucleotide linkages were afforded by oxidation with aqueous
iodine. Phosphorothioate internucleotide linkages were generated by
sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide
(Beaucage Reagent) in anhydrous acetonitrile-. Standard
base-protected beta-cyanoethyl-di-isopropyl phosphoramidites were
purchased from commercial vendors (e.g. PE-Applied Biosystems,
Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard
nucleosides are synthesized as per published methods. They are
utilized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0117] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
[0118] A series of oligonucleotides were designed to target
different regions of the human RhoA RNA, using published sequences
(GenBank accession number X05026, incorporated herein as SEQ ID NO:
1). The oligonucleotides are shown in Table 10. Target sites are
indicated by nucleotide numbers, as given in the sequence source
reference (Genbank accession no. X05026), to which the
oligonucleotide binds.
[0119] All compounds in Table 10 are oligodeoxynucleotides with
phosphorothioate backbones (internucleoside linkages) throughout.
All compounds in Table 11 are chimeric oligonucleotides ("gapmers")
18 nucleotides in length, composed of a central "gap" region
consisting of ten 2'-deoxynucleotides, which is flanked on both
sides (5' and 3' directions) by four-nucleotide "wings." The wings
are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P=S)
throughout the oligonucleotide. Cytidine residues in the 2'-MOE
wings are 5-methylcytidines.
11TABLE 10 Nucleotide Sequences of Human RhoA Phosphorothioate
Oligodeoxynucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE
ID NUCLEOTIDE TARGET NO. (5' -> 3') NO: CO-ORDINATES.sup.1
REGION 25544 AGAGAACCGACGGAGCAC 23 0030-0047 5'-UTR 25545
GTCGACTAATGAGAGAAC 24 0041-0058 5'-UTR 25546 GACCGTCCACTAATCAGA 25
0045-0062 5'-UTR 25547 AGCTGAAGACCAGACCGT 26 0057-0074 5'-UTR 25548
AGTCGCAAACTCGGAGAC 4 0085-0102 5'-UTR 25549 AATCCGAGTCCAGCCTCT 27
0128-0145 5'-UTR 25550 AACGAATCCGAGTCCACC 28 0132-0149 5'-UTR 25551
TCAGGCAACGAATCCCAG 29 0138-0155 5'-UTR 25552 CACCAACAATCACCAGTT 30
0178-0195 Coding 25553 AAGACTATGAGCAAGCAT 31 0215-0232 Coding 25554
ATACACCTCTGGGAACTG 32 0243-0260 Coding 25555 ACATAGTTCTCAAACACT 33
0269-0286 Coding 25556 ACTCTACCTGCTTTCCAT 34 0304-0321 Coding 25557
CACAAACCCAACTCTACC 35 0314-0331 Coding 25558 AACATCGCTATCTGGGTA 36
0378-0395 Coding 25559 TTCTGGGATGTTTTCTAA 37 0432-0449 Coding 25560
GGACAGAAATGCTTGACT 38 0464-0481 Coding 25561 GTGCTCATCATTCCGAAG 39
0519-0536 Coding 25562 CTTCTCTGCTCATCATTC 40 0524-0541 Coding 25563
TAGCTCCCGCCTTGTGTG 41 0534-0551 Coding 25564 CCAATCCTGTTTGCCATA 42
0596-0613 Coding 25565 GTCTTTCCTGAACACTCC 43 0629-0646 Coding 25566
AAAACCTCTCTCACTCCA 44 0653-0670 Coding 25567 AAGACAAGGCAACCAGAT 45
0719-0736 Coding 25568 TTTCACAAGACAAGGCAA 46 0725-0742 Stop 25569
GCAAGGTTTCACAAGACA 47 0731-0748 Stop 25570 ATTAACCGCATAAGGGCT 48
0758-0775 3'-UTR 25571 TAATAAACAGCACTTCAA 49 0777-0794 3'-UTR 25572
CCAGTAATCATACACTAA 50 0798-0815 3'-UTR 25573 ATGACTTCTGATTTGTAA 51
0847-0864 3'-UTR 25574 TAGCAAGATGACTTCTGA 52 0854-0871 3'-UTR 25575
CTGGTAGOAAGATGACTT 53 0858-0875 3'-UTR 25576 CTAAATACTGGTAGCAAG 54
0865-0882 3'-UTR 25577 TTGGCTTCTAAATACTGG 55 0872-0889 3'-UTR 25578
TCATAGTTGGCTTCTAAA 56 0878-0895 3'-UTR 25579 AATAATCATAGTTGGCTT 57
0883-0900 3'-UTR 25580 TCAAAAGGACCCTGGTGG 58 0923-0940 3'-UTR 25581
GTGCAGAGGAGGGCTGTT 59 0950-0967 3'-UTR 25582 CCAACTGTTTCTCTTTCT 60
1026-1043 3'-UTR 25583 AAGTAGTTACAGCCTAAT 61 1056-1073 3'-UTR
.sup.1All cytidines are 5-methyl-cytidines; all linkages are
phosphorothioate linkages. .sup.2Co-Cordinates from Genbank
Accession No. X05026, locus name "HSRHOB"; SEQ ID NO. 1.
Example 9
Total RNA Isolation
[0120] Total mRNA was isolated using an RNEASY 96 kit and buffers
purchased from Qiagen Inc. (Valencia Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 100 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 100 .mu.L of 70% ethanol was then added to each well and
the contents mixed by pippeting three times up and down. The
samples were then transferred to the RNEASY 96 well plate attached
to a QIAVAC manifold fitted with a waste collection tray and
attached to a vacuum source. Vacuum was applied for 15 seconds. 1
mL of Buffer RW1 was added to each well of the RNEASY 96 plate and
the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was
then added to each well of the RNEASY 96 plate and the vacuum
applied for a period of 15 seconds. The Buffer RPE wash was then
repeated and the vacuum was applied for an additional 10 minutes.
The plate was then removed from the QIAVAC manifold and blotted dry
on paper towels. The plate was then re-attached to the QIAVAC
manifold fitted with a collection tube rack containing 1.2 mL
collection tubes. RNA was then eluted by pipetting 60 .mu.L water
into each well, incubating 1 minute, and then applying the vacuum
for 30 seconds. The elution step was repeated with an additional 60
.mu.L water.
[0121] Poly(A)+ mRNA may be isolated according to Miura et al.,
Clin. Chem., 42, 1758 (1996). Other methods for poly(A)+ mRNA
isolation are taught in, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3,
John Wiley & Sons, Inc., (1993). Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 ml cold PBS. 60 ml lysis buffer (10 mM
Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 ml of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 ml of
wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After
the final wash, the plate was blotted on paper towels to remove
excess wash buffer and then air-dried for 5 minutes. 60 ml of
elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70.degree. C.
was added to each well, the plate was incubated on a 90.degree. hot
plate for 5 minutes, and the eluate was then transferred to a fresh
96-well plate.
[0122] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 10
Real-Time Quantitative PCR Analysis of RhoA mRNA Levels
[0123] Quantitation of RhoA mRNA levels was determined by real-time
quantitative PCR using the ABI PRISM 7700 Sequence Detection System
(PE-Applied Biosystems, Foster City, Calif.) according to
manufacturer's instructions. This is a closed-tube, non-gel-based,
fluorescence detection system which allows high-throughput
quantitation of polymerase chain reaction (PCR) products in
real-time. As opposed to standard PCR, in which amplification
products are quantitated after the PCR is completed, products in
real-time quantitative PCR are quantitated as they accumulate. This
is accomplished by including in the PCR reaction an oligonucleotide
probe that anneals specifically between the forward and reverse PCR
primers, and contains two fluorescent dyes. A reporter dye (e.g.,
JOE or FAM, obtained from either Operon Technologies Inc., Alameda,
Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached
to the 5' end of the probe and a quencher dye (e.g., TAMRA,
obtained from either Operon Technologies Inc., Alameda, Calif. or
PE-Applied Biosystems, Foster City, Calif.) is attached to the 3'
end of the probe. When the probe and dyes are intact, reporter dye
emission is quenched by the proximity of the 3' quencher dye.
During amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular (six-second)
intervals by laser optics built into the ABI PRISM 7700 Sequence
Detection System. In each assay, a series of parallel reactions
containing serial dilutions of mRNA from untreated control samples
generates a standard curve that is used to quantitate the percent
inhibition after antisense oligonucleotide treatment of test
samples.
[0124] PCR reagents were obtained from PE-Applied Biosystems,
Foster City, Calif.. RT-PCR reactions were carried out by adding 25
.mu.L PCR cocktail (1.times.TAQMAN buffer A, 5.5 mM MgCl.sub.2, 300
.mu.M each of dATP, dCTP and dGTP, 600 .mu.M of dUTP, 100 nM each
of forward primer, reverse primer, and probe, 20 Units RNAse
inhibitor, 1.25 Units AMPLITAQ GOLD, and 12.5 Units MuLV reverse
transcriptase) to 96 well plates containing 25 .mu.L poly(A) mRNA
solution. The RT reaction was carried out by incubation for 30
minutes at 48.degree. C. Following a 10 minute incubation at
95.degree. C. to activate the AMPLITAQ GOLD, 40 cycles of a
two-step PCR protocol were carried out: 95.degree. C. for 15
seconds (denaturation) followed by 60.degree. C. for 1.5 minutes
(annealing/extension). RhoA probes and primers were designed to
hybridize to the human RhoA sequence, using published sequence
information (GenBank accession number X05026, incorporated herein
as SEQ ID NO: 1).
12 For RhoA the PCR primers were: forward primer:
GGCTGGACTCGGATTCGTT (SEQ ID NO: 62) reverse primer:
CCATCACCAACAATCACCAGTT (SEQ ID NO: 63) and the PCR probe was:
FAM-CCTGAGCAATGGCTGCCATCCG-TAMRA
[0125] (SEQ ID NO: 64) where FAM (PE-Applied Biosystems, Foster
City, Calif.) is the fluorescent reporter dye) and TAMRA
(PE-Applied Biosystems, Foster City, Calif.) is the quencher
dye.
13 For GAPDH the PCR primers were: forward primer:
GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 65) reverse primer:
GAAGATGGTGATGGGATTTC (SEQ ID NO: 66) and the PCR probe was: 5'
JOE-CAAGCTTCCCGTTCTCAGCC- TAMPA 3' (SEQ ID NO: 67)
[0126] ID NO: 67) where JOE (PE-Applied Biosystems, Foster City,
Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied
Biosystems, Foster City, Calif.) is the quencher dye.
Example 11
Antisense Inhibition of RhoA Expression-Phosphorothioate
Oligodeoxynucleotides
[0127] In accordance with the present invention, a series of
oligonucleotides were designed to target different regions of the
human RhoA RNA, using published sequences (GenBank accession number
X05026, incorporated herein as SEQ ID NO: 1). The oligonucleotides
are shown in Table 10. Target sites are indicated by nucleotide
numbers, as given in the sequence source reference (Genbank
accession no. X05026), to which the oligonucleotide binds. All
compounds in Table 10 are oligodeoxynucleotides with
phosphorothioate backbones (internucleoside linkages) throughout.
The compounds were analyzed for effect on RhoA mRNA levels by
quantitative real-time PCR as described in other examples herein.
Data are shown in Table 11 and are averages from three experiments.
If present, "N.D." indicates "no data".
14TABLE 11 Inhibition of RhoA mRNA levels by phosphorothioate
oligodeoxynucleotides SEQ TARGET % Inhi- ID ISIS# REGION SITE
SEQUENCE bition NO. 25544 5' UTR 30 AGACAACCGACGGAGGAC 47 23 25545
5' UTR 41 GTGGACTAATGAGAGAAC 0 24 25546 5' UTR 45
GACCGTGGACTAATGAGA 40 25 25547 5' UTR 57 AGCTGAAGACCAGACCGT 76 26
25548 5' UTR 85 AGTCGCAAACTCGGAGAC 36 4 25549 5' UTR 128
AATCCGAGTCCAGCCTCT 67 27 25550 5' UTR 132 AACGAATCCGAGTCCAGC 34 28
25551 5' UTR 138 TCAGGCAACGAATCCGAG 59 29 25552 CODING 178
CACCAACAATCACCAGTT 47 30 25553 CODING 215 AAGACTATGAGCAAGCAT 36 31
25554 CODING 243 ATACACCTCTGGGAACTG 74 32 25555 CODING 269
ACATAGTTCTCAAACACT 31 33 25556 CODING 304 ACTCTACCTGCTTTCCAT 64 34
25557 CODING 314 CACAAAGCCAACTCTACC 25 35 25558 CODING 378
AACATCGGTATCTGGGTA 35 36 25559 CODING 432 TTCTGGGATGTTTTCTAA 21 37
25560 CODING 464 GGACAGAAATGCTTGACT 64 38 25561 CODING 519
GTGCTCATCATTCCGAAG 71 39 25562 CODING 524 CTTGTGTGCTCATCATTC 38 40
25563 CODING 534 TAGCTCCCGCCTTGTGTG 78 41 25564 CODING 596
CCAATCCTGTTTGCCATA 82 42 25565 CODING 629 GTCTTTGCTGAACACTCC 56 43
25566 CODING 653 AAAACCTCTCTCACTCCA 68 44 25567 CODING 719
AAGACAAGGCAACCAGAT 55 45 25568 STOP 725 TTTCACAAGACAAGGCAA 0 46
25569 STOP 731 GCAAGGTTTCACAAGACA 37 47 25570 3' UTR 758
ATTAACCGCATAAGGGCT 77 48 25571 3' UTR 777 TAATAAACAGCACTTCAA 19 49
25572 3' UTR 798 CCAGTAATCATACACTAA 26 50 25573 3' UTR 847
ATGACTTCTGATTTGTAA 27 51 25574 3' UTR 854 TAGCAAGATGACTTCTGA 62 52
25575 3' UTR 858 CTGGTAGCAAGATGACTT 59 53 25576 3' UTR 865
CTAAATACTGGTAGCAAG 29 54 25577 3' UTR 872 TTGGCTTCTAAATACTGG 57 55
25578 3' UTR 878 TCATAGTTGGCTTCTAAA 60 56 25579 3' UTR 883
AATAATCATAGTTGGCTT 33 57 25580 3' UTR 923 TCAAAAGGACCCTGGTGG 25 58
25581 3' UTR 950 GTGCAGAGGAGGGCTGTT 68 59 25582 3' UTR 1026
CCAACTGTTTCTCTTTCT 52 60 25583 3' UTR 1056 AAGTAGTTACAGCCTAAT 26
61
[0128] As shown in Table 11, SEQ ID NOs 23, 26, 27, 29, 30, 32, 34,
38, 39, 41, 42, 43, 44, 45, 48, 52, 53, 56, 57, 59 and 60
demonstrated at least 45% inhibition of RhoA expression in this
assay and are therefore preferred.
