U.S. patent application number 10/476962 was filed with the patent office on 2004-09-30 for antisense modulation of src-c expression.
Invention is credited to Bennett, C Frank, Watt, Andrew T.
Application Number | 20040191904 10/476962 |
Document ID | / |
Family ID | 25333295 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040191904 |
Kind Code |
A1 |
Bennett, C Frank ; et
al. |
September 30, 2004 |
Antisense modulation of src-c expression
Abstract
Antisense compounds, compositions and methods are provided for
modulating the expression of src-c. The compositions comprise
antisense compounds, particularly antisense oligonucleotides,
targeted to nucleic acids encoding src-c. Methods of using these
compounds for modulation of src-c expression and for treatment of
diseases associated with expression of src-c are provided.
Inventors: |
Bennett, C Frank; (Carlsbad,
CA) ; Watt, Andrew T; (Vista, CA) |
Correspondence
Address: |
LICATA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Family ID: |
25333295 |
Appl. No.: |
10/476962 |
Filed: |
November 5, 2003 |
PCT Filed: |
May 16, 2002 |
PCT NO: |
PCT/US02/15684 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10476962 |
Nov 5, 2003 |
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09860473 |
May 18, 2001 |
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6656732 |
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Current U.S.
Class: |
435/375 ;
514/44A; 536/23.1 |
Current CPC
Class: |
C12N 15/1137 20130101;
C12N 2310/321 20130101; C12N 2310/3341 20130101; C12N 2310/346
20130101; C12N 2310/315 20130101; Y02P 20/582 20151101; A61K 38/00
20130101; C12N 2310/341 20130101; C12N 2310/321 20130101; C12N
2310/3525 20130101 |
Class at
Publication: |
435/375 ;
514/044; 536/023.1 |
International
Class: |
A61K 048/00; C07H
021/04; C12N 005/00 |
Claims
What is claimed is:
1. A compound 8 to 50 nucleobases in length targeted to a nucleic
acid molecule encoding a 5'UTR, 3'UTR, coding region, intron
region, exon region, stop codon, intron:exon junction, exon:exon
junction, or 5' mRNA variant of src-c, wherein said compound
specifically hybridizes with and inhibits the expression of
src-c.
2. The compound of claim 1 which is an antisense
oligonucleotide.
3. The compound of claim 2 wherein the antisense oligonucleotide
has a sequence comprising SEQ ID NO: 29, 30, 35, 44, 45, 46, 48,
53, 54, 56, 57, 58, 60, 61, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73,
76, 77, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95,
96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 52, 68, 78, 108, 110,
113, 118, 120, 121, 124, 125, 128, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 143, 145, 146, 147, 148, 151, 155, 157, 158,
163, 164, 167, 168 or 169.
4. The compound of claim 2 wherein the antisense oligonucleotide
comprises at least one modified internucleoside linkage.
5. The compound of claim 4 wherein the modified internucleoside
linkage is a phosphorothioate linkage.
6. The compound of claim 2 wherein the antisense oligonucleotide
comprises at least one modified sugar moiety.
7. The compound of claim 6 wherein the modified sugar moiety is a
2'-O-methoxyethyl sugar moiety.
8. The compound of claim 2 wherein the antisense oligonucleotide
comprises at least one modified nucleobase.
9. The compound of claim 8 wherein the modified nucleobase is a
5-methylcytosine.
10. The compound of claim 2 wherein the antisense oligonucleotide
is a chimeric oligonucleotide.
11. A compound 8 to 50 nucleobases in length which specifically
hybridizes with at least an 8-nucleobase portion of an active site
on a nucleic acid molecule encoding src-c.
12. A composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier or diluent.
13. The composition of claim 12 further comprising a colloidal
dispersion system.
14. The composition of claim 12 wherein the compound is an
antisense oligonucleotide.
15. A method of inhibiting the expression of src-c in cells or
tissues comprising contacting said cells or tissues with the
compound of claim 1 so that expression of src-c is inhibited.
16. A method of treating an animal having a disease or condition
associated with src-c comprising administering to said animal a
therapeutically or prophylactically effective amount of the
compound of claim 1 so that expression of src-c is inhibited.
17. The method of claim 16 wherein the disease or condition is a
hyperproliferative disorder.
18. The method of claim 17 wherein the hyperpriliferative disorder
is cancer.
19. The method of claim 18 wherein the cancer is breast, colon,
pancreatic, lung, ovarian, esophageal, neuroblastoma,
retinoblastoma or Kaposi's sarcoma.
20. The method of claim 16 wherein the disease or condition is
aberrant bone remodeling.
Description
FIELD OF THE INVENTION
[0001] The present invention provides compositions and methods for
modulating the expression of src-c. In particular, this invention
relates to compounds, particularly oligonucleotides, specifically
hybridizable with nucleic acids encoding src-c. Such compounds have
been shown to modulate the expression of src-c.
BACKGROUND OF THE INVENTION
[0002] Cells in higher animals normally divide only when they are
stimulated by growth factors produced by other cells and act by
binding to receptor tyrosine kinases on dividing cells. Cancer
cells proliferate excessively because, as a result of accumulated
mutations, they are able to divide without stimulation from other
cells and therefore are no longer subject to normal controls on
cell proliferation. The mutated genes which led to excessive
proliferation were originally called oncogenes before their origin
as normal genes was understood. The normal genes, from which they
arise, are thus often referred to as proto-oncogenes.