Example 12
Antisense Inhibition of RhoA Expression-Phosphorothioate 2'-MOE
Gapmer Oligonucleotides
[0129] In accordance with the present invention, a second series of
oligonucleotides targeted to human RhoA were synthesized. The
oligonucleotide sequences are shown in Table 12. Target sites are
indicated by nucleotide numbers, as given in the sequence source
reference (Genbank accession no. X05026), to which the
oligonucleotide binds.
[0130] All compounds in Table 12 are chimeric oligonucleotides
("gapmers") 18 nucleotides in length, composed of a central "gap"
region consisting of ten 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by four-nucleotide "wings". The
wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P=S)
throughout the oligonucleotide. Cytidine residues in the 2'-MOE
wings are 5-methylcytidines.
15TABLE 12 Nucleotide Sequences of Human RhoA Gapmer
Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID
NUCLEOTIDE TARGET NO. (5' -> 3') NO: CO-ORDINATES.sup.1 REGION
25584 AGAGAACCGACGGAGGAC 23 0030-0047 5'-UTR 25585
GTGGACTAATGAGAGAAC 24 0041-0058 5'-UTR 25586 GACCGTGGACTAATGAGA 25
0045-0062 5'-UTR 25587 AGCTGAAGACCAGACCGT 26 0057-0074 5'-UTR 25588
AGTCGCAAACTCGGAGAC 4 0085-0102 5'-UTR 25589 AATCCGAGTCCAGCCTCT 27
0128-0145 5'-UTR 25590 AACGAATCCGAGTCCAGC 28 0132-0149 5'-UTR 25591
TCAGGCAACGAATCCGAG 29 0138-0155 5'-UTR 25592 CACCAACAATCACCAGTT 30
0178-0195 Coding 25593 AAGACTATGAGCAAGCAT 31 0215-0232 Coding 25594
ATACACCTCTGGGAACTG 32 0243-0260 Coding 25595 ACATAGTTCTCAAACACT 33
0269-0286 Coding 25596 ACTCTACCTGCTTTCCAT 34 0304-0321 Coding 25597
CACAAAGCCAACTCTACC 35 0314-0331 Coding 25598 AACATCGGTATCTGGGTA 36
0378-0395 Coding 25599 TTCTGGGATGTTTTCTAA 37 0432-0449 Coding 25600
GGACAGAAATGCTTGACT 38 0464-0481 Coding 25601 GTGCTCATCATTCCGAAG 39
0519-0536 Coding 25602 CTTGTGTGCTCATCATTC 40 0524-0541 Coding 25603
TAGCTCCCGCCTTGTGTG 41 0534-0551 Coding 25604 CCAATCCTGTTTGCCATA 42
0596-0613 Coding 25605 GTCTTTGCTGAACACTCC 43 0629-0646 Coding 25606
AAAACCTCTCTCACTCCA 44 0653-0670 Coding 25607 AAGACAACCCAACCAGAT 45
0719-0736 Coding 25608 TTTCACAAGACAAGGCAA 46 0725-0742 Stop 25609
GCAACCTTTCACAAGACA 47 0731-0748 Stop 25610 ATTAACCCCATAACGGCT 48
0758-0775 3'-UTR 25611 TAATAAACAGCACTTCAA 49 0777-0794 3'-UTR 25612
CCAGTAATCATACACTAA 50 0798-0815 3'-UTR 25613 ATGACTTCTGATTTGTAA 51
0847-0864 3'-UTR 25614 TAGCAAGATGACTTCTGA 52 0854-0871 3'-UTR 25615
CTGGTAGCAAGATGACTT 53 0858-0875 3'-UTR 25616 CTAAATACTGGTAGCAAG 54
0865-0882 3'-UTR 25617 TTGGCTTCTAAATACTGG 55 0872-0889 3'-UTR 25618
TCATAGTTCGCTTCTAAA 56 0878-0895 3'-UTR 25619 AATAATCATAGTTGGCTT 57
0883-0900 3'-UTR 25620 TCAAAAGGACCCTCGTGG 58 0923-0940 3'-UTR 25621
GTGCAGAGGAGGCCTGTT 59 0950-0967 3'-UTR 25622 CCAACTCTTTCTCTTTCT 60
1026-1043 3'-UTR 25623 AAGTAGTTACAGCCTAAT 61 1056-1073 3'-UTR
.sup.1Emboldened residues are 2'-methoxyethoxy residues (others are
2'-deoxy-). All 2'-methoxyethoxy cytidines and cytidines are
5-methyl-cytidines; all linkages are phosphorothioate linkages.
.sup.2Co-ordinates from Genbank Accession No. X05026, locus name
"HSRHOB" SEQ ID NO. 1.
[0131] The oligonucleotides shown in Table 12 were tested by
real-time quantitative PCR as described in other examples herein
and data are shown in Table 13 (averaged from three experiments).
If present, "N.D." indicates "no data".
16TABLE 13 Inhibition of RhoA mRNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap SEQ TARGET % Inhi- ID ISIS# REGION SITE SEQUENCE bition NO.
25584 5' UTR 30 AGAGAACCGACGGAGGAC 44 23 25585 5' UTR 41
GTGGACTAATGAGAGAAC 35 24 25586 5' UTR 45 GACCGTGGACTAATGAGA 53 25
25587 5' UTR 57 AGCTGAAGACCAGACCGT 62 26 25588 5' UTR 85
AGTCGCAAACTCGGAGAC 54 4 25589 5' UTR 128 AATCCGAGTCCAGCCTCT 38 27
25590 5' UTR 132 AACGAATCCGAGTCCAGC 47 28 25591 5' UTR 138
TCAGGCAACGAATCCGAG 31 29 25592 CODING 178 CACCAACAATCACCAGTT 0 30
25593 CODING 215 AAGACTATGAGCAAGCAT 43 31 25594 CODING 243
ATACACCTCTGGGAACTG 23 32 25595 CODING 269 ACATAGTTCTCAAACACT 16 33
25596 CODING 304 ACTCTACCTGCTTTCCAT 0 34 25597 CODING 314
CACAAAGCCAACTCTACC 0 35 25598 CODING 378 AACATCGGTATCTGGGTA 65 36
25599 CODING 432 TTCTGGGATGTTTTCTAA 53 37 25600 CODING 464
GGACAGAAATGCTTGACT 50 38 25601 CODING 519 GTGCTCATCATTCCGAAG 45 39
25602 CODING 524 CTTGTGTGCTCATCATTC 26 40 25603 CODING 534
TAGCTCCCGCCTTGTGTG 59 41 25604 CODING 596 CCAATCCTGTTTGCCATA 40 42
25605 CODING 629 GTCTTTGCTGAACACTCC 47 43 25606 CODING 653
AAAACCTCTCTCACTCCA 30 44 25607 CODING 719 AAGACAAGGCAACCAGAT 0 45
25608 STOP 725 TTTCACAAGACAAGGCAA 7 46 25609 STOP 731
GCAAGGTTTCACAAGACA 53 47 25610 3' UTR 758 ATTAACCGCATAAGGGCT 56 48
25611 3' UTR 777 TAATAAACAGCACTTCAA 7 49 25612 3' UTR 798
CCAGTAATCATACACTAA 41 50 25613 3' UTR 847 ATGACTTCTGATTTGTAA 53 51
25614 3' UTR 854 TAGCAAGATGACTTCTGA 59 52 25615 3' UTR 858
CTGGTAGCAAGATGACTT 67 53 25616 3' UTR 865 CTAAATACTGGTAGCAAG 65 54
25617 3' UTR 872 TTGGCTTCTAAATACTGG 74 55 25618 3' UTR 878
TCATAGTTGGCTTCTAAA 52 56 25619 3' UTR 883 AATAATCATACTTGGCTT 49 57
25620 3' UTR 923 TCAAAAGGACCCTGGTGG 58 58 25621 3' UTR 950
GTGCAGAGGAGGGCTGTT 60 59 25622 3' UTR 1026 CCAACTGTTTCTCTTTCT 62 60
25623 3' UTR 1056 AAGTAGTTACACCCTAAT 44 61
[0132] As shown in Table 13, SEQ ID NOs 23, 24, 25, 26, 4, 27, 28,
31, 36, 37, 38, 39, 41, 42, 43, 47, 48, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60 and 61 demonstrated at least 35% inhibition of RhoA
expression in this experiment and are therefore preferred.
Example 13
Synthesis of RhoB Antisense Oligonucleotide Sequences
[0133] Oligonucleotide sequences were synthesized as described in
previous examples. Antisense oligonucleotides were designed to
target human RhoB. Target sequence data are from the RhoB cDNA
sequence published by Chardin, P., et al. (Nucleic Acids Res. 1988,
16, 2717); Genbank accession number X06820, provided herein as SEQ
ID NO: 68.
17TABLE 14 Nucleotide Sequences of Human RhoB Phosphorothioate
Oligodeoxynucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE
ID NUCLEOTIDE TARGET NO. (5' -> 3') NO: CO-ORDINATES.sup.1
REGION 25384 CCACCACCACCTTCTTCC 69 0014-0031 Coding 25385
CCCTCCCCCACCACCACC 70 0024-0041 Coding 25386 GCACGTCTTGCCACACGC 71
0043-0060 Coding 25387 ACTGAACACGATCAGCAG 72 0061-0078 Coding 25388
TTACTGAACACGATCAGC 73 0063-0080 Coding 25389 CCTTACTGAACACGATCA 74
0065-0082 Coding 25390 GTCCTTACTGAACACGAT 75 0067-0084 Coding 25391
CTCGTCCTTACTGAACAC 76 0070-0087 Coding 25392 AACTCGTCCTTACTGAAC 77
0072-0089 Coding 25393 CATAGTTCTCGAAGACGG 78 0110-0127 Coding 25394
TCGCCCACATAGTTCTCG 79 0117-0134 Coding 25395 CCGTCCACCTCAATGTCG 80
0132-0149 Coding 25396 AAGCACATGAGAATGACG 81 0234-0251 Coding 25397
GAGTCCGGGCTGTCCACC 82 0255-0272 Coding 25398 ATGTTCTCCAGCGAGTCC 83
0267-0284 Coding 25399 GGGATGTTCTCCAGCGAG 84 0270-0287 Coding 25400
GACATGCTCGTCGCTGCG 85 0364-0381 Coding 25401 CCGACATGCTCGTCGCTG 86
0366-0383 Coding 25402 TGTGCGGACATGCTCGTC 87 0370-0387 Coding 25403
CTCTGTGCGGACATGCTC 88 0373-0390 Coding 25404 CCAGCTCTGTGCGGACAT 89
0377-0394 Coding 25405 CGCCCCACCTCTGTGCGG 90 0381-0398 Coding 25406
TGCGGGCCAGCTCTGTGC 91 0383-0400 Coding 25407 GTTCCTGCTTCATGCGGG 92
0395-0412 Coding 25408 ACGGGTTCCTGCTTCATG 93 0399-0416 Coding 25409
GTAGTCGTAGGCTTGGAT 94 0451-0468 Coding 25410 CGACGTAGTCGTAGCCTT 95
0455-0472 Coding 25411 GTCTTCCCACACCACTCG 96 0471-0488 Coding 25412
ACCTCGCGCACGCCTTCC 97 0492-0509 Coding 25413 AGACCTCGCGCACGCCTT 98
0494-0511 Coding 25414 CGAAGACCTCCCGCACGC 99 0497-0514 Coding 25415
CTCGAAGACCTCGCGCAC 100 0499-0516 Coding 25416 GCCGTCTCGAAGACCTCG
101 0504-0521 Coding 25417 CCTGGCCGTCTCGAACAC 102 0508-0525 Coding
25418 GTTCTGGGAGCCGTAGCG 103 0544-0561 Coding 25419
GCCGTTCTGGGAGCCGTA 104 0547-0564 Coding 25420 GATGCAGCCGTTCTGGGA
105 0553-0570 Coding 25421 GTTGATGCAGCCGTTCTG 106 0556-0573 Coding
25422 CAGCAGTTGATCCAGCCG 107 0561-0578 Coding 25423
AGCACCTTGCAGCAGTTG 108 0570-0587 Coding .sup.1All cytidines are
5-methyl-cytidines; all linkages are phosphorothioate linkages.
.sup.2Co-ordinates from Genbank Accession No. X06820, locus name
"HSRHOB6" SEQ ID NO. 68.
Example 14
Antisense Inhibition of RhoB Expression-Phosphorothioate
Oligodeoxynucleotides
[0134] In accordance with the present invention, the
oligonucleotides shown in Table 14 were analyzed for effect on RhoB
mRNA levels by quantitative real-time PCR as described in examples
herein. Data are averages from three experiments. If present,
"N.D." indicates "no data".