[0003] The v-src gene encoded by the Rous sarcoma virus was the
first discovered as a transmissible agent found to induce tumors in
chickens (Irby and Yeatman, Oncogene, 2000, 19, 5636-5642). The
protein product of this gene, v-src, is a tyrosine kinase with a
cellular homolog known as src-c (also known as c-src, SRC and
pp60c-src) (Irby and Yeatman, Oncogene, 2000, 19, 5636-5642). The
structure of the two proteins is similar but the regulatory
carboxyl-terminus of v-src is truncated (Irby and Yeatman,
Oncogene, 2000, 19, 5636-5642). Found in normal cells and presumed
to be a proto-oncogene, src-c is a tyrosine kinase which regulates
cell growth via phosphorylation of transcription factors, members
of signal transduction cascades and growth factor receptors (Irby
and Yeatman, Oncogene, 2000, 19, 5636-5642).
[0004] While elevation of src-c protein levels is common to a large
number of cancers, this elevation is often modest when compared to
the increases in src-c kinase activity that have been observed
(Irby and Yeatman, Oncogene, 2000, 19, 5636-5642). These data
indicate the importance of src-c activation in human tumor
development and progression.
[0005] Several mechanisms of activation of src-c in cancer have
been proposed including: (i) activation of src-c by receptor
tyrosine kinases such as epidermal growth factor receptor (EGFR)
and human growth factor (HGF) (Mao et al., Oncogene, 1997, 15,
3083-3090), (ii) post translational activation (for example, via
insufficient phosphorylation of src-c by protein tyrosine kinase
Csk) (Lutz et al., Biochem. Biophys. Res. Conmmun., 1998, 243,
503-508), (iii) activation via mutations (Irby et al., Nat. Genet.,
1999, 21, 187-190) and (iv) loss of function of src-c regulatory
proteins (Irby and Yeatman, Oncogene, 2000, 19, 5636-5642).
[0006] Activated src-c has been implicated in the progression and
metastasis of many human cancers. Increased src-c activity has been
shown to increase the growth rate of cells (Mao et al., Oncogene,
1997, 15, 3083-3090), reduce the adhesive contacts between cells
(Hamaguchi et al., Embo J., 1993, 12, 307-314) and increase the
metastatic potential of cells (Irby et al., Nat. Genet., 1999, 21,
187-190).
[0007] In human mammary carcinomas, src-c kinase activity has been
detected at levels 4-20 times higher than that found in normal
tissue (Irby and Yeatman, Oncogene, 2000, 19, 5636-5642). Likewise,
elevation of src-c kinase activity is increased 5-8 fold in the
majority of colon tumors (Irby and Yeatman, Oncogene, 2000, 19,
5636-5642). Activated src-c was found to be instrumental in
promoting the growth of pancreatic tumor cells by increasing the
number of insulin-like growth factor receptor molecules and thus
enhancing insulin growth factor-dependent growth (Flossmann-Kast et
al., Cancer Res., 1998, 58, 3551-3554). Additional cancers with
elevated src-c protein levels have been reported in neuroblastomas,
retinoblastomas, Kaposi's sarcoma, as well as lung, ovarian, and
esophageal cancers (Irby and Yeatman, Oncogene, 2000, 19,
5636-5642).
[0008] Due to the prevalence of activated src-c in cancer, src-c
has been the subject of investigations aimed at inhibition of src-c
expression and/or activity.
[0009] Investigations of src-c null mice indicate that src-c is not
required for general cell viability but does have an essential role
in osteoclast function and bone remodeling. It is thus possible
that other tyrosine kinases related to src-c may provide overlap in
function and reduce the severity of the phenotype (Soriano et al.,
Cell, 1991, 64, 693-702).
[0010] Examples of inhibition of human src-c expression by vectors
containing antisense src-c fragments of src-c have been described
in a mouse models (Karni et al., Oncogene, 1999, 18, 4654-4662;
Wiener et al., Clin. Cancer Res., 1999, 5, 2164-2170), colon cancer
cell lines (Ellis et al., J. Biol. Chem., 1998, 273, 1052-1057;
Fleming et al., Surgery, 1997, 122, 501-507; Rajala et al.,
Biochem. Biophys. Res. Commun., 2000, 273, 1116-1120; Staley et
al., Cell Growth Differ., 1997, 8, 269-274) and leukemia cell lines
(Kitanaka et al., Biochem. Biophys. Res. Commun., 1994, 201,
1534-1540; Waki et al., Biochem. Biophys. Res. Commun., 1994, 201,
1001-1007; Yamaguchi et al., Leukemia, 1997, 11, 497-503).
[0011] Inhibition of expression of src-c by antisense
phosphorothioate oligonucleotides targeting the start codon of
human and mouse src-c has been observed in osteoclasts, osteoblasts
and vascular endothelial cells (Chellaiah et al., J. Biol. Chem.,
1998, 273, 11908-11916.; Marzia et al., J. Cell Biol., 2000, 151,
311-320; Naruse et al., FEBS Lett., 1998, 441, 111-115; Tanaka et
al., Nature, 1996, 383, 528-531).
[0012] A 60-mer oligonucleotide targeting the 18-nucleotide
brain-specific insert of rat src-c was used to map the expression
levels of brain-specific src-c in various brain structures (Ross et
al., Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 9831-9835).
[0013] An expression construct comprising a tumor suppressor gene
and an antisense src-c gene directed to the use of genetic therapy
is claimed in PCT publication WO 99/47690 (Almond et al.,
1999).
[0014] An antisense molecule inhibiting the expression of src-c in
combination with a lipid formulation containing other compounds
used for treatment of hyperproliferative disease in humans is
claimed in PCT WO/71096 (Ramesh et al., 2000).
[0015] A therapeutic composition including an antisense
oligonucleotide specific for src-c and at least one second
antisense oligonucleotide specific for a nuclear oncogene is
claimed in U.S. Pat. No. 5,734,039 (Calabretta and Skorski,
1998).