18TABLE 15 Inhibition of RhoB mRNA levels by phosphorothioate
oligodeoxynucleotides SEQ TARGET % Inhibi- ID ISIS# REGION SITE
SEQUENCE tion NO. 25384 Coding 14 CCACCACCAGCTTCTTGC 0 69 25385
CODING 24 CCGTCGCCCACCACCACC 0 70 25386 CODING 43
GCACGTCTTGCCACACGC 0 71 25387 CODING 61 ACTGAACACGATCAGCAG 0 72
25388 CODING 63 TTACTGAACACGATCAGC 0 73 25389 CODING 65
CCTTACTGAACACGATCA 0 74 25390 CODING 67 GTCCTTACTGAACACGAT 5 75
25391 CODING 70 CTCGTCCTTACTGAACAC 1 76 25392 CODING 72
AACTCGTCCTTACTGAAC 30 77 25393 CODING 110 CATAGTTCTCGAAGACGG 0 78
25394 CODING 117 TCGGCCACATAGTTCTCG 13 79 25395 CODING 132
CCGTCCACCTCAATGTCG 0 80 25396 CODING 234 AAGCACATGAGAATGACG 0 81
25397 CODING 255 GAGTCCGGGCTGTCCACC 0 82 25398 CODING 267
ATGTTCTCCAGCGAGTCC 0 83 25399 CODING 270 GGGATGTTCTCCAGCGAG 33 84
25400 CODING 364 GACATGCTCGTCGCTGCG 0 85 25401 CODING 366
CGGACATGCTCGTCGCTG 0 86 25402 CODING 370 TGTGCGGACATGCTCGTC 0 87
25403 CODING 373 CTCTGTGCGGACATGCTC 39 88 25404 CODING 377
CCAGCTCTGTGCGGACAT 21 89 25405 CODING 381 CGGGCCAGCTCTGTGCGG 38 90
25406 CODING 383 TGCGGGCCAGCTCTGTGC 31 91 25407 CODING 395
GTTCCTGCTTCATGCGGG 27 92 25408 CODING 399 ACGGGTTCCTGCTTCATG 0 93
25409 CODING 451 GTAGTCGTAGGCTTGGAT 29 94 25410 CODING 455
CGAGGTAGTCGTAGGCTT 39 95 25411 CODING 471 GTCTTGGCAGAGCACTCG 20 96
25412 CODING 492 ACCTCGCGCACGCCTTCC 0 97 25413 CODING 494
AGACCTCGCGCACGCCTT 16 98 25414 CODING 497 CGAAGACCTCGCGCACGC 0 99
25415 CODING 499 CTCGAAGACCTCGCGCAC 0 100 25416 CODING 504
GCCGTCTCGAAGACCTCG 0 101 25417 CODING 508 CGTGGCCGTCTCGAAGAC 0 102
25418 CODING 544 GTTCTGGGAGCCGTAGCG 36 103 25419 CODING 547
GCCGTTCTGGGAGCCGTA 0 104 25420 CODING 553 GATGCAGCCGTTCTGGGA 0 105
25421 CODING 556 GTTGATGCAGCCGTTCTG 7 106 25422 CODING 561
CAGCAGTTGATGCAGCCG 31 107 25423 CODING 570 AGCACCTTGCAGCAGTTG 0
108
[0135] As shown in Table 15, SEQ ID Nos 77, 84, 88, 90, 91, 92, 94,
95, 103 and 107 demonstrated at least 25% inhibition of RhoB
expression in this assay and are therefore preferred.
Example 15
Antisense Inhibition of RhoB Expression-Phosphorothioate 2'-MOE
Gapmer Oligonucleotides
[0136] In accordance with the present invention, a second series of
oligonucleotides targeted to human RhoB were synthesized. The
oligonucleotide sequences are shown in Table 16. Target sites are
indicated by nucleotide numbers, as given in the sequence source
reference (Genbank accession no. X06820), to which the
oligonucleotide binds.
[0137] All compounds in Table 16 are chimeric oligonucleotides
("gapmers") 18 nucleotides in length, composed of a central "gap"
region consisting of ten 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by four-nucleotide "wings". The
wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P=S)
throughout the oligonucleotide. Cytidine residues in the 2'-MOE
wings are 5-methylcytidines.
19TABLE 16 Nucleotide Sequences of Human RhoB Gapmer
Oligonucleotides NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID
NUCLEOTIDE TARGET NO. (5'.fwdarw.3') NO: CO-ORDINATES.sup.1 REGION
25424 CCACCACCAGCTTCTTGC 69 0014-0031 Coding 25425
CCGTCGCCCACCACCACC 70 0024-0041 Coding 25426 GCACGTCTTGCCACACGC 71
0043-0060 Coding 25427 ACTGAACACGATCAGCAG 72 0061-0078 Coding 25428
TTACTGAACACGATCAGC 73 0063-0080 Coding 25429 CCTTACTGAACACGATCA 74
0065-0082 Coding 25430 GTCCTTACTGAACACGAT 75 0067-0084 Coding 25431
CTCGTCCTTACTGAACAC 76 0070-0087 Coding 25432 AACTCGTCCTTACTGAAC 77
0072-0089 Coding 25433 CATAGTTCTCGAAGACGG 78 0110-0127 Coding 25434
TCGGCCACATAGTTCTCG 79 0117-0134 Coding 25435 CCGTCCACCTCAATGTCG 80
0132-0149 Coding 25436 AAGCACATGAGAATGACG 81 0234-0251 Coding 25437
GAGTCCGGGCTGTCCACC 82 0255-0272 Coding 25438 ATGTTCTCCAGCGAGTCC 83
0267-0284 Coding 25439 GGGATGTTCTCCAGCGAG 84 0270-0287 Coding 25440
GACATGCTCGTCGCTGCG 85 0364-0381 Coding 25441 CGGACATGCTCGTCGCTG 86
0366-0383 Coding 25442 TGTGCGGACATGCTCGTC 87 0370-0387 Coding 25443
CTCTGTGCGGACATGCTC 88 0373-0390 Coding 25444 CCAGCTCTGTGCGGACAT 89
0377-0394 Coding 25445 CGGGCCAGCTCTGTGCGG 90 0381-0393 Coding 25446
TGCGGGCCAGCTCTGTGC 91 0383-0400 Coding 25447 GTTCCTGCTTCATGCGGG 92
0395-0412 Coding 25448 ACGGGTTCCTCCTTCATG 93 0399-0416 Coding 25449
GTAGTCGTAGGCTTGGAT 94 0451-0468 Coding 25450 CGAGGTAGTCGTAGGCTT 95
0455-0472 Coding 25451 GTCTTGGCAGAGCACTCG 96 0471-0488 Coding 25452
ACCTCGCGCACGCCTTCC 97 0492-0509 Coding 25453 AGACCTCGCGCACGCCTT 98
0494-0511 Coding 25454 CGAAGACCTCGCGCACGC 99 0497-0514 Coding 25455
CTCGAAGACCTCGCGCAC 100 0499-0516 Coding 25456 GCCGTCTCGAAGACCTCG
101 0504-0521 Coding 25457 CGTGGCCGTCTCGAAGAC 102 0508-0525 Coding
25458 GTTCTGGGAGCCGTAGCG 103 0544-0561 Coding 25459
GCCGTTCTGGGAGCCGTA 104 0547-0564 Coding 25460 GATGCACCCGTTCTGGGA
105 0553-0570 Coding 25461 GTTGATGCAGCCGTTCTG 106 0556-0573 Coding
25462 CAGCAGTTGATGCAGCCG 107 0561-0578 Coding 25463
AGCACCTTCCACCAGTTG 108 0570-0587 Coding .sup.1Emboldened residues
are 2'-methoxyethoxy residues (others are 2'-deoxy-). All
2'-methoxyethoxy cytidines and cytidines are 5-methyl-cytidines;
all linkages are phosphorothioate linkages. .sup.2Co-ordinates from
Genbank Accession No. X06820, locus name "HSRHOB6" SEQ ID NO.
68.
[0138] Data for the compounds in Table 16 were obtained by
real-time quantitative PCR as described in other examples herein
and are averaged from three experiments. Results are shown in Table
17. If present, "N.D." indicates "no data".
20TABLE 17 Inhibition of RhoB mRNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap % TARGET Inhi- SEQ ID ISIS# REGION SITE SEQUENCE bition NO.
25424 Coding 14 CCACCACCAGCTTCTTGC 29 69 25425 CODING 24
CCGTCGCCCACCACCACC 23 70 25426 CODING 43 GCACGTCTTGCCACACGC 46 71
25427 CODING 61 ACTGAACACGATCAGCAG 37 72 25428 CODING 63
TTACTGAACACGATCAGC 47 73 25429 CODING 65 CCTTACTGAACACGATCA 7 74
25430 CODING 67 GTCCTTACTGAACACGAT 46 75 25431 CODING 70
CTCGTCCTTACTGAACAC 52 76 25432 CODING 72 AACTCGTCCTTACTGAAC 35 77
25433 CODING 110 CATAGTTCTCGAAGACGG 29 78 25434 CODING 117
TCGGCCACATAGTTCTCG 65 79 25435 CODING 132 CCGTCCACCTCAATGTCG 40 80
25436 CODING 234 AAGCACATGAGAATGACG 44 81 25437 CODING 255
GAGTCCGGGCTGTCCACC 36 82 25438 CODING 267 ATGTTCTCCAGCGAGTCC 28 83
25439 CODING 270 GGGATGTTCTCCAGCGAG 54 84 25440 CODING 364
GACATGCTCGTCGCTGCG 49 85 25441 CODING 366 CGGACATGCTCGTCGCTG 46 86
25442 CODING 370 TGTGCGGACATGCTCGTC 65 87 25443 CODING 373
CTCTGTGCGGACATGCTC 39 88 25444 CODING 377 CCAGCTCTGTGCGGACAT 19 89
25445 CODING 381 CGGGCCAGCTCTGTGCGG 21 90 25446 CODING 383
TGCGGGCCAGCTCTGTGC 9 91 25447 CODING 395 GTTCCTGCTTCATGCGGG 16 92
25448 CODING 399 ACGGGTTCCTGCTTCATG 7 93 25449 CODING 451
GTAGTCGTAGGCTTGGAT 38 94 25450 CODING 455 CGAGGTAGTCGTAGGCTT 0 95
25451 CODING 471 GTCTTGGCAGAGCACTCG 42 96 25452 CODING 492
ACCTCGCGCACGCCTTCC 9 97 25453 CODING 494 AGACCTCGCGCACGCCTT 7 98
25454 CODING 497 CGAAGACCTCGCGCACGC 12 99 25455 CODING 499
CTCGAAGACCTCGCGCAC 23 100 25456 CODING 504 GCCGTCTCGAAGACCTCG 34
101 25457 CODING 508 CGTGGCCGTCTCGAAGAC 27 102 25458 CODING 544
GTTCTGGGAGCCGTAGCG 58 103 25459 CODING 547 GCCGTTCTGGGAGCCGTA 63
104 25460 CODING 553 GATGCAGCCGTTCTGGGA 17 105 25461 CODING 556
GTTGATGCAGCCGTTCTG 21 106 25462 CODING 561 CAGCAGTTGATGCAGCCG 50
107 25463 CODING 570 AGCACCTTGCAGCAGTTG 55 108
[0139] As shown in Table 17, SEQ ID Nos 71, 62, 63, 75, 76, 77, 79,
80, 81, 82, 84, 85, 86, 87, 88, 94, 96, 101, 103, 104, 107 and 108
demonstrated at least 30% inhibition of RhoB expression in this
experiment and are therefore preferred.
Example 16
Synthesis of RhoC Antisense Oligonucleotide Sequences
[0140] Oligonucleotide sequences were synthesized as described in
previous examples. Antisense oligonucleotides were designed to
target human RhoC. Target sequence data are from the RhoC cDNA
sequence determined by Fagan, K. P., et al.; Genbank accession
number L25081, provided herein as SEQ ID NO: 109.
21TABLE 18 Nucleotide Sequences of Human RhoC Phosphorothioate
Oligonucleotides NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID
NUCLEOTIDE TARGET NO. (5'.fwdarw.3') NO: CO-ORDINATES.sup.1 REGION
25304 GAGCTGAGATGAAGTCAA 110 0004-0021 5'-UTR 25305
GCTGAAGTTCCCAGGCTG 111 0044-0061 5'-UTR 25306 CCGGCTGAAGTTCCCAGG
112 0047-0064 5'-UTR 25307 GGCACCATCCCCAACGAT 113 0104-0121 Coding
25308 AGGCACCATCCCCAACGA 114 0105-0122 Coding 25309
TCCCACAGGCACCATCCC 115 0111-0128 Coding 25310 AGGTCTTCCCACAGGCAC
116 0117-0134 Coding 25311 ATGAGGAGGCACCTCTTC 117 0127-0144 Coding
25312 TTGCTGAAGACGATGAGG 118 0139-0156 Coding 25313
TCAAAGACAGTAGGGACG 119 0173-0195 Coding 25314 TTCTCAAAGACAGTAGGG
120 0181-0193 Coding 25315 ACTTCTCAAAGACAGTAG 121 0183-0200 Coding
25316 TGTTTTCCAGGCTGTCAG 122 0342-0359 Coding 25317
TCGTCTTGCCTCAGGTCC 123 0433-0450 Coding 25318 GTGTGCTCGTCTTGCCTC
124 0439-0456 Coding 25319 CTCCTGGTGTGCTCGTCT 125 0445-0462 Coding
25320 CAGACCCAACGGGCTCCT 126 0483-0500 Coding 25321
TTCCTCAGACCGAACGGG 127 0488-0505 Coding 25322 ACTCAAGGTAGCCAAAGG
128 0534-0551 Coding 25323 CTCCCGCACTCCCTCCTT 129 0566-0583 Coding
25324 CTCAAACACCTCCCGCAC 130 0575-0592 Coding 25325
GGCCATCTCAAACACCTC 131 0581-0598 Coding 25326 CTTGTTCTTGCGGACCTG
132 0614-0631 Coding 25327 CCCCTCCGACGCTTGTTC 133 0625-0642 Coding
25328 GTATGGAGCCCTCAGGAG 134 0737-0754 3'-UTR 25329
GAGCCTTCAGTATGGAGC 135 0746-0763 3'-UTR 25330 GAAAATGGAGCCTTCAGT
136 0753-0770 3'-UTR 25331 GGAACTGAAAATGGAGCC 137 0759-0776 3'-UTR
25332 GGAGGGAACTGAAAATGG 138 0763-0780 3'-UTR 25333
GCAGGAGGGAACTGAAAA 139 0766-0783 3'-UTR 25334 AGGGCAGGGCATAGGCGT
140 0851-0868 3'-UTR 25335 GGAAGGGCAGGGCATAGG 141 0854-0871 3'-UTR
25336 CATGAGGAAGGGCAGGGC 142 0859-0876 3'-UTR 25337
TAAAGTGCTGGTGTGTGA 143 0920-0937 3'-UTR 25338 CCTGTGAGCCAGAAGTGT
144 0939-0956 3'-UTR 25339 TTCCTGTGAGCCAGAAGT 145 0941-0958 3'-UTR
25340 CACTTTCCTGTGAGCCAG 146 0945-0962 3'-UTR 25341
AGACACTTTCCTGTGAGC 147 0948-0965 3'-UTR 25342 ACTCTGGGTCCCTACTGC
148 0966-0983 3'-UTR 25343 TGCAGAAACAACTCCAGG 149 0992-1009 3'-UTR
.sup.1All cytidines are 5'-methyl-cytidines; all linkages are
phosphorothioate linkages. .sup.2Co-ordinates from Genbank
Accession No. L25081, locus name "HUMRHOCA" SEQ ID NO. 109.
[0141] The compounds shown in Table 18 were analyzed for effect on
RhoC mRNA levels by quantitative real-time PCR as described in
examples herein. Data are shown in Table 19 and are averages from
three experiments. If present, "N.D." indicates "no data".