[0016] Antisense oligonucleotides corresponding to src-c and in
combination with at least one other antisense oligonucleotide
corresponding to a different gene are claimed in PCT publication WO
99/13886 (Nyce, 1999).
[0017] A therapeutic agent composed of a nucleic acid construct
containing antisense RNA for disrupting expression of src-c is
claimed in PCT publication WO 01/00791 (Lee, 2001).
[0018] Methods for producing recombinant viral vectors containing
antisense constructs of src-c are claimed in PCT publication WO
99/27123 and WO 00/32754 (Fang et al., 1999; Zhang et al.,
2000).
[0019] Disclosed and claimed in U.S. Pat. Nos. 5,264,423 and
5,276,019 are oligodeoxynucleotide compounds capable of inhibiting
the replication or apoptotic effect of a foreign nucleic acid in a
host. In U.S. Pat. No. 5,264,423, a foreign nucleic acid includes
an oncogene nucleic acid (Cohen et al., 1993; Cohen et al.,
1994).
[0020] Antisense technology is emerging as an effective means of
reducing the expression of specific gene products and may therefore
prove to be uniquely useful in a number of therapeutic, diagnostic
and research applications involving modulation of src-c
expression.
[0021] The present invention provides compositions and methods for
modulating src-c expression including modulation of the currently
disclosed 5' UTR variant of src-c.
SUMMARY OF THE INVENTION
[0022] The present invention is directed to compounds, particularly
antisense oligonucleotides, which are targeted to a nucleic acid
encoding src-c, and which modulate the expression of src-c.
Pharmaceutical and other compositions comprising the compounds of
the invention are also provided. Further provided are methods of
modulating the expression of src-c in cells or tissues comprising
contacting said cells or tissues with one or more of the antisense
compounds or compositions of the invention. Further provided are
methods of treating an animal, particularly a human, suspected of
having or being prone to a disease or condition associated with
expression of src-c by administering a therapeutically or
prophylactically effective amount of one or more of the antisense
compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention employs oligomeric compounds,
particularly antisense oligonucleotides, for use in modulating the
function of nucleic acid molecules encoding src-c, ultimately
modulating the amount of src-c produced. This is accomplished by
providing antisense compounds which specifically hybridize with one
or more nucleic acids encoding src-c. As used herein, the terms
"target nucleic acid" and "nucleic acid encoding src-c" encompass
DNA encoding src-c, RNA (including pre-mRNA and mRNA) transcribed
from such DNA, and also cDNA derived from such RNA. The specific
hybridization of an oligomeric compound with its target nucleic
acid interferes with the normal function of the nucleic acid. This
modulation of function of a target nucleic acid by compounds which
specifically hybridize to it is generally referred to as
"antisense". The functions of DNA to be interfered with include
replication and transcription. The functions of RNA 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 or facilitated by the RNA. The overall effect of such
interference with target nucleic acid function is modulation of the
expression of src-c. In the context of the present invention,
"modulation" means either an increase (stimulation) or a decrease
(inhibition) in the expression of a gene. In the context of the
present invention, inhibition is the preferred form of modulation
of gene expression and mRNA is a preferred target.
[0024] It is preferred to target specific nucleic acids for
antisense. "Targeting" an antisense compound to a particular
nucleic acid, in the context of this invention, is a multistep
process. The process usually begins with the identification of a
nucleic acid sequence whose function is to be modulated. This may
be, for example, a cellular gene (or mRNA transcribed from the
gene) whose expression is associated with a particular disorder or
disease state, or a nucleic acid molecule from an infectious agent.
In the present invention, the target is a nucleic acid molecule
encoding src-c. The targeting process also includes determination
of a site or sites within this gene for the antisense interaction
to occur such that the desired effect, e.g., detection or
modulation of expression of the protein, will result. Within the
context of the present invention, a preferred intragenic site is
the region encompassing the translation initiation or termination
codon of the open reading frame (ORF) of the gene. 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 (in 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
src-c, regardless of the sequence(s) of such codons.
[0025] 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" 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. 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.
[0026] 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 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.
[0027] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a transcript before it is translated. 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., 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. 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.
[0028] Once one or more target 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 effect.
[0029] In the context of this invention, "hybridization" means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases. For example, adenine and thymine are
complementary nucleobases which pair through the formation of
hydrogen bonds. "Complementary," as used herein, refers to the
capacity for precise pairing between two nucleotides. For example,
if a nucleotide at a certain position of an oligonucleotide is
capable of hydrogen bonding with a nucleotide at the same position
of a DNA or RNA molecule, then the oligonucleotide and the DNA or
RNA are considered to be complementary to each other at that
position. The oligonucleotide and the DNA or RNA are complementary
to each other when a sufficient number of corresponding positions
in each molecule are occupied by nucleotides which can hydrogen
bond with each other. Thus, "specifically hybridizable" and
"complementary" are terms which are used to indicate a sufficient
degree of complementarity or precise pairing such that stable and
specific binding occurs between the oligonucleotide and the DNA or
RNA target. It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. An antisense
compound is specifically hybridizable when binding of the compound
to the target DNA or RNA molecule interferes with the normal
function of the target DNA or RNA to cause a loss of utility, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound 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, and in the case of in vitro assays, under
conditions in which the assays are performed.
[0030] Antisense and other compounds of the invention which
hybridize to the target and inhibit expression of the target are
identified through experimentation, and the sequences of these
compounds are hereinbelow identified as preferred embodiments of
the invention. The target sites to which these preferred sequences
are complementary are hereinbelow referred to as "active sites" and
are therefore preferred sites for targeting. Therefore another
embodiment of the invention encompasses compounds which hybridize
to these active sites.
[0031] Antisense compounds are commonly used as research reagents
and diagnostics. For example, antisense oligonucleotides, which are
able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes. Antisense compounds are also used, for example,
to distinguish between functions of various members of a biological
pathway. Antisense modulation has, therefore, been harnessed for
research use.