22TABLE 19 Inhibition of RhoC mRNA levels by phosphorothioate
oligodeoxynucleotides % TARGET Inhi- SEQ ID ISIS# REGION SITE
SEQUENCE bition NO. 25304 5' UTR 4 GAGCTGAGATGAAGTCAA 29 110 25305
5' UTR 44 GCTGAAGTTCCCAGGCTG 25 111 25306 5' UTR 47
CCGGCTGAAGTTCCCAGG 42 112 25307 CODING 104 GGCACCATCCCCAACGAT 81
113 25308 CODING 105 AGGCACCATCCCCAACGA 81 114 25309 CODING 111
TCCCACAGGCACCATCCC 70 115 25310 CODING 117 AGGTCTTCCCACAGGCAC 40
116 25311 CODING 127 ATGAGGAGGCAGGTCTTC 41 117 25312 CODING 139
TTGCTGAAGACGATGAGG 23 118 25313 CODING 178 TCAAAGACAGTAGGGACG 0 119
25314 CODING 181 TTCTCAAAGACAGTAGGG 2 120 25315 CODING 183
AGTTCTCAAAGACAGTAG 38 121 25316 CODING 342 TGTTTTCCAGGCTGTCAG 59
122 25317 CODING 433 TCGTCTTGCCTCAGGTCC 79 123 25318 CODING 439
GTGTGCTCGTCTTGCCTC 67 124 25319 CODING 445 CTCCTGGTGTGCTCGTCT 67
125 25320 CODING 483 CAGACCGAACGGGCTCCT 65 126 25321 CODING 488
TTCCTCAGACCGAACGGG 57 127 25322 CODING 534 ACTCAAGGTAGCCAAAGG 33
128 25323 CODING 566 CTCCCGCACTCCCTCCTT 91 129 25324 CODING 575
CTCAAACACCTCCCGCAC 34 130 25325 CODING 581 GGCCATCTCAAACACCTC 64
131 25326 CODING 614 CTTGTTCTTGCGGACCTG 72 132 25327 CODING 625
CCCCTCCGACGCTTGTTC 66 133 25328 3' UTR 737 GTATGGAGCCCTCAGGAG 60
134 25329 3' UTR 746 GAGCCTTCAGTATGGAGC 63 135 25330 3' UTR 753
GAAAATGGAGCCTTCAGT 24 136 25331 3' UTR 759 GGAACTGAAAATGGAGCC 2 137
25332 3' UTR 763 GGAGGGAACTGAAAATGG 13 138 25333 3' UTR 766
GCAGGAGGGAACTGAAAA 27 139 25334 3' UTR 851 AGGGCAGGGCATAGGCGT 31
140 25335 3' UTR 854 GGAAGGGCAGGGCATAGG 21 141 25336 3' UTR 859
CATGAGGAAGGGCAGGGC 0 142 25337 3' UTR 920 TAAAGTGCTGGTGTGTGA 39 143
25338 3' UTR 939 CCTGTGAGCCAGAAGTGT 69 144 25339 3' UTR 941
TTCCTGTGAGCCAGAAGT 69 145 25340 3' UTR 945 CACTTTCCTGTGAGCCAG 82
146 25341 3' UTR 948 AGACACTTTCCTGTGAGC 69 147 25342 3' UTR 966
ACTCTGGGTCCCTACTGC 20 148 25343 3' UTR 992 TGCAGAAACAACTCCAGG 0
149
[0142] As shown in Table 19, SEQ ID NOs 113, 114, 115, 122, 123,
124, 125, 126, 127, 129, 131, 132, 133, 134, 135, 144, 145, 146 and
147 demonstrated at least 50% inhibition of RhoC expression in this
assay and are therefore preferred.
Example 17
Antisense Inhibition of RhoC Expression-Phosphorothioate 2'-MOE
Gapmer Oligonucleotides
[0143] In accordance with the present invention, a second series of
oligonucleotides targeted to human RhoC were synthesized. The
oligonucleotide sequences are shown in Table 20. Target sites are
indicated by nucleotide numbers, as given in the sequence source
reference (Genbank accession no. L25081), to which the
oligonucleotide binds.
[0144] All compounds in Table 20 are chimeric oligonucleotides
("gapmers") 18 nucleotides in length, composed of a central "gap"
region consisting of ten 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by four-nucleotide "wings". The
wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P=S)
throughout the oligonucleotide. Cytidine residues in the 2'-MOE
wings are 5-methylcytidines.
23TABLE 20 Nucleotide Sequences of Human RhoC Gapmer
Oligonucleotides NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID
NUCLEOTIDE TARGET NO. (5'.fwdarw.3') NO: CO-ORDINATES.sup.1 REGION
25344 GAGCTGAGATGAAGTCAA 110 0004-0021 5'-UTR 25345
GCTGAAGTTCCCAGGCTG 111 0044-0061 5'-UTR 25346 CCGGCTGAAGTTCCCAGG
112 0047-0064 5'-UTR 25347 GGCACCATCCCCAACGAT 113 0104-0121 Coding
25348 AGGCACCATCCCCAACGA 114 0105-0122 Coding 25349
TCCCACAGGCACCATCCC 115 0111-0128 Coding 25350 AGGTCTTCCCACAGGCAC
116 0117-0134 Coding 25351 ATGAGGAGGCAGGTCTTC 117 0127-0144 Coding
25352 TTGCTGAAGACGATGAGG 118 0139-0156 Coding 25353
TCAAAGACAGTAGGGACG 119 0178-0195 Coding 25354 TTCTCAAAGACAGTAGGG
120 0181-0198 Coding 25355 AGTTCTCAAAGACAGTAG 121 0183-0200 Coding
25356 TGTTTTCCAGGCTGTCAG 122 0342-0359 Coding 25357
TCGTCTTGCCTCAGGTCC 123 0433-0450 Coding 25358 GTGTCCTCGTCTTGCCTC
124 0439-0456 Coding 25359 CTCCTGGTGTGCTCGTCT 125 0445-0462 Coding
25360 CAGACCGAACGGCCTCCT 126 0483-0500 Coding 25361
TTCCTCAGACCGAACGGG 127 0488-0505 Coding 25362 ACTCAAGGTAGCCAAAGG
128 0534-0551 Coding 25363 CTCCCGCACTCCCTCCTT 129 0566-0583 Coding
25364 CTCAAACACCTCCCGCAC 130 0575-0592 Coding 25365
GGCCATCTCAAACACCTC 131 0581-0598 Coding 25366 CTTGTTCTTGCGGACCTG
132 0614-0631 Coding 25367 CCCCTCCGACGCTTGTTC 133 0625-0642 Coding
25368 GTATGGAGCCCTCAGGAG 134 0737-0754 3'-UTR 25369
GAGCCTTCAGTATGGAGC 135 0746-0763 3'-UTR 25370 GAAAATGGAGCCTTCAGT
136 0753-0770 3'-UTR 25371 GGAACTGAAAATGGAGCC 137 0759-0776 3'-UTR
25372 GGAGGGAACTGAAAATGG 138 0763-0780 3'-UTR 25373
GCAGGAGGGAACTGAAAA 139 0766-0783 3'-UTR 25374 AGGGCAGGGCATAGGCGT
140 0851-0868 3'-UTR 25375 GGAAGGGCAGGGCATAGG 141 0854-0871 3'-UTR
25376 CATGAGGAAGGGCAGGGC 142 0859-0876 3'-UTR 25377
TAAAGTGCTGGTGTGTGA 143 0920-0937 3'-UTR 25378 CCTGTGAGCCAGAAGTGT
144 0939-0956 3'-UTR 25379 TTCCTGTGAGCCAGAAGT 145 0941-0958 3'-UTR
25380 CACTTTCCTGTGAGCCAG 146 0945-0962 3'-UTR 25381
AGACACTTTCCTGTGAGC 147 0948-0965 3'-UTR 25382 ACTCTGGGTCCCTACTGC
148 0966-0983 3'-UTR 25383 TGCAGAAACAACTCCAGG 149 0992-1009 3'-UTR
.sup.1Emboldened residues are 2'-methoxyethoxy residues (others are
2'-deoxy-). All 2'-methoxyethoxy cytidines and cytidines are
5-methyl-cytidines; all linkages are phosphorothioate linkages.
.sup.2Co-ordinates from Genbank Accession No. L25081, locus name
"HUMRHOCA" SEQ ID NO. 109.
[0145] RhoC inhibition data for these compounds were obtained by
real-time quantitative PCR as described in other examples herein
and are averaged from three experiments. Data are shown in Table
21. If present, "N.D." indicates "no data".
24TABLE 21 Inhibition of RhoC mRNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap % TARGET Inhi- SEQ ID ISIS# REGION SITE SEQUENCE bition NO.
25344 5' UTR 4 GAGCTGAGATGAAGTCAA 0 110 25345 5' UTR 44
GCTGAAGTTCCCAGGCTG 35 111 25346 5' UTR 47 CCGGCTGAAGTTCCCAGG 53 112
25347 Coding 104 GGCACCATCCCCAACGAT 50 113 25348 Coding 105
AGGCACCATCCCCAACGA 56 114 25349 Coding 111 TCCCACAGGCACCATCCC 4 115
25350 Coding 117 AGGTCTTCCCACAGGCAC 11 116 25351 Coding 127
ATGAGGAGGCAGGTCTTC 6 117 25352 Coding 139 TTGCTGAAGACGATGAGG 15 118
25353 Coding 178 TCAAAGACAGTAGGGACG 32 119 25354 Coding 181
TTCTCAAAGACAGTAGGG 7 120 25355 Coding 183 AGTTCTCAAAGACAGTAG 39 121
25356 Coding 342 TGTTTTCCAGGCTGTCAG 59 122 25357 Coding 433
TCGTCTTGCCTCAGGTCC 48 123 25358 Coding 439 GTGTGCTCGTCTTGCCTC 36
124 25359 Coding 445 CTCCTCGTGTGCTCGTCT 61 125 25360 Coding 483
CAGACCGAACGGGCTCCT 50 126 25361 Coding 488 TTCCTCAGACCGAACGGG 14
127 25362 Coding 534 ACTCAAGGTAGCCAAAGG 32 128 25363 Coding 566
CTCCCGCACTCCCTCCTT 21 129 25364 Coding 575 CTCAAACACCTCCCGCAC 9 130
25365 Coding 581 GGCCATCTCAAACACCTC 66 131 25366 Coding 614
CTTGTTCTTGCGGACCTG 61 132 25367 Coding 625 CCCCTCCGACGCTTGTTC 0 133
25368 3' UTR 737 GTATGGAGCCCTCAGGAG 28 134 25369 3' UTR 746
GAGCCTTCAGTATGGAGC 32 135 25370 3' UTR 753 GAAAATGGAGCCTTCAGT 0 136
25371 3' UTR 759 GGAACTGAAAATGGAGCC 40 137 25372 3' UTR 763
GGAGGGAACTGAAAATGG 45 133 25373 3' UTR 766 GCAGGAGGGAACTGAAAA 35
139 25374 3' UTR 351 AGGGCAGGGCATAGGCGT 5 140 25375 3' UTR 854
GGAAGGGCAGGGCATAGG 0 141 25376 3' UTR 859 CATGAGGAAGGGCAGGGC 0 142
25377 3' UTR 920 TAAAGTGCTGGTGTGTGA 20 143 25378 3' UTR 939
CCTGTGAGCCAGAAGTGT 67 144 25379 3' UTR 941 TTCCTGTGAGCCAGAAGT 61
145 25380 3' UTR 945 CACTTTCCTGTGAGCCAG 80 146 25381 3' UTR 943
AGACACTTTCCTGTGAGC 0 147 25382 3' UTR 966 ACTCTGGGTCCCTACTGC 0 148
25383 3' UTR 992 TGCAGAAACAACTCCAGG 0 149
[0146] As shown in Table 21, SEQ ID NOs 111, 112, 113, 114, 119,
121, 122, 123, 124, 125, 126, 128, 131, 132, 134, 135, 137, 138,
139, 144, 145 and 146 demonstrated at least 25% inhibition of RhoC
expression in this experiment and are therefore preferred.
Example 18
Synthesis of RhoG Antisense Oligonucleotide Sequences
[0147] Oligonucleotide sequences designed to target human RhoG were
synthesized as described in previous examples and are shown in
Table 22. Target sequence data are from the RhoG cDNA sequence
published by Vincent, S., et al. (Mol. Cell. Biol. 1992, 12,
3138-3148); Genbank accession number X61587, provided herein as SEQ
ID NO: 150.
25TABLE 22 Nucleotide Sequences of Human RhoG Phosphorothioate
Oligodeoxynucleotide NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ
ID NUCLEOTIDE TARGET NO. (5'.fwdarw.3') NO: CO-ORDINATES.sup.1
REGION 25464 GACCTGGTGCCCCTCCCG 151 0048-0065 5'-UTR 25465
TCTTCTGGACCCCTCTGG 152 0073-0090 5'-UTR 25466 GGCAGTGCCTCCTCTCTC
153 0089-0106 5'-UTR 25467 GTGCAGTTGCTGTAGTGA 154 0107-0124 5'-UTR
25468 GCATCGTGGGTGCAGTTG 155 0116-0133 AUG 25469 CCACCACGCACTTGATGC
156 0137-0154 Coding 25470 TTGTGTAGCAGATGAGCA 157 0185-0202 Coding
25471 AAAGCGTTAGTTGTGTAG 158 0195-0212 Coding 25472
GCGCGCTGTAATTGTCGA 159 0239-0256 Coding 25473 GGTTCACTGTGCGCCCGT
160 0269-0286 Coding 25474 GTCCCACAGGTTCAGGTT 161 0283-0300 Coding
25475 TGTACGGAGGCGGTCATA 162 0319-0336 Coding 25476
ACGTTGGTCTGAGGGTAG 163 0342-0359 Coding 25477 CAATGGAGAAACAGATGA
164 0365-0382 Coding 25478 CATAGGACGGCGGACTGG 165 0383-0400 Coding
25479 CGCACGTTCTCATAGGAC 166 0393-0410 Coding 25480
ACCTCTGGATGCCACTTG 167 0414-0431 Coding 25481 AGGGCAGTGGTGGCACAC
168 0430-0447 Coding 25482 CAGCAGGATGGCCACATC 169 0448-0465 Coding
25483 GGGTGTCAGGCTGGGCTC 170 0488-0505 Coding 25484
CCCTGCTGCGGTGTGATG 171 0537-0554 Coding 25485 CGCGAGTGCCTGGCCCTG
172 0550-0567 Coding 25486 GTAGCGCACAGCGTGGAT 173 0574-0591 Coding
25487 CATTCGAGGTAGCGCACA 174 0582-0599 Coding 25488
ACACCATCCTGTTGCAGG 175 0606-0623 Coding 25489 GAACACTTCCTTGACACC
176 0619-0636 Coding 25490 ACAGCCTCGGCGAACACT 177 0630-0647 Coding
25491 AAGAGGATGCAGGACCGC 178 0684-0701 Coding 25492
GCAGCCTCCAAGCCAAGT 179 0713-0730 3'-UTR 25493 AAAAGGCATTCAGGGAAC
180 0818-0835 3'-UTR 25494 GGGTCCAACCTTGGCTTG 181 0936-0953 3'-UTR
25495 GTCAGTAGCGGAAAATGG 182 0984-1001 3'-UTR 25496
AGCTGGATGAACTGGTCA 183 0998-1015 3'-UTR 25497 AACTGTGTGGAAAGCTGG
184 1010-1027 3'-UTR 25498 ACCACAATAGGCAGCAAC 185 1028-1045 3'-UTR
25499 GAGGGCAGAGGTTAGAGA 186 1074-1091 3'-UTR 25500
CAATTCCAAGAGCAGCGA 187 1090-1107 3'-UTR 25501 TGGAGAAGGGAGAGAGCA
188 1119-1136 3'-UTR 25502 ACATTCACCTTCTCAGGA 189 1154-1171 3'-UTR
25503 GTCAGCAAATGCGTAAGG 190 1199-1216 3'-UTR .sup.1All cytidines
are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
.sup.2Co-ordinates from Genbank Accession No. X61587, locus name
"HSRHOG" SEQ ID NO. 150.