[0032] For use in kits and diagnostics, the antisense compounds of
the present invention, either alone or in combination with other
antisense compounds or therapeutics, can be used as tools in
differential and/or combinatorial analyses to elucidate expression
patterns of a portion or the entire complement of genes expressed
within cells and tissues.
[0033] Expression patterns within cells or tissues treated with one
or more antisense compounds are compared to control cells or
tissues not treated with antisense compounds and the patterns
produced are analyzed for differential levels of gene expression as
they pertain, for example, to disease association, signaling
pathway, cellular localization, expression level, size, structure
or function of the genes examined. These analyses can be performed
on stimulated or unstimulated cells and in the presence or absence
of other compounds which affect expression patterns.
[0034] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression)(Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein
arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (reviewed in (To, Comb.
Chem. High Throughput Screen, 2000, 3, 235-41).
[0035] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense oligonucleotides have been employed as therapeutic
moieties in the treatment of disease states in animals and man.
Antisense oligonucleotide drugs, including ribozymes, have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that
oligonucleotides can be useful therapeutic modalities that can be
configured to be useful in treatment regimes for treatment of
cells, tissues and animals, especially humans.
[0036] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) or mimetics thereof. This term includes
oligonucleotides composed of naturally-occurring nucleobases,
sugars and covalent internucleoside (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
affinity for nucleic acid target and increased stability in the
presence of nucleases.
[0037] While antisense oligonucleotides are a preferred form of
antisense compound, the present invention comprehends other
oligomeric antisense compounds, including but not limited to
oligonucleotide mimetics such as are described below. The antisense
compounds in accordance with this invention preferably comprise
from about 8 to about 50 nucleobases (i.e. from about 8 to about 50
linked nucleosides). Particularly preferred antisense compounds are
antisense oligonucleotides, even more preferably those comprising
from about 12 to about 30 nucleobases. Antisense compounds include
ribozymes, external guide sequence (EGS) oligonucleotides
(oligozymes), and other short catalytic RNAs or catalytic
oligonucleotides which hybridize to the target nucleic acid and
modulate its expression.
[0038] 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.
[0039] 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.
[0040] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriest- ers,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Preferred oligonucleotides having inverted
polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be a basic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0041] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos. 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; 5,194,599; 5,565,555;
5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are
commonly owned with this application, and each of which is herein
incorporated by reference.
[0042] 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; riboacetyl 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.
[0043] Representative United States 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; 5,792,608;
5,646,269 and 5,677,439, certain of which are commonly owned with
this application, and each of which is herein incorporated by
reference.
[0044] 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 United States 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, each of
which is herein incorporated by reference. Further teaching of PNA
compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0045] 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.
[0046] 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--, S--, or N-alkenyl; O--, S--or N-alkynyl; or O-alkyl-O-- alkyl,
wherein the alkyl., alkenyl and alkynyl may be substituted or
unsubstituted C, 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.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.nCH.su- b.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, alkenyl, alkynyl, 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 examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples hereinbelow.
[0047] A further preferred modification includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. The linkage is preferably a methelyne (--CH.sub.2--).sub.n
group bridging the 2' oxygen atom and the 4' carbon atom wherein n
is 1 or 2. LNAs and preparation thereof are described in WO
98/39352 and WO 99/14226.
[0048] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH=CH.sub.2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 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 United States patents that
teach the preparation of such modified sugar 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;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0049] 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), 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
(--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 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, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further modified nucleobases include tricyclic
pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b]
[1,4]benzoxazin-2(3H)-one), phenothiazine cytidine
(1H-pyrimido[5,4-b] [1,4]benzothiazin-2(3H)-one), G-clamps such as
a substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b] [1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2- -one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2- -one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. 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, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0050] Representative United States 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; 5,645,985; 5,830,653;
5,763,588; 6,005,096; and 5,681,941, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference, and U.S. Pat. No. 5,750,692, which is
commonly owned with the instant application and also herein
incorporated by reference.
[0051] 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. The
compounds of the invention can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugates groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve oligomer
uptake, enhance oligomer resistance to degradation, and/or
strengthen sequence-specific hybridization with RNA. Groups that
enhance the pharmacokinetic properties, in the context of this
invention, include groups that improve oligomer uptake,
distribution, metabolism or excretion. Representative conjugate
groups are disclosed in International Patent Application
PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which
is incorporated herein by reference. Conjugate 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. Let.,
1994, 4, 1053-1060), 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 triethyl-ammonium 1,2-di-O-hexadecyl-rac-gly-
cero-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. Oligonucleotides of the
invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) which is incorporated herein by reference in its
entirety.
[0052] Representative United States 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, certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference.
[0053] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. 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
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0054] Chimeric antisense compounds of the invention may be formed
as composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative United States patents
that teach the preparation of such hybrid structures include, but
are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference in its entirety.
[0055] The antisense compounds 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, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0056] The antisense compounds of the invention are synthesized in
vitro and do not include antisense compositions of biological
origin, or genetic vector constructs designed to direct the in vivo
synthesis of antisense molecules. The compounds of the invention
may also be admixed, encapsulated, conjugated or otherwise
associated with other molecules, molecule structures or mixtures of
compounds, as for example, liposomes, receptor targeted molecules,
oral, rectal, topical or other formulations, for assisting in
uptake, distribution and/or absorption. Representative United
States patents that teach the preparation of such uptake,
distribution and/or absorption assisting formulations include, but
are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
[0057] The antisense compounds of the invention 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 prodrugs and
pharmaceutically acceptable salts of the compounds of the
invention, pharmaceutically acceptable salts of such prodrugs, and
other bioequivalents.