[0148] The compounds in Table 22 were analyzed for effect on RhoG
mRNA levels by quantitative real-time PCR as described in other
examples herein. Data, shown in Table 23, are averages from three
experiments. If present, "N.D." indicates "no data".
26TABLE 23 Inhibition of RhoG mRNA levels by phosphorothioate
oligodeoxynucleotides % TARGET Inhi- SEQ ID ISIS# REGION SITE
SEQUENCE bition NO. 25464 5' UTR 48 GACCTGGTGCCCCTCCCG 35 151 25465
5' UTR 73 TCTTCTGGACCCCTCTGG 36 152 25466 5' UTR 89
GGCAGTGCCTCCTCTCTC 35 153 25467 5' UTR 107 GTGCAGTTGCTGTAGTGA 10
154 25468 5' UTR 116 GCATCGTGGGTGCAGTTG 47 155 25469 CODING 137
CCACCACGCACTTGATGC 14 156 25470 CODING 185 TTGTGTAGCAGATGAGCA 35
157 25471 CODING 195 AAAGCGTTAGTTGTGTAG 0 158 25472 CODING 239
GCGCGCTGTAATTGTCGA 36 159 25473 CODING 269 GGTTCACTGTGCGCCCGT 16
160 25474 CODING 283 GTCCCACAGGTTCAGGTT 31 161 25475 CODING 319
TGTACGGAGGCGGTCATA 37 162 25476 CODING 342 ACGTTGGTCTGAGGGTAG 38
163 25477 CODING 365 CAATGGAGAAACAGATGA 0 164 25478 CODING 383
CATAGGACGGCGGACTGG 17 165 25479 CODING 393 CGCACGTTCTCATAGGAC 24
166 25480 CODING 414 ACCTCTGGATGCCACTTG 35 167 25481 CODING 430
AGGGCAGTGGTGGCACAC 15 168 25482 CODING 448 CAGCAGGATGGGCACATC 20
169 25483 CODING 488 GGGTGTCAGGCTGGGCTC 15 170 25484 CODING 537
CCCTGCTGCGGTGTGATG 44 171 25464 5' UTR 48 GACCTGGTGCCCCTCCCG 35 151
25465 5' UTR 73 TCTTCTGGACCCCTCTGG 36 152 25466 5' UTR 89
GGCAGTGCCTCCTCTCTC 35 153 25485 CODING 550 CGCGAGTGCCTGGCCCTG 9 172
25486 CODING 574 GTAGCGCACAGCGTGGAT 35 173 25487 CODING 582
CATTCGAGGTAGCGCACA 39 174 25488 CODING 606 ACACCATCCTGTTGCAGG 23
175 25489 CODING 619 GAACACTTCCTTGACACC 31 176 25490 CODING 630
ACAGCCTCGGCGAACACT 6 177 25491 CODING 684 AAGAGGATGCAGGACCGC 18 178
25492 3' UTR 713 GCAGCCTCCAAGCCAAGT 42 179 25493 3' UTR 818
AAAAGGCATTCAGGGAAC 0 180 25494 3' UTR 936 GGGTCCAACCTTGGCTTG 58 181
25495 3' UTR 984 GTCAGTAGCGGAAAATGG 0 182 25496 3' UTR 998
AGCTGGATGAACTGGTCA 23 183 25497 3' UTR 1010 AACTGTGTGGAAAGCTGG 8
184 25498 3' UTR 1028 ACCACAATAGGCAGCAAC 31 185 25499 3' UTR 1074
GAGGGCAGAGGTTAGAGA 21 186 25500 3' UTR 1090 CAATTCCAAGAGCAGCGA 18
187 25501 3' UTR 1119 TGGAGAAGGGAGAGAGCA 32 188 25502 3' UTR 1154
ACATTCACCTTCTCAGGA 20 189 25503 3' UTR 1199 GTCAGCAAATGCGTAAGG 24
190
[0149] As shown in Table 23, SEQ ID NOs 151, 152, 153, 155, 157,
159, 161, 162, 163, 167, 171, 173, 174, 176, 179, 181, 185 and 188
demonstrated at least 25% inhibition of RhoG expression in this
assay and are therefore preferred.
Example 19
Antisense Inhibition of RhoG Expression-Phosphorothioate 2'-MOE
Gapmer Oligonucleotides
[0150] In accordance with the present invention, a second series of
oligonucleotides targeted to human RhoG were synthesized. The
oligonucleotide sequences are shown in Table 24. Target sites are
indicated by nucleotide numbers, as given in the sequence source
reference (Genbank accession no. X61587), to which the
oligonucleotide binds.
[0151] All compounds in Table 24 are chimeric oligonucleotides
("gapmers") 18 nucleotides in length, composed of a central "gap"
region consisting of ten 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by four-nucleotide "wings". The
wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P=S)
throughout the oligonucleotide. Cytidine residues in the 2'-MOE
wings are 5-methylcytidines.
27TABLE 24 Nucleotide Sequences of Human RhoG Gapmer
Oligonucleotides NUCLEOTIDE TARGET GENE GENE ISIS SEQUENCE SEQ ID
NUCLEOTIDE TARGET NO. (5'.fwdarw.3') NO: CO-ORDINATES.sup.1 REGION
25504 GACCTGGTGCCCCTCCCG 151 0048-0065 5'-UTR 25505
TCTTCTGGACCCCTCTGG 152 0073-0090 5'-UTR 25506 GGCAGTGCCTCCTCTCTC
153 0089-0106 5'-UTR 25507 GTGCAGTTGCTCTAGTGA 154 0107-0124 5'-UTR
25508 GCATCGTGGGTGCAGTTG 155 0116-0133 AUG 25509 CCACCACGCACTTGATGC
156 0137-0154 Coding 25510 TTGTGTAGCACATGAGCA 157 0185-0202 Coding
25511 AAAGCGTTAGTTGTGTAG 158 0195-0212 Coding 25512
GCGCGCTGTAATTGTCGA 159 0239-0256 Coding 25513 GGTTCACTGTGCGCCCGT
160 0269-0286 Coding 25514 GTCCCACAGGTTCAGGTT 161 0283-0300 Coding
25515 TGTACGGAGGCGGTCATA 162 0319-0336 Coding 25516
ACGTTGGTCTGAGGGTAG 163 0342-0359 Coding 25517 CAATGGAGAAACAGATGA
164 0365-0382 Coding 25518 CATAGGACGGCGGACTGG 165 0383-0400 Coding
25519 CGCACGTTCTCATAGGAC 166 0393-0410 Coding 25520
ACCTCTGGATGCCACTTG 167 0414-0431 Coding 25521 AGGGCAGTGGTGGCACAC
168 0430-0447 Coding 25522 CAGCAGGATGCGCACATC 169 0448-0465 Coding
25523 GGGTGTCAGGCTGGGCTC 170 0488-0505 Coding 25524
CCCTGCTGCGGTGTGATG 171 0537-0554 Coding 25525 CGCGAGTGCCTGGCCCTG
172 0550-0567 Coding 25526 GTAGCGCACAGCGTGGAT 173 0574-0591 Coding
25527 CATTCGAGGTAGCGCACA 174 0582-0599 Coding 25528
ACACCATCCTGTTGCAGG 175 0606-0623 Coding 25529 GAACACTTCCTTGACACC
176 0619-0636 Coding 25530 ACAGCCTCGGCGAACACT 177 0630-0647 Coding
25531 AAGAGGATGCAGGACCGC 178 0684-0701 Coding 25532
GCAGCCTCCAAGCCAAGT 179 0713-0730 3'-UTR 25533 AAAAGGCATTCAGGGAAC
180 0818-0835 3'-UTR 25534 GGGTCCAACCTTGGCTTG 181 0936-0953 3'-UTR
25535 GTCAGTAGCGGAAAATGG 182 0984-1001 3'-UTR 25536
AGCTGGATGAACTGGTCA 183 0998-1015 3'-UTR 25537 AACTGTGTGGAAAGCTGG
184 1010-1027 3'-UTR 25538 ACCACAATAGGCAGCAAC 185 1028-1045 3'-UTR
25539 GAGGGCAGAGGTTAGAGA 186 1074-1091 3'-UTR 25540
CAATTCCAAGAGCAGCGA 187 1090-1107 3'-UTR 25541 TGGAGAAGGGAGAGAGCA
188 1119-1136 3'-UTR 25542 ACATTCACCTTCTCAGGA 189 1154-1171 3'-UTR
25543 GTCAGCAAATGCGTAAGG 190 1199-1216 3'-UTR .sup.1Emboldened
residues are 2'-methoxyethoxy residues (others are 2'-deoxy-). All
2'-methoxyethoxy cytidines and cytidines are 5-methyl-cytidines;
all linkages are phosphorothioate linkages. Co-ordinates from
Genbank Accession No. X61587, locus name "HSRHOG" SEQ ID NO.
150.
[0152] RhoG inhibition data for compounds in Table 24 were obtained
by real-time quantitative PCR as described in other examples herein
and are averaged from three experiments. Data are shown in Table
25. If present, "N.D." indicates "no data".
28TABLE 25 Inhibition of RhoG mBNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap TAR- SEQ GET % ID ISIS# REGION SITE SEQUENCE Inhibition NO.
25504 5'UTR 48 GACCTGGTGCCCCTCCCG 0 151 25505 5'UTR 73
TCTTCTGGACCCCTCTGG 32 152 25506 5'UTR 89 GGCAGTGCCTCCTCTCTC 28 153
25507 5'UTR 107 GTGCAGTTGCTGTAGTGA 0 154 25508 5'UTR 116
GCATCGTGGGTGCAGTTG 12 155 25509 Coding 137 CCACCACGCACTTGATGC 0 156
25510 Coding 185 TTGTGTAGCAGATGAGCA 0 157 25511 Coding 195
AAAGCGTTAGTTGTGTAG 33 158 25512 Coding 239 GCGCGCTGTAATTGTCGA 0 159
25513 Coding 269 GGTTCACTGTCCGCCCGT 82 160 25514 Coding 283
GTCCCACAGGTTCAGGTT 0 161 25515 Coding 319 TGTACGCACGCCGTCATA 13 162
25516 Coding 342 ACGTTGGTCTCAGGGTAG 53 163 25517 Coding 365
CAATGGAGAAACAGATGA 0 164 25518 Coding 383 CATAGGACGGCGGACTGG 55 165
25519 Coding 393 CGCACGTTCTCATAGGAC 9 166 25520 Coding 414
ACCTCTCGATGCCACTTG 56 167 25521 Coding 430 AGGGCAGTGGTGGCACAC 0 168
25522 Coding 448 CAGCAGGATGGGCACATC 0 169 25523 Coding 488
GGGTCTCAGGCTGGGCTC 27 170 25524 Coding 537 CCCTGCTGCGGTGTGATG 55
171 25525 Coding 550 CGCGAGTGCCTGGCCCTG 41 172 25526 Coding 574
GTAGCGCACAGCGTGGAT 41 173 25527 Coding 582 CATTCGAGGTAGCGCACA 0 174
25528 Coding 606 ACACCATCCTCTTGCAGG 37 175 25529 Coding 619
GAACACTTCCTTCACACC 23 176 25530 Coding 630 ACAGCCTCGGCGAACACT 59
177 25531 Coding 684 AAGAGGATGCAGGACCGC 39 178 25532 3'UTR 713
GCAGCCTCCAAGCCAAGT 13 179 25533 3'UTR 818 AAAAGGCATTCAGGGAAC 43 180
25534 3'UTR 936 GGGTCCAACCTTGGCTTG 78 181 25535 3'UTR 984
GTCAGTAGCGGAAAATGG 54 182 25536 3'UTR 998 AGCTGGATGAACTGGTCA 54 183
25537 3'UTR 1010 AACTCTGTGCAAAGCTGG 59 184 25538 3'UTR 1028
ACCACAATAGGCAGCAAC 43 185 25539 3'UTR 1074 GAGGGCAGAGGTTAGAGA 0 186
25540 3'UTR 1090 CAATTCCAAGAGCAGCGA 26 187 25541 3'UTR 1119
TGGAGAACGGAGAGAGCA 0 188 25542 3'UTR 1154 ACATTCACCTTCTCAGGA 26 189
25543 3'UTR 1199 GTCACCAAATGCGTAAGG 73 190
[0153] As shown in Table 25, SEQ ID NOs 152, 158, 160, 163, 165,
167, 171, 172, 173, 175, 177, 178, 180, 181, 182, 183, 184, 185 and
190 demonstrated at least 30% inhibition of RhoG expression in this
experiment and are therefore preferred.
Example 20
Human Rac1 Oligonucleotide Sequences
[0154] Antisense oligonucleotides were designed to target human
Rac1. Target sequence data are from the Rac1 cDNA sequence
published by Didsbury, J., et al. (J. Biol. Chem. 1989, 264,
16378-16382); Genbank accession number M29870, provided herein as
SEQ ID NO: 191. Oligonucleotides were synthesized primarily with
phosphorothioate linkages. Oligonucleotide sequences are shown in
Table 26.
[0155] Cells were cultured, treated with oligonucleotides, and mRNA
was isolated and quantitated as described in Example 2. A 45-mer
antisense oligonucleotide to Rac1
(5'-ATAGAATGTGAGTCTGAACTCTTACATTTAGAACAAACAAAACCT- -3' SEQ ID NO.
192) was used as a probe as described in Didsbury, J., et al. (J.
Biol. Chem. 1989, 264, 16378-16382).
[0156] An initial screen of Rac1 specific antisense
oligonucleotides was performed using a oligonucleotide
concentration of 300 nM.
[0157] Results are shown in Table 27. Oligonucleotides 16052 (SEQ
ID NO. 195), 16056 (SEQ ID NO. 199), 16058 (SEQ ID NO. 201), 16062
(SEQ ID NO. 204) and 16063 (SEQ ID NO. 205) gave better than 50%
inhibition of Rac1 mRNA levels. Oligonucleotides 16052 (SEQ ID NO.
195), 16058 (SEQ ID NO. 201) and 16062 (SEQ ID NO. 204) gave better
than 75% inhibition.
29TABLE 26 Nucleotide Sequences of Rac-1 Phosphorothioate
Oligonucleotides TARGET GENE GENE NUCLEOTIDE SEQUENCE NUCLEOTIDE
TARGET ISIS NO. (5' ->3') SEQ ID NO: CO-ORDINATES.sup.1 REGION
16050 CAAATGATGCAGGACTCACA 193 0252-0271 Coding 16051
CACCACCACACACTTGATC 194 0009-0027 Coding 16052 ATAAGCCCAGATTCACCG
195 0149-0166 Coding 16053 TCTTTGCGGATAGGATAGG 196 0207-0225 Coding
16054 GCTTCTTCTCCTTCAGTTTCTC 197 0379-0400 Coding 16055
CAGCACCAATCTCCTTAGC 198 0436-0454 Coding 16056 CTCTTCCTCTTCTTCACCC
199 0542-0560 Coding 16057 CCTAAGATCAAGTTTAGTTC 200 0341-0360
Coding 16058 CGCACCTCAGGATACCACTT 201 0286-0305 Coding 16059
ATCTACCATAACATTGGCAG 202 0122-0141 Coding 16060
TAATTGTCAAAGACAGTAGG 203 0100-0119 Coding 16062
GAGCGCCGAGCACTCCAGGT 204 0461-0480 Coding 16063
CTCAAACACTGTCTTGAGGC 205 0491-0510 Coding 16143
ATAGAATGTGAGTCTCAACT 206 unknown.sup.3 3'-UTR 16144
CTTACATTTAGAACAAACAAAACCT 207 unknown.sup.3 3'-UTR 16849
CCCAGCTAAGAATTCCGCTC 208 16058 control 16850 TAAACGCCGAATCTACGC 209
16052 control .sup.1all linkages are phosphorothioate linkages.
.sup.2Co-ordinates from Genbank Accession No. M29870, locus name
"HUMRACA" SEQ ID NO. 191. .sup.3These oligonucleotides were
designed based on a probe to the 3'-UTR region of Rac1 (Didsbury,
J., et al., J. Biol. Chem. 1989, 264, 16378.gtoreq.16382)
[0158]
30TABLE 27 Activities of Phosphorothioate Oligonucleotides Targeted
to Human Rac1 SEQ GENE ISIS ID TARGET % mRNA % mRNA No: NO: REGION
EXPRESSION INHIBITION LIPOFECTIN -- -- 100.0% 0.0% only 16051 194
Coding 77.1% 22.9% 16052 195 Coding 3.7% 96.3% 16053 196 Coding
68.4% 31.6% 16054 197 Coding 67.6% 32.4% 16055 198 Coding 70.8%
29.2% 16056 199 Coding 48.0% 52.0% 16057 200 Coding 97.3% 2.7%
16058 201 Coding 22.2% 77.8% 16059 202 Coding 57.7% 42.3% 16060 203
Coding 91.6% 8.4% 16062 204 Coding 21.7% 78.3% 16063 205 Coding
32.4% 67.6% 16143 206 3'-UTR 56.1% 43.9% 16144 207 3'-UTR 72.9%
27.1%
Example 21
Dose Response and Specificity of Antisense Oligonucleotide Effects
on Human Rac1 mRNA Levels in A549 Cells
[0159] Oligonucleotides 16050 (SEQ ID NO. 193), 16052 (SEQ ID NO.
195)16058 (SEQ ID NO. 201), 16062 (SEQ ID NO. 204) and 16143 (SEQ
ID NO. 206) were chosen for dose response studies. Oligonucleotide
16057 (SEQ ID NO. 200) was chosen as a negative control because it
was inactive in the initial screen. Results are shown in Table 28.
Oligonucleotides 16050, 16052, 16058 and 16062 inhibited Rac1 mRNA
expression in a dose dependent manner with maximum expression of
65% to 82%.
[0160] The specificity of oligonucleotides 16052 and 16058 was
tested using scrambled controls. Results are shown in Table 29.
Both sequences inhibited Rac1 mRNA expression in a dose dependent
manner and were significantly better than their scrambled
controls.
31TABLE 28 Dose Response of A549 Cells to Rac1 Antisense
Oligonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO:
Target Dose Expression Inhibition control -- LIPOFECTIN -- 100% 0%
only 16050 193 coding 75 nM 71.1% 28.9% 16050 193 " 150 nM 53.6%
46.4% 16050 193 " 300 nM 33.6% 66.4% 16052 195 coding 75 nM 68.2%
31.8% 16052 195 " 150 nM 40.5% 59.5% 16052 195 " 300 nM 28.3% 71.7%
16057 200 coding 75 nM 81.7% 18.3% 16057 200 " 150 nM 80.2% 19.8%
16057 200 " 300 nM 85.8% 14.2% 16058 201 coding 75 nM 60.1% 39.9%
16058 201 " 150 nM 42.9% 57.1% 16058 201 " 300 nM 17.7% 82.3% 16062
204 coding 75 nM 50.5% 49.5% 16062 204 " 150 nM 40.2% 59.8% 16062
204 " 300 nM 25.2% 74.8% 16143 206 3'-UTR 75 nM 294.8% -- 16143 206
" 150 nM 100.8% -- 16143 206 " 300 nM 88.6% 11.4%
[0161]
32TABLE 29 Specificity of Rac1 Antisense Oligonucleotides (ASOs) in
A549 Cells SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose
Expression Inhibition control -- LIPOFECTIN -- 100% 0% only 16052
195 coding 75 nM 86.6% 13.4% 16052 195 " 150 nM 52.8% 47.2% 16052
195 " 300 nM 18.5% 81.5% 16850 209 control 75 nM 88.9% 11.1% 16850
209 " 150 nM 97.2% 2.8% 16850 209 " 300 nM 107.4% -- 16058 201
coding 75 nM 82.7% 17.3% 16058 201 " 150 nM 36.8% 63.2% 16058 201 "
300 nM 21.1% 78.9% 16849 208 control 75 nN 90.7% 9.3% 16849 208 "
150 nM 70.2% 29.8% 16849 208 " 300 nM 68.2% 31.8%
Example 22
Design and Testing of Chimeric (Deoxy Gapped) 2'-O-Methoxyethyl
Rac1 Antisense Oligonucleotides on Rac1 mRNA Levels in A549
Cells
[0162] Oligonucleotides targeted to Rac1 were synthesized as a
uniformly phosphorothioate or mixed phosphorothioate/phosphodiester
chimeric oligonucleotides having variable regions of
2'-O-methoxyethyl (2'-MOE) nucleotides and deoxynucleotides. All
2'-MOE cytosines were 5-methyl-cytosines. Additionally, some
oligonucleotides were synthesized with deoxycytosines as
5-methyl-cytosines. Additional oligonucleotides were synthesized,
with similar chemistries, as scrambled controls. Oligonucleotide
sequences and chemistries are shown in Tables 30 and 31. A dose
response experiment was performed using a number of these
oligonucleotides as described in Example 3.
[0163] Results are shown in Table 32. All of the chimeric
oligonucleotides tested showed a dose dependent effect and showed
inhibition of Rac mRNA levels comparable to that of the
phosphorothioate oligodeoxynucleotide.
33TABLE 30 Nucleotide Sequences of Rac1 Gapmer Oligonucleotides SEQ
TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO.
(5' -> 3') NO: CO-ORDINATES.sup.1 REGION 16899
ATAAGCCCAGATTCACCG 195 0149-0166 Coding 16900 CAAATGATGCAGGACTCACA
193 0252-0271 Coding 16901 CGCACCTCAGGATACCACTT 201 0286-0305
Coding 17161 ATAAGCCCAGATTCACCG 195 0149-0166 Coding 17162
ATAAGCCCAGATTCACCG 195 0149-0166 Coding 17163 ATAAGCCCAGATTCACCG
195 0149-0166 Coding 17164 ATAAGCCCAGATTCACCG 195 0149-0166 Coding
18540 ATAAGCCCTGATTCACCG 210 16899 mismatch 18541
ATACGCCCTGATTCACCG 211 16899 mismatch 18542 ATACGCCCTGATTAACCG 212
16899 mismatch 18549 TAAACGCCGAATCTACGC 213 16899 control
.sup.1Emboldened residues are 2'-methoxyethoxy residues (others are
2'-deoxy-). All 2'-methoxyethoxy cytidines are 5-methyl-cytidines;
all linkages are phosphorothioate linkages. .sup.2Co-ordinates from
Genbank Accession No. M29870, locus name "HUMRACA" SEQ ID NO.
191.
[0164]
34TABLE 31 Nucleotide Sequences of Rac1 Mixed Backbone
Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID
NUCLEOTIDE TARGET NO. (5' ->3') NO: CO-ORDINATES.sup.1 REGION
17814 ToAoAoAoCoGoCoCoGsAsAsTsCs- TsAsCsGsC 213 16899 control 17815
AoToAoAoGoCoCoCoAsGsAsTsTsCsAsCs- CsC 195 0149-0166 Coding 17816
CoAoAoAoToGsAsTsGsCsAsGsGsAsCsToCoA- oCoA 193 0252-0271 Coding
17817 AoAoAoCoToGsCsTsGsAsAsGsTsAsCsGoCoA- oCoA 214 17816 control
24686 ToAoAoAoCoGoCoCoGoAoAoToCoToAoCoGoC 213 16899 control 24687
TsAsAsAsCsGsCsCsGsAsAsTsCsTsAsCsGsC 213 16899 control
.sup.1Emboldened residues are 2'-methoxyethoxy residues (others are
2'-deoxy-). All 2'-methoxyethoxy cytidines and 2'-deoxy cytidines
are 5-methyl-cytidines; "s" linkages are phosphorothioate linkages,
"o" linkages are phosphodiester linkages. .sup.2Co-ordinates from
Cenbank Accession No. M29870, locus name "HUMRACA" SEQ ID NO.
191.
[0165]
35TABLE 32 Dose Response of A549 Cells to Rac1 Antisense Gapmer
Oligonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO:
Target Dose Expression Inhibition control -- LIPOFECTIN -- 100 0.0%
only 16899 195 coding 75 nM 79.9% 20.1% " " " 150 nM 40.8% 59.2% "
" " 300 nM 21.8% 78.2% 17161 195 coding 75 nM 31.3% 68.7% " " " 150
nM 16.9% 83.1% " " " 300 nM 12.3% 87.7% 17162 195 coding 75 nM
89.2% 10.8% " " " 150 nM 63.0% 37.0% " " " 300 nM 18.4% 81.6% 17163
195 coding 75 nM 93.4% 6.6% " " " 150 nM 67.3% 32.7% " " " 300 nM
34.4% 65.6% 17164 195 coding 75 nM 94.7% 5.3% " " " 150 nM 65.9%
34.1% " " " 300 nM 36.2% 63.8%
Example 23
Human cdc42 Chimeric (Deoxy Gapped) 2'-O-methoxyethyl
Oligonucleotide Sequences
[0166] Antisense oligonucleotides were designed to target human
cdc42. Target sequence data are from the cdc42 cDNA sequence
published by Shinjo, K. et al. (Proc. Natl. Acad. Sci. U.S.A. 1990,
87, 9853-9857); Genbank accession number M57298, provided herein as
SEQ ID NO: 215. Oligonucleotides were synthesized as uniformly
phosphorothioate chimeric oligonucleotides having a centered deoxy
gap of eight nucleotides flanked by 2'-O-methoxyethyl (2'-MOE)
regions. All 2'-MOE cytosines were 5-methyl-cytosines.
Oligonucleotide sequences are shown in Table 33.
[0167] A549 cells were cultured and treated with oligonucleotide as
described in Example 2. Quantitation of cdc42 mRNA levels was
determined by real-time PCR (RT-PCR) as described in previous
examples.
[0168] For cdc42 the PCR primers were:
36 For cdc42 the PCR primers were: Forward:
5'-TTCAGCAATGCACACAATTAAGTGT-3' SEQ ID NO. 216 Reverse:
5'-TGTTGTGTAGGATATCAGGAGACATGT-3' SEQ ID NO. 217 and the PCR probe
was: FAM-TTGTGGGCGATGGTGCTGTTGGTA-TAMRA
[0169] (SEQ ID NO. 218) where FAM or JOE (PE-Applied Biosystems,
Foster City, Calif.) is the fluorescent reporter dye) and TAMRA
(PE-Applied Biosystems, Foster City, Calif.) is the quencher
dye.
[0170] For GAPDH the PCR primers were:
37 For GAPDH the PCR primers were: Forward primer:
5'-GAAGGTGAAGGTCGGAGTC-3' SEQ ID NO. 65 Reverse primer:
5'-GAAGATGGTGATGGGATTTC-3' SEQ ID NO. 66 and the PCR probe was: 5'
JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3'
[0171] (SEQ ID NO. 67) where FAM or JOE (PE-Applied Biosystems,
Foster City, Calif.) is the fluorescent reporter dye) and TAMRA
(PE-Applied Biosystems, Foster City, Calif.) is the quencher
dye.
[0172] Results are shown in Table 34. All oligonucleotides tested
gave greater than 40% inhibition of cdc42 mRNA expression.
38TABLE 33 Nucleotide Sequences of cdc42 Oligonucleotides SEQ
TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO.