[0058] The term "prodrug" 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 December 9, 1993 or in WO 94/26764 and
U.S. Pat. No. 5,770,713 to Imbach et al.
[0059] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds of the invention: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto.
[0060] Pharmaceutically acceptable base addition salts are formed
with metals or amines, such as alkali and alkaline earth metals or
organic amines. Examples of metals used as cations are sodium,
potassium, magnesium, calcium, and the like. Examples of suitable
amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, dicyclohexylamine, ethylenediamine,
N-methylglucamine, and procaine (see, for example, Berge et al.,
"Pharmaceutical Salts," J. of Pharma Sci., 1977, 66, 1-19). The
base addition salts of said acidic compounds are prepared by
contacting the free acid form with a sufficient amount of the
desired base to produce the salt in the conventional manner. The
free acid form may be regenerated by contacting the salt form with
an acid and isolating the free acid in the conventional manner. The
free acid forms differ from their respective salt forms somewhat in
certain physical properties such as solubility in polar solvents,
but otherwise the salts are equivalent to their respective free
acid for purposes of the present invention. As used herein, a
"pharmaceutical addition salt" includes a pharmaceutically
acceptable salt of an acid form of one of the components of the
compositions of the invention. These include organic or inorganic
acid salts of the amines. Preferred acid salts are the
hydrochlorides, acetates, salicylates, nitrates and phosphates.
Other suitable pharmaceutically acceptable salts are well known to
those skilled in the art and include basic salts of a variety of
inorganic and organic acids, such as, for example, with inorganic
acids, such as for example hydrochloric acid, hydrobromic acid,
sulfuric acid or phosphoric acid; with organic carboxylic,
sulfonic, sulfo or phospho acids or N-substituted sulfamic acids,
for example acetic acid, propionic acid, glycolic acid, succinic
acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric
acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic
acid, glucaric acid, glucuronic acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic
acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid,
nicotinic acid or isonicotinic acid; and with amino acids, such as
the 20 alpha-amino acids involved in the synthesis of proteins in
nature, for example glutamic acid or aspartic acid, and also with
phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,
2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,
benzenesulfonic acid, 4-methylbenzenesulfonic acid,
naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or
3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid
(with the formation of cyclamates), or with other acid organic
compounds, such as ascorbic acid. Pharmaceutically acceptable salts
of compounds may also be prepared with a pharmaceutically
acceptable cation. Suitable pharmaceutically acceptable cations are
well known to those skilled in the art and include alkaline,
alkaline earth, ammonium and quaternary ammonium cations.
Carbonates or hydrogen carbonates are also possible.
[0061] For oligonucleotides, preferred 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.
[0062] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. For therapeutics, an animal, preferably a human,
suspected of having a disease or disorder which can be treated by
modulating the expression of src-c is treated by administering
antisense compounds in accordance with this invention. The
compounds of the invention can be utilized in pharmaceutical
compositions by adding an effective amount of an antisense compound
to a suitable pharmaceutically acceptable diluent or carrier. Use
of the antisense compounds and methods of the invention may also be
useful prophylactically, e.g., to prevent or delay infection,
inflammation or tumor formation, for example.
[0063] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding src-c, enabling sandwich and other assays to
easily be constructed to exploit this fact. Hybridization of the
antisense oligonucleotides of the invention with a nucleic acid
encoding src-c can be detected by means known in the art. Such
means may include conjugation of an enzyme to the oligonucleotide,
radiolabelling of the oligonucleotide or any other suitable
detection means. Kits using such detection means for detecting the
level of src-c in a sample may also be prepared.
[0064] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention. 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 and
to mucous membranes including vaginal and rectal delivery),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; 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] Pharmaceutical compositions and 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. Preferred
topical formulations include those in which the oligonucleotides of
the invention are in admixture with a topical delivery agent such
as lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Preferred lipids and liposomes
include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl
choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Preferred fatty acids and esters
include but are not limited arachidonic acid, oleic acid,
eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic
acid, palmitic acid, stearic acid, linoleic acid, linolenic acid,
dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-10 alkyl ester (e.g. isopropylmyristate IPM),
monoglyceride, diglyceride or pharmaceutically acceptable salt
thereof. Topical formulations are described in detail in U.S.
patent application Ser. No. 09/315,298 filed on May 20, 1999 which
is incorporated herein by reference in its entirety.
[0066] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Preferred surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Prefered bile acids/salts
include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic
acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid,
glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic
acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusid- ate,
sodium glycodihydrofusidate,. Prefered fatty acids include
arachidonic acid, undecanoic acid, oleic acid, lauric acid,
caprylic acid, capric acid, myristic acid, palmitic acid, stearic
acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a monoglyceride, a diglyceride or a pharmaceutically acceptable
salt thereof (e.g. sodium). Also prefered are combinations of
penetration enhancers, for example, fatty acids/salts in
combination with bile acids/salts. A particularly prefered
combination is the sodium salt of lauric acid, capric acid and
UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the invention may be delivered orally in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. Oligonucleotide complexing agents
include poly-amino acids; polyimines; polyacrylates;
polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates;
cationized gelatins, albumins, starches, acrylates,
polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates;
DEAE-derivatized polyimines, pollulans, celluloses and starches.
Particularly preferred complexing agents include chitosan,
N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,
polyspermines, protamine, polyvinylpyridine,
polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.
p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),
poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),
poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,
DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG). Oral formulations for oligonucleotides
and their preparation are described in detail in U.S. applications
Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul.
1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May
21, 1998) and 09/315,298 (filed May 20, 1999) each of which is
incorporated herein by reference in their entirety.