(5' ->3') NO: CO-ORDINATES.sup.1 REGION 17203
TAATTGTCTGCATTGCTGAA 219 0063-0082 AUG 17209 TTACCAACAGCACGATCGCC
220 0097-0116 Coding 17210 CCACCAATCATAACTGTGAC 221 0193-0212
Coding 17211 GTGGATAACTCAGCGGTCGT 222 0270-0289 Coding 17212
GAAGATGGAGAGACCACTGA 223 0316-0335 Coding 17213
GTGAGTTATCTCAGGCACCC 224 0359-0378 Coding 17214
GCTTCTGTTTGTTCTTGGCA 225 0456-0475 Coding 17215
TGACAGCCTTCAGGTCACGG 226 0507-0526 Coding 17216
CACCTGCGGCTCTTCTTCGG 227 0613-0632 Coding 17217
TTGTCTCACACGAGTGCATG 228 0774-0793 3'-UTR 17218
TTCTGACAATACAATTACTC 229 0844-0863 3'-UTR 17219
TTACAGAGTCATCCACAAGC 230 0961-0980 3'-UTR 20457
CGATAGTCTCCACGTGAGGC 231 17215 control 21668 CGATAGTCTCCACGTGAGGC
231 17215 control 21917 GTAACGCTCCTATGGCCAGG 232 17215 control
21918 AGACTGACTGCTCGTCGCGA 233 17215 control .sup.1Emboldened
residues are 2'-methoxyethoxy residues (others are 2'-deoxy-) . All
2'-methoxyethoxy cytidines are 5-methyl-cytidines, underlined "C"
residues are 5-methyl-cytidines; all linkages are phosphorothioate
linkages. .sup.2Co-ordinates from Genbank Accession No. M57298,
locus name "HUMGPG25K" SEQ ID NO. 215.
[0173]
39TABLE 34 Activities of Phosphorothioate Oligonucleotides Targeted
to Human Cdc42 SEQ GENE ISIS ID TARGET % mRNA 5 mRNA No: NO: REGION
EXPRESSION INHIBITION LIPOFECTIN -- -- 100% 0% only 17208 219 AUG
40.6% 59.4% 17209 220 Coding 43.4% 56.6% 17210 221 Coding 55.4%
44.6% 17211 222 Coding 25.5% 74.5% 17212 223 Coding 31.1% 68.9%
17213 224 Coding 14.0% 86.0% 17214 225 Coding 27.4% 72.6% 17215 226
Coding 16.9% 83.1% 17216 227 Coding 26.0% 74.0% 17217 228 3'-UTR
28.4% 71.6% 17218 229 3'-UTR 17.2% 82.8% 17219 230 3'-UTR 20.2%
79.8%
Example 24
Dose Response of Antisense Oligonucleotide Effects on Human cdc42
mRNA Levels in A549 Cells
[0174] Oligonucleotides 17213 (SEQ ID NO. 224), 17215 (SEQ ID NO.
226), 17218 (SEQ ID NO. 229), and 17219 (SEQ ID NO. 230) were
chosen for dose response studies. Results are shown in Table
35.
40TABLE 35 Dose Response of A549 Cells to Cdc42 Antisense
Oligonucleotides (ASOs) SEQ ID ASO Gene % mRNA % mRNA ISIS # NO:
Target Dose Expression Inhibition control -- LIPOFECTIN -- 100% 0%
only 17213 224 coding 75 nM 158% -- 17213 " " 300 nM 16% 84% 17215
226 coding 75 nM 90% 10% 17215 " " 300 nM 21% 79% 17218 229 3'-UTR
75 nM 53% 47% 17218 " " 300 nM 38% 62% 17219 230 3'-UTR 75 nM 102%
-- 17219 " " 300 nM 41% 59%
Example 25
Additional cdc42 Chimeric Oligonucleotides
[0175] Oligonucleotides having SEQ ID NO: 226 were synthesized as
mixed phosphorothioate/phosphodiester chimeric oligonucleotides
having variable wing regions of 2'-O-methoxyethyl (2'-MOE)
nucleotides and a central stretch of nine deoxynucleotides. All
2'-MOE cytosines were 5-methyl-cytosines. Oligonucleotide sequences
and chemistries are shown in Table 36.
41TABLE 36 Nucleotide Sequence of 17215 Analog SEQ TARGET GENE GENE
ISIS NUCLEOTIDE SEQUENCE ID NUCLEOTIDE TARGET NO. (5' ->3') NO:
CO-ORDINATES.sup.1 REGION 22276
ToGoAoCoAoGsCsCsTsTsCsAsGsGsTsCoAoCoGoG 226 0507-0526 Coding 22277
CoGoAoToAoGsTsCsTsCsCsAsCsGsTsGoAoGoGoC 231 22276 control
.sup.1Emboldened residues are 2'-methoxyethoxy residues (others are
2'-deoxy). All 2'-methoxyethoxy cytidines are 5-methyl-cytidines;
"s" linkages are phosphorothioate linkages, "o" linkages are
phosphodiester linkages. .sup.2Co-ordinates from Cenbank Accession
No. M57298, locus name "HUMCPC25K" SEQ ID NO. 215.
[0176]
Sequence CWU 1
1
233 1 1074 DNA Homo sapiens 1 gaattcgggc taccctcgcc ccgcccgcgg
tcctccgtcg gttctctcat tagtccacgg 60 tctggtcttc agctacccgc
cttcgtctcc gagtttgcga ctcgcgggac cggcgtcccc 120 ggcgcgaaga
ggctggactc ggattcgttg cctgagcaat ggctgccatc cggaagaaac 180
tggtgattgt tggtgatgga gcctgtggaa agacatgctt gctcatagtc ttcagcaagg
240 accagttccc agaggtgtat gtgcccacag tgtttgagaa ctatgtggca
gatatcgagg 300 tggatggaaa gcaggtagag ttggctttgt gggacacagc
tgggcaggaa gattatgatc 360 gcctgaggcc cctctcctac ccagataccg
atgttatact gatgtgtttt tccatcgaca 420 gccctgatag tttagaaaac
atcccagaaa agtggacccc agaagtcaag catttctgtc 480 ccaacgtgcc
catcatcctg gttgggaata agaaggatct tcggaatgat gagcacacaa 540
ggcgggagct agccaagatg aagcaggagc cggtgaaacc tgaagaaggc agagatatgg
600 caaacaggat tggcgctttt gggtacatgg agtgttcagc aaagaccaaa
gatggagtga 660 gagaggtttt tgaaatggct acgagagctg ctctgcaagc
tagacgtggg aagaaaaaat 720 ctggttgcct tgtcttgtga aaccttgctg
caagcacagc ccttatgcgg ttaattttga 780 agtgctgttt attaatctta
gtgtatgatt actggccttt ttcatttatc tataatttac 840 ctaagattac
aaatcagaag tcatcttgct accagtattt agaagccaac tatgattatt 900
aacgatgtcc aacccgtctg gcccaccagg gtccttttga cactgctcta acagccctcc
960 tctgcactcc cacctgacac accaggcgct aattcaagga atttcttaac
ttcttgcttc 1020 tttctagaaa gagaaacagt tggtaacttt tgtcaattag
gctgtaacta cttt 1074 2 19 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 2 tgcaagcaca gcccttatg 19 3 19 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 3
tgtcaaaagg accctggtg 19 4 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 4 agtcgcaaac tcggagac 18 5 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 5
ttgctcaggc aacgaatc 18 6 22 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 6 ctgaagacta tgagcaagca tg 22 7 19 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 7
ctcatcattc cgaagatcc 19 8 22 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 8 ccaatcctgt ttgccatatc tc 22 9 22 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 9
ccatctttgg tctttgctga ac 22 10 21 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 10 gcagagcagc
tctcgtagcc a 21 11 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 11 tcacaagaca aggcaaccag 20 12 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 12
aggccagtaa tcatacacta 20 13 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 13 gttggcttct aaatactggt 20 14 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
14 ggctgttaga gcagtgtcaa 20 15 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 15 agcgcctggt
gtgtcaggtg 20 16 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 16 tagttacagc ctaattgaca 20 17 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 17
ggcacctgtt gggtgagctg 20 18 22 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 18 acactcttgc ttaccgtacc tt 22 19
20 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 19 tgcggtaagt gcggtatcaa 20 20 19 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 20 gtcgttagtc
gaaatgagg 19 21 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 21 agcttgtgaa cgagtgtcga 20 22 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 22
tgcagttggc agagtctgaa 20 23 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 23 agagaaccga cggaggac 18 24 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 24
gtggactaat gagagaac 18 25 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 25 gaccgtggac taatgaga 18 26 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 26
agctgaagac cagaccgt 18 27 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 27 aatccgagtc cagcctct 18 28 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 28
aacgaatccg agtccagc 18 29 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 29 tcaggcaacg aatccgag 18 30 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 30
caccaacaat caccagtt 18 31 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 31 aagactatga gcaagcat 18 32 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 32
atacacctct gggaactg 18 33 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 33 acatagttct caaacact 18 34 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 34
actctacctg ctttccat 18 35 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 35 cacaaagcca actctacc 18 36 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 36
aacatcggta tctgggta 18 37 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 37 ttctgggatg ttttctaa 18 38 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 38
ggacagaaat gcttgact 18 39 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 39 gtgctcatca ttccgaag 18 40 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 40
cttgtgtgct catcattc 18 41 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 41 tagctcccgc cttgtgtg 18 42 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 42
ccaatcctgt ttgccata 18 43 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 43 gtctttgctg aacactcc 18 44 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 44
aaaacctctc tcactcca 18 45 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 45 aagacaaggc aaccagat 18 46 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 46
tttcacaaga caaggcaa 18 47 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 47 gcaaggtttc acaagaca 18 48 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 48
attaaccgca taagggct 18 49 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 49 taataaacag cacttcaa 18 50 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 50
ccagtaatca tacactaa 18 51 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 51 atgacttctg atttgtaa 18 52 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 52
tagcaagatg acttctga 18 53 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 53 ctggtagcaa gatgactt 18 54 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 54
ctaaatactg gtagcaag 18 55 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 55 ttggcttcta aatactgg 18 56 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 56
tcatagttgg cttctaaa 18 57 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 57 aataatcata gttggctt 18 58 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 58
tcaaaaggac cctggtgg 18 59 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 59 gtgcagagga gggctgtt 18 60 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 60
ccaactgttt ctctttct 18 61 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 61 aagtagttac agcctaat 18 62 19 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 62
ggctggactc ggattcgtt 19 63 22 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 63 ccatcaccaa caatcaccag tt 22 64
22 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 64 cctgagcaat ggctgccatc cg 22 65 19 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 65
gaaggtgaag gtcggagtc 19 66 20 DNA Homo sapiens Description of
Artificial SequenceSynthetic 66 gaagatggtg atgggatttc 20 67 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 67
caagcttccc gttctcagcc 20 68 591 DNA Homo sapiens Description of
Artificial SequenceSynthetic 68 atggcggcca tccgcaagaa gctggtggtg
gtgggcgacg gcgcgtgtgg caagacgtgc 60 ctgctgatcg tgttcagtaa
ggacgagttc cccgaggtgt acgtgcccac cgtcttcgag 120 aactatgtgg
ccgacattga ggtggacggc aagcaggtgg agctggcgct gtgggacacg 180
gcgggccagg aggactacga ccgcctgcgg ccgctctcct acccggacac cgacgtcatt
240 ctcatgtgct tctcggtgga cagcccggac tcgctggaga acatccccga
gaagtgggtc 300 cccgaggtga agcacttctg tcccaatgtg cccatcatcc
tggtggccaa caaaaaagac 360 ctgcgcagcg acgagcatgt ccgcacagag
ctggcccgca tgaagcagga acccgtgcgc 420 acggatgacg gccgcgccat
ggccgtgcgc atccaagcct acgactacct cgagtgctct 480 gccaagacca
aggaaggcgt gcgcgaggtc ttcgagacgg ccacgcgcgc cgcgctgcag 540
aagcgctacg gctcccagaa cggctgcatc aactgctgca aggtgctatg a 591 69 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
69 ccaccaccag cttcttgc 18 70 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 70 ccgtcgccca ccaccacc 18 71 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 71
gcacgtcttg ccacacgc 18 72 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 72 actgaacacg atcagcag 18 73 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 73
ttactgaaca cgatcagc 18 74 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 74 ccttactgaa cacgatca 18 75 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 75
gtccttactg aacacgat 18 76 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 76 ctcgtcctta ctgaacac 18 77 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 77
aactcgtcct tactgaac 18 78 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 78 catagttctc gaagacgg 18 79 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 79
tcggccacat agttctcg 18 80 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 80 ccgtccacct caatgtcg 18 81 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 81
aagcacatga gaatgacg 18 82 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 82 gagtccgggc tgtccacc 18 83 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 83
atgttctcca gcgagtcc 18 84 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 84 gggatgttct ccagcgag 18 85 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 85
gacatgctcg tcgctgcg 18 86 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 86 cggacatgct cgtcgctg 18 87 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 87
tgtgcggaca tgctcgtc 18 88 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 88 ctctgtgcgg acatgctc 18 89 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 89
ccagctctgt gcggacat 18 90 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 90 cgggccagct ctgtgcgg 18 91 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 91
tgcgggccag ctctgtgc 18 92 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 92 gttcctgctt catgcggg 18 93 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 93
acgggttcct gcttcatg 18 94 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 94 gtagtcgtag gcttggat 18 95 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 95
cgaggtagtc gtaggctt 18 96 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 96 gtcttggcag agcactcg 18 97 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 97
acctcgcgca cgccttcc 18 98 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 98 agacctcgcg cacgcctt 18 99 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 99
cgaagacctc gcgcacgc 18 100 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 100 ctcgaagacc tcgcgcac 18 101 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
101 gccgtctcga agacctcg 18 102 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 102 cgtggccgtc tcgaagac
18 103 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 103 gttctgggag ccgtagcg 18 104 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 104 gccgttctgg
gagccgta 18 105 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 105 gatgcagccg ttctggga 18 106 18 DNA
Artificial Sequence Description of
Artificial SequenceSynthetic 106 gttgatgcag ccgttctg 18 107 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 107
cagcagttga tgcagccg 18 108 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 108 agcaccttgc agcagttg 18 109 1058
DNA Homo sapiens 109 gccttgactt catctcagct ccagagcccg ccctctcttc
ctgcagcctg ggaacttcag 60 ccggctggag cccaccatgg ctgcaatccg
aaagaagctg gtgatcgttg gggatggtgc 120 ctgtgggaag acctgcctcc
tcatcgtctt cagcaaggat cagtttccgg aggtctacgt 180 ccctactgtc
tttgagaact atattgcgga cattgaggtg gacggcaagc aggtggagct 240
ggctctgtgg gacgcggaca ttgaggtgga cggcaagcag gtggagctgg ctctgtggga
300 cgacactgat gtcatcctca tgtgcttctc catcgacagc cctgacagcc
tggaaaacat 360 tcctgagaag tggaccccag aggtgaagca cttctgcccc
aacgtgccca tcatcctggt 420 ggggaataag aaggacctga ggcaagacga