[0067] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0068] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0069] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0070] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0071] In one embodiment of the present invention the
pharmaceutical compositions may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product. The
preparation of such compositions and formulations is generally
known to those skilled in the pharmaceutical and formulation arts
and may be applied to the formulation of the compositions of the
present invention.
[0072] Emulsions
[0073] The compositions of the present invention may be prepared
and formulated as emulsions. Emulsions are typically heterogenous
systems of one liquid dispersed in another in the form of droplets
usually exceeding 0.1 .mu.m in diameter. (Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p.
335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often
biphasic systems comprising of two immiscible liquid phases
intimately mixed and dispersed with each other. In general,
emulsions may be either water-in-oil (w/o) or of the oil-in-water
(o/w) variety. When an aqueous phase is finely divided into and
dispersed as minute droplets into a bulk oily phase the resulting
composition is called a water-in-oil (w/o) emulsion. Alternatively,
when an oily phase is finely divided into and dispersed as minute
droplets into a bulk aqueous phase the resulting composition is
called an oil-in-water (o/w) emulsion. Emulsions may contain
additional components in addition to the dispersed phases and the
active drug which may be present as a solution in either the
aqueous phase, oily phase or itself as a separate phase.
Pharmaceutical excipients such as emulsifiers, stabilizers, dyes,
and anti-oxidants may also be present in emulsions as needed.
Pharmaceutical emulsions may also be multiple emulsions that are
comprised of more than two phases such as, for example, in the case
of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w)
emulsions. Such complex formulations often provide certain
advantages that simple binary emulsions do not. Multiple emulsions
in which individual oil droplets of an o/w emulsion enclose small
water droplets constitute a w/o/w emulsion. Likewise a system of
oil droplets enclosed in globules of water stabilized in an oily
continuous provides an o/w/o emulsion.
[0074] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0075] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants may be classified into different classes
based on the nature of the hydrophilic group: nonionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0076] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0077] A large variety of non-emulsifying materials are also
included in emulsion formulations and contribute to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0078] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0079] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0080] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for
oral delivery have been very widely used because of reasons of ease
of formulation, efficacy from an absorption and bioavailability
standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 199). Mineral-oil base laxatives,
oil-soluble vitamins and high fat nutritive preparations are among
the materials that have commonly been administered orally as o/w
emulsions.
[0081] In one embodiment of the present invention, the compositions
of oligonucleotides and nucleic acids are formulated as
microemulsions. A microemulsion may be defined as a system of
water, oil and amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically
microemulsions are systems that are prepared by first dispersing an
oil in an aqueous surfactant solution and then adding a sufficient
amount of a fourth component, generally an intermediate
chain-length alcohol to form a transparent system. Therefore,
microemulsions have also been described as thermodynamically
stable, isotropically clear dispersions of two immiscible liquids
that are stabilized by interfacial films of surface-active
molecules (Leung and Shah, in: Controlled Release of Drugs:
Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0082] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0083] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (S0750),
decaglycerol decaoleate (DA0750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized C8-C10
glycerides, vegetable oils and silicone oil.
[0084] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or oligonucleotides. Microemulsions
have also been effective in the transdermal delivery of active
components in both cosmetic and pharmaceutical applications. It is
expected that the microemulsion compositions and formulations of
the present invention will facilitate the increased systemic
absorption of oligonucleotides and nucleic acids from the
gastrointestinal tract, as well as improve the local cellular
uptake of oligonucleotides and nucleic acids within the
gastrointestinal tract, vagina, buccal cavity and other areas of
administration.
[0085] Microemulsions of the present invention may also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
oligonucleotides and nucleic acids of the present invention.
Penetration enhancers used in the microemulsions of the present
invention may be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
[0086] Liposomes
[0087] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0088] Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous
interior. The aqueous portion contains the composition to be
delivered. Cationic liposomes possess the advantage of being able
to fuse to the cell wall. Non-cationic liposomes, although not able
to fuse as efficiently with the cell wall, are taken up by
macrophages in vivo.
[0089] In order to cross intact mammalian skin, lipid vesicles must
pass through a series of fine pores, each with a diameter less than
50 nm, under the influence of a suitable transdermal gradient.
Therefore, it is desirable to use a liposome which is highly
deformable and able to pass through such fine pores.
[0090] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0091] Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes. As the merging of the liposome and cell progresses, the
liposomal contents are emptied into the cell where the active agent
may act.
[0092] Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
[0093] Several reports have detailed the ability of liposomes to
deliver agents including high-molecular weight DNA into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin. The
majority of applications resulted in the targeting of the upper
epidermis.
[0094] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et al., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
[0095] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0096] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0097] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g. as a solution or as an emulsion) were ineffective (Weiner et
al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0098] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome.TM. I
(glyceryl dilaurate/cholesterol/po- lyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
[0099] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.M1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
[0100] Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside GM1,
galactocerebroside sulfate and phosphatidylinositol to improve
blood half-lives of liposomes. These findings were expounded upon
by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949).
U.S. Pat. Nos. 4,837,028 and WO 88/04924, both to Allen et al.,
disclose liposomes comprising (1) sphingomyelin and (2) the
ganglioside G.sub.M1 or a galactocerebroside sulfate ester. U.S.
Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising
sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-
idylcholine are disclosed in WO 97/13499 (Lim et al.).
[0101] Many liposomes comprising lipids derivatized with one or
more hydrophilic polymers, and methods of preparation thereof, are
known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53,
2778) described liposomes comprising a nonionic detergent,
2C.sub.1215G, that contains a PEG moiety. Illum et al. (FEBS Lett.,
1984, 167, 79) noted that hydrophilic coating of polystyrene
particles with polymeric glycols results in significantly enhanced
blood half-lives. Synthetic phospholipids modified by the
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)
are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899).
Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments
demonstrating that liposomes comprising phosphatidylethanolamine
(PE) derivatized with PEG or PEG stearate have significant
increases in blood circulation half-lives. Blume et al. (Biochimica
et Biophysica Acta, 1990, 1029, 91) extended such observations to
other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from
the combination of distearoylphosphatidylethanolamine (DSPE) and
PEG. Liposomes having covalently bound PEG moieties on their
external surface are described in European Patent No. EP 0 445 131
B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20
mole percent of PE derivatized with PEG, and methods of use
thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556
and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 B1). Liposomes comprising a number
of other lipid-polymer conjugates are disclosed in WO 91/05545 and
U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073
(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids
are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos.
5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe
PEG-containing liposomes that can be further derivatized with
functional moieties on their surfaces.
[0102] A limited number of liposomes comprising nucleic acids are
known in the art. WO 96/40062 to Thierry et al. discloses methods
for encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al.
describes certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising antisense oligonucleotides targeted to the raf gene.
[0103] Transfersomes are yet another type of liposomes, and are
highly deformable lipid aggregates which are attractive candidates
for drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g. they are self-optimizing (adaptive to the shape of pores
in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
[0104] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0105] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical and cosmetic products and are usable
over a wide range of pH values. In general their HLB values range
from 2 to about 18 depending on their structure. Nonionic
surfactants include nonionic esters such as ethylene glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols, and ethoxylated/propoxylated block polymers
are also included in this class. The polyoxyethylene surfactants
are the most popular members of the nonionic surfactant class.
[0106] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0107] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0108] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0109] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).
[0110] Penetration Enhancers
[0111] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides, to the skin of animals. Most
drugs are present in solution in both ionized and nonionized forms.
However, usually only lipid soluble or lipophilic drugs readily
cross cell membranes. It has been discovered that even
non-lipophilic drugs may cross cell membranes if the membrane to be
crossed is treated with a penetration enhancer. In addition to
aiding the diffusion of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs.
[0112] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
p.92). Each of the above mentioned classes of penetration enhancers
are described below in greater detail.
[0113] Surfactants: In connection with the present invention,
surfactants (or "surface-active agents") are chemical entities
which, when dissolved in an aqueous solution, reduce the surface
tension of the solution or the interfacial tension between the
aqueous solution and another liquid, with the result that
absorption of oligonucleotides through the mucosa is enhanced. In
addition to bile salts and fatty acids, these penetration enhancers
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, p.92); and perfluorochemical emulsions, such as FC-43.
Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
[0114] Fatty acids: Various fatty acids and their derivatives which
act as penetration enhancers include, for example, oleic acid,
lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,
caprylic acid, arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,
1992, 44, 651-654).
[0115] Bile salts: The physiological role of bile includes 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, 1996, pp. 934-935). Various natural
bile salts, and their synthetic derivatives, act as penetration
enhancers. Thus the term "bile salts" includes any of the naturally
occurring components of bile as well as any of their synthetic
derivatives. The bile salts of the invention include, for example,
cholic acid (or its pharmaceutically acceptable sodium salt, sodium
cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic
acid (sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
[0116] Chelating Agents: Chelating agents, as used in connection
with the present invention, can be defined as compounds that remove
metallic ions from solution by forming complexes therewith, with
the result that absorption of oligonucleotides through the mucosa
is enhanced. With regards to their use as penetration enhancers in
the present invention, chelating agents have the added advantage of
also serving as DNase inhibitors, as most characterized DNA
nucleases require a divalent metal ion for catalysis and are thus
inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,
315-339). Chelating agents of the invention 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).
[0117] Non-chelating non-surfactants: As used herein, non-chelating
non-surfactant penetration enhancing compounds can be defined as
compounds that demonstrate insignificant activity as chelating
agents or as surfactants but that nonetheless enhance absorption of
oligonucleotides through the alimentary mucosa (Muranishi, Critical
Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This
class of penetration enhancers 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).
[0118] Agents that enhance uptake of oligonucleotides at the
cellular level may also be added to the pharmaceutical and other
compositions of the present invention. For example, cationic
lipids, such as lipofectin (Junichi et al, U.S. Pat. No.
5,705,188), cationic glycerol derivatives, and polycationic
molecules, such as polylysine (Lollo et al., PCT Application WO
97/30731), are also known to enhance the cellular uptake of
oligonucleotides.
[0119] Other agents may be utilized to enhance the penetration of
the administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
[0120] Carriers
[0121] Certain compositions of the present invention also
incorporate carrier compounds in the formulation. As used herein,
"carrier compound" or "carrier" can refer 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. For example, the recovery of a
partially phosphorothioate oligonucleotide in hepatic tissue can be
reduced when it is coadministered with polyinosinic acid, dextran
sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
[0122] Excipients
[0123] In contrast to a carrier compound, a "pharmaceutical
carrier" or "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 excipient
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
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone 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.); disintegrants
(e.g., starch, sodium starch glycolate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc.).
[0124] Pharmaceutically acceptable organic or inorganic excipient
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can also be used to
formulate the compositions of the present invention. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the
like.
[0125] Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
[0126] Suitable pharmaceutically acceptable excipients include, but
are not limited to, water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose,
polyvinylpyrrolidone and the like.
[0127] Other Components
[0128] 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, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions of the 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 present invention. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0129] Aqueous suspensions may contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0130] Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more antisense compounds and (b)
one or more other chemotherapeutic agents which function by a
non-antisense mechanism. 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,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphor- amide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, 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. 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). Anti-inflammatory drugs, including but not
limited to nonsteroidal anti-inflammatory drugs and
corticosteroids, and antiviral drugs, including but not limited to
ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. See, generally, The
Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
Other non-antisense chemotherapeutic agents are also within the
scope of this invention. Two or more combined compounds may be used
together or sequentially.