gcacaccagg agagagctgg ccaagatgaa 480 gcaggagccc gttcggtctg
aggaaggccg ggacatggcg aaccggatca gtgcctttgg 540 ctaccttgag
tgctcagcca agaccaagga gggagtgcgg gaggtgtttg agatggccac 600
tcgggctggc ctccaggtcc gcaagaacaa gcgtcggagg ggctgtccca ttctctgaga
660 tccccccaaa gggccctttt cctacatgcc ccctcccttc acaggggtac
agaaattatc 720 cccctacaac cccagcctcc tgagggctcc atactgaagg
ctccattttc agttccctcc 780 tgcccaggac tgcattgttt tctagccccg
aggtgtggca cgggccctcc ctcccagcgc 840 tctgggagcc acgcctatgc
cctgcccttc ctcatgggcc cctggggatc ttgccccttt 900 gaccttcccc
aaaggatggt cacacaccag cactttatac acttctggct cacaggaaag 960
tgtctgcagt agggacccag agtcccaggc ccctggagtt gtttctgcag gggccttgtc
1020 tctcactgca tttggtcagg ggggcatgaa taaaggct 1058 110 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 110
gagctgagat gaagtcaa 18 111 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 111 gctgaagttc ccaggctg 18 112 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
112 ccggctgaag ttcccagg 18 113 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 113 ggcaccatcc ccaacgat
18 114 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 114 aggcaccatc cccaacga 18 115 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 115 tcccacaggc
accatccc 18 116 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 116 aggtcttccc acaggcac 18 117 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 117
atgaggaggc aggtcttc 18 118 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 118 ttgctgaaga cgatgagg 18 119 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
119 tcaaagacag tagggacg 18 120 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 120 ttctcaaaga cagtaggg
18 121 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 121 agttctcaaa gacagtag 18 122 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 122 tgttttccag
gctgtcag 18 123 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 123 tcgtcttgcc tcaggtcc 18 124 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 124
gtgtgctcgt cttgcctc 18 125 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 125 ctcctggtgt gctcgtct 18 126 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
126 cagaccgaac gggctcct 18 127 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 127 ttcctcagac cgaacggg
18 128 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 128 actcaaggta gccaaagg 18 129 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 129 ctcccgcact
ccctcctt 18 130 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 130 ctcaaacacc tcccgcac 18 131 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 131
ggccatctca aacacctc 18 132 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 132 cttgttcttg cggacctg 18 133 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
133 cccctccgac gcttgttc 18 134 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 134 gtatggagcc ctcaggag
18 135 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 135 gagccttcag tatggagc 18 136 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 136 gaaaatggag
ccttcagt 18 137 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 137 ggaactgaaa atggagcc 18 138 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 138
ggagggaact gaaaatgg 18 139 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 139 gcaggaggga actgaaaa 18 140 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
140 agggcagggc ataggcgt 18 141 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 141 ggaagggcag ggcatagg
18 142 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 142 catgaggaag ggcagggc 18 143 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 143 taaagtgctg
gtgtgtga 18 144 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 144 cctgtgagcc agaagtgt 18 145 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 145
ttcctgtgag ccagaagt 18 146 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 146 cactttcctg tgagccag 18 147 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
147 agacactttc ctgtgagc 18 148 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 148 actctgggtc cctactgc
18 149 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 149 tgcagaaaca actccagg 18 150 1284 DNA Homo
sapiens 150 gcttctcgag cccggagccg ctgccgccgc ccccagctcc cccgcctcgg
gaggggcacc 60 aggtcactgc agccagaggg gtccagaaga gagaggaggc
actgcctcac tacagcaact 120 gcacccacga tgcagagcat caagtgcgtg
gtggtgggtg atggggctgt gggcaagacg 180 tgcctgctca tctgctacac
aactaacgct ttccccaaag agtacatccc caccgtgttc 240 gacaattaca
gcgcgcagag cgcagttgac gggcgcacag tgaacctgaa cctgtgggac 300
actgcgggcc aggaggagta tgaccgcctc cgtacactct cctaccctca gaccaacgtt
360 ttcgtcatct gtttctccat tgccagtccg ccgtcctatg agaacgtgcg
gcacaagtgg 420 catccagagg tgtgccacca ctgccctgat gtgcccatcc
tgctggtggg caccaagaag 480 gacctgagag cccagcctga caccctacgg
cgcctcaagg agcagagcca ggcgcccatc 540 acaccgcagc agggccaggc
actcgcgaaa cagatccacg ctgtgcgcta cctcgaatgc 600 tcagccctgc
aacaggatgg tgtcaaggaa gtgttcgccg aggctgtccg ggctgtgctc 660
aaccccacgc cgatcaagcg tgggcggtcc tgcatcctct tgtgaccctg gcacttggct
720 tggaggctgc ccctgccctc cccccaccag ttgtgccttg gtgccttgtc
cgcctcagct 780 gtgccttaag gactaattct ggcacccctt tccaggggtt
ccctgaatgc ctttttctct 840 gagtgccttt ttctccttaa ggaggcctgc
agagaaaggg gctttgggct ctgcccctct 900 ggcttgggaa cactgggtat
tctcatgagc tcatccaagc caaggttgga cccctcccca 960 agaggccaac
ccagtgcccc ctcccatttt ccgctactga ccagttcatc cagctttcca 1020
cacagttgtt gctgcctatt gtggtgccgc ctcaggttag gggctctcag ccatctctaa
1080 cctctgccct cgctgctctt ggaattgcgc ccccaagatg ctctctccct
tctccaatga 1140 gggagccaca gaatcctgag aaggtgaatg taccctaacc
tgctcctctg tgcctaggcc 1200 ttacgcattt gctgactgac tcagccccca
tgcttctggg gacctttcct acccccatca 1260 gcatcaataa aacctcctgt ctcc
1284 151 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 151 gacctggtgc ccctcccg 18 152 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 152 tcttctggac
ccctctgg 18 153 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 153 ggcagtgcct cctctctc 18 154 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 154
gtgcagttgc tgtagtga 18 155 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 155 gcatcgtggg tgcagttg 18 156 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
156 ccaccacgca cttgatgc 18 157 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 157 ttgtgtagca gatgagca
18 158 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 158 aaagcgttag ttgtgtag 18 159 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 159 gcgcgctgta
attgtcga 18 160 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 160 ggttcactgt gcgcccgt 18 161 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 161
gtcccacagg ttcaggtt 18 162 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 162 tgtacggagg cggtcata 18 163 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
163 acgttggtct gagggtag 18 164 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 164 caatggagaa acagatga
18 165 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 165 cataggacgg cggactgg 18 166 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 166 cgcacgttct
cataggac 18 167 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 167 acctctggat gccacttg 18 168 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 168
agggcagtgg tggcacac 18 169 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 169 cagcaggatg ggcacatc 18 170 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
170 gggtgtcagg ctgggctc 18 171 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 171 ccctgctgcg gtgtgatg
18 172 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 172 cgcgagtgcc tggccctg 18 173 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 173 gtagcgcaca
gcgtggat 18 174 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 174 cattcgaggt agcgcaca 18 175 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 175
acaccatcct gttgcagg 18 176 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 176 gaacacttcc ttgacacc 18 177 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
177 acagcctcgg cgaacact 18 178 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 178 aagaggatgc aggaccgc
18 179 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 179 gcagcctcca agccaagt 18 180 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 180 aaaaggcatt
cagggaac 18 181 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 181 gggtccaacc ttggcttg 18 182 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 182
gtcagtagcg gaaaatgg 18 183 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 183 agctggatga actggtca 18 184 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
184 aactgtgtgg aaagctgg 18 185 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 185 accacaatag gcagcaac
18 186 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 186 gagggcagag gttagaga 18 187 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 187 caattccaag
agcagcga 18 188 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 188 tggagaaggg agagagca 18 189 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 189
acattcacct tctcagga 18 190 18 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 190 gtcagcaaat gcgtaagg 18 191 579
DNA Artificial Sequence Description of Artificial SequenceSynthetic
191 atgcaggcca tcaagtgtgt ggtggtggga gacggagctg taggtaaaac
ttgcctactg 60 atcagttaca caaccaatgc atttcctgga gaatatatcc
ctactgtctt tgacaattat 120 tctgccaatg ttatggtaga tggaaaaccg
gtgaatctgg gcttatggga tacagctgga 180 caagaagatt atgacagatt
acgcccccta tcctatccgc aaacagatgt gttcttaatt 240 tgcttttccc
ttgtgagtcc tgcatcattt gaaaatgtcc gtgcaaagtg gtatcctgag 300
gtgcggcacc actgtcccaa cactcccatc atcctagtgg gaactaaact tgatcttagg
360 gatgataaag acacgatcga gaaactgaag gagaagaagc tgactcccat
cacctatccg 420 cagggtctag ccatggctaa ggagattggt gctgtaaaat
acctggagtg ctcggcgctc 480 acacagcgag gcctcaagac agtgtttgac
gaagcgatcc gagcagtcct ctgcccgcct 540 cccgtgaaga agaggaagag
aaaatgcctg ctgttgtaa 579 192 45 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 192 atagaatgtg agtctgaact
cttacattta gaacaaacaa aacct 45 193 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 193 caaatgatgc
aggactcaca 20 194 19 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 194 caccaccaca cacttgatg 19 195 18 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 195
ataagcccag attcaccg 18 196 19 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 196 tgtttgcgga taggatagg 19 197 22
DNA Artificial Sequence Description of Artificial SequenceSynthetic
197 gcttcttctc cttcagtttc tc 22 198 19 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 198 cagcaccaat
ctccttagc 19 199 19 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 199 ctcttcctct tcttcacgg
19 200 20 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 200 cctaagatca agtttagttc 20 201 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 201
cgcacctcag gataccactt 20 202 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 202 atctaccata acattggcag 20 203 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
203 taattgtcaa agacagtagg 20 204 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 204 gagcgccgag
cactccaggt 20 205 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 205 gtcaaacact gtcttgaggc 20 206 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
206 atagaatgtg agtctgaact 20 207 25 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 207 cttacattta
gaacaaacaa aacct 25 208 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 208 cccagctaag aattccgctc 20 209 18
DNA Artificial Sequence Description of Artificial SequenceSynthetic
209 taaacgccga atctacgc 18 210 18 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 210 ataagccctg attcaccg
18 211 18 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 211 atacgccctg attcaccg 18 212 18 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 212 atacgccctg
attaaccg 18 213 18 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 213 taaacgccga atctacgc 18 214 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 214
aaactgctga agtacgcaca 20 215 1175 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 215 ccccggtgga
gaagctgagg tcatcatcag atttgaaata tttaaagtgg atacaaaatt 60
atttcagcaa tgcagacaat taagtgtgtt gttgtgggcg atggtgctgt tggtaaaaca
120 tgtctcctga tatcctacac aacaaacaaa tttccatcgg aatatgtacc
gactgttttt 180 gacaactatg cagtcacagt tatgattggt ggagaaccat
atactcttgg actttttgat 240 actgcagggc aagaggatta tgacagatta
cgaccgctga gttatccaca aacagatgta 300 tttctagtct gtttttcagt
ggtctctcca tcttcatttg aaaacgtgaa agaaaagtgg 360 gtgcctgaga
taactcacca ctgtccaaag actcctttct tgcttgttgg gactcaaatt 420
gatctcagag atgacccctc tactattgag aaacttgcca agaacaaaca gaagcctatc
480 actccagaga ctgctgaaaa gctggcccgt gacctgaagg ctgtcaagta
tgtggagtgt 540 tctgcactta cacagaaagg cctaaagaat gtatttgacg
aagcaatatt ggctgccctg 600 gagcctccag aaccgaagaa gagccgcagg
tgtgtgctgc tatgaacatc tctccagagc 660 cctttctgca cagctggtgt
cggcatcata ctaaaagcaa tgtttaaatc aaactaaaga 720 ttaaaaatta
aaattcgttt ttgcaataat gacaaatgcc ctgcacctac ccacatgcac 780
tcgtgtgaga caaggcccat aggtatggcc ccccccttcc ccctcccagt actagttaat
840 tttgagtaat tgtattgtca gaaaagtgat tagtactatt tttttttgtt
gtttcaaaaa 900 aaaaattttt gtgtgtctgt tttttttttt tttttttttt
gttgtttaaa aggaaggcat 960 gcttgtggat gactctgtaa cagactaatt
ggaattgttg aagctgctcc ctggttccac 1020 tctggagagt aatctgggac
atcttagtgt tttgttttgt ttttttccct cctctttttt 1080 ttggggggga
gtgtgtgggg ggtttgtttt ttagtcttgt ttttttaatt cattaaccag 1140
tggttaagcc cttaagggag gaggacggat tgatt 1175 216 25 DNA Artificial
Sequence Description of Artificial SequenceSynthetic 216 ttcagcaatg
cagacaatta agtgt 25 217 27 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 217 tgttgtgtag gatatcagga gacatgt 27
218 24 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 218 ttgtgggcga tggtgctgtt ggta 24 219 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 219
taattgtctg cattgctgaa 20 220 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 220 ttaccaacag caccatcgcc 20 221 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
221 ccaccaatca taactgtgac 20 222 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 222 gtggataact
cagcggtcgt 20 223 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 223 gaagatggag agaccactga 20 224 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
224 gtgagttatc tcaggcaccc 20 225 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 225 gcttctgttt
gttcttggca 20 226 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 226 tgacagcctt caggtcacgg 20 227 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
227 cacctgcggc tcttcttcgg 20 228 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 228 ttgtctcaca
cgagtgcatg 20 229 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 229 ttctgacaat acaattactc 20 230 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
230 ttacagagtc atccacaagc 20 231 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 231 cgatagtctc
cacgtgaggc 20 232 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 232 gtaacgctcc tatggccagg 20 233 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
233 agactgactg ctcgtcgcga 20
* * * * *