[0131] In another related embodiment, compositions of the invention
may contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Numerous examples of antisense compounds are known in the
art. Two or more combined compounds may be used together or
sequentially.
[0132] 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 in vitro and in vivo animal models. In general, dosage
is from 0.01 ug 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 ug to 100 g per kg of body
weight, once or more daily, to once every 20 years.
[0133] While the present invention has been described with
specificity in accordance with certain of its preferred
embodiments, the following examples serve only to illustrate the
invention and are not intended to limit the same.
EXAMPLES
Example 1
Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and
2'-alkoxy amidites
[0134] 2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl
phosphoramidites were purchased from commercial sources (e.g.
Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.).
Other 2'-O-alkoxy substituted nucleoside amidites are prepared as
described in U.S. Pat. No. 5,506,351, herein incorporated by
reference. For oligonucleotides synthesized using 2'-alkoxy
amidites, the standard cycle for unmodified oligonucleotides was
utilized, except the wait step after pulse delivery of tetrazole
and base was increased to 360 seconds.
[0135] Oligonucleotides containing 5-methyl-2'-deoxycytidine
(5-Me-C) nucleotides were synthesized according to published
methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21,
3197-3203] using commercially available phosphoramidites (Glen
Research, Sterling Va. or ChemGenes, Needham Mass.).
2'-Fluoro amidites
2'-Fluorodeoxyadenosine amidites
[0136] 2'-fluoro oligonucleotides were synthesized as described
previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841]
and U.S. Pat. No. 5,670,633, herein incorporated by reference.
Briefly, the protected nucleoside
N6-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-trityl group. Thus
N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively
protected in moderate yield as the 3',5'-ditetrahydropyranyl (THP)
intermediate. Deprotection of the THP and N6-benzoyl groups was
accomplished using standard methodologies and standard methods were
used to obtain the 5'-dimethoxytrityl-(DMT) and
51-DMT-3'-phosphoramidite intermediates.
2'-Fluorodeoxyguanosine
[0137] The synthesis of 2'-deoxy-2'-fluoroguanosine was
accomplished using tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosylguani- ne 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.
2'-Fluorouridine
[0138] Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by
the modification of a literature procedure in which
2,2'-anhydro-1-beta-D-ara- binofuranosyluracil was treated with 70%
hydrogen fluoride-pyridine. Standard procedures were used to obtain
the 5'-DMT and 5'-DMT-3'phosphoramidites.
2'-Fluorodeoxycytidine
[0139] 2'-deoxy-2'-fluorocytidine was synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were
used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.
2'-O-(2-Methoxyethyl) modified amidites
[0140] 2'-O-Methoxyethyl-substituted nucleoside amidites are
prepared as follows, or alternatively, as per the methods of
Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.
2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]
[0141] 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 that was
crushed to a light tan powder (57 g, 85% crude yield). The NMR
spectrum was consistent with the structure, contaminated with
phenol as its sodium salt (ca. 5%). The material was used as is for
further reactions (or it can be purified further by column
chromatography using a gradient of methanol in ethyl acetate
(10-25%) to give a white solid, mp 222-4.degree. C.).
2'-O-Methoxyethyl-5-methyluridine
[0142] 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.
Additional material was obtained by reworking impure fractions.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0143] 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 dimethoxytrityl
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%).
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0144] 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%). An additional 1.5 g
was recovered from later fractions.
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleurid-
ine
[0145] 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 latter
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.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0146] A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxy-trityl-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 NH3 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.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-cytidine
[0147] 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.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-cytidine-3'-ami-
dite
[0148]
N4-Benzoyl-21-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-cytidine
(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/hexane (3:1)
as the eluting solvent. The pure fractions were combined to give
90.6 g (87%) of the title compound.
2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites
[0149] 2'-(Dimethylaminooxyethoxy) nucleoside amidites
2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the
art as 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are
prepared as described in the following paragraphs. Adenosine,
cytidine and guanosine nucleoside amidites are prepared similarly
to the thymidine (5-methyluridine) except the exocyclic amines are
protected with a benzoyl moiety in the case of adenosine and
cytidine and with isobutyryl in the case of guanosine.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
[0150] O.sup.2-2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese,
Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013
eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient
temperature under an argon atmosphere and with mechanical stirring.
tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458
mmol) was added in one portion. The reaction was stirred for 16 h
at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a
complete reaction. The solution was concentrated under reduced
pressure to a thick oil. This was partitioned between
dichloromethane (1 L) and saturated sodium bicarbonate (2.times.1
L) and brine (1 L). The organic layer was dried over sodium sulfate
and concentrated under reduced pressure to a thick oil. The oil was
dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600
mL) and the solution was cooled to
[0151] -10.degree. C. The resulting crystalline product was
collected by filtration, washed with ethyl ether (3.times.200 mL)
and dried (40.degree. C., 1 mm Hg, 24 h) to 149 g (74.8%) of white
solid. TLC and NMR were consistent with pure product.
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
[0152] In a 2 L stainless steel, unstirred pressure reactor was
added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the
fume hood and with manual stirring, ethylene glycol (350 mL,
excess) was added cautiously at first until the evolution of
hydrogen gas subsided.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
(149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were
added with manual stirring. The reactor was sealed and heated in an
oil bath until an internal temperature of 160.degree. C. was
reached and then maintained for 16 h (pressure<100 psig). The
reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for
desired product and Rf 0.82 for ara-T side product, ethyl acetate)
indicated about 70% conversion to the product. In order to avoid
additional side product formation, the reaction was stopped,
concentrated under reduced pressure (10 to 1 mm Hg) in a warm water
bath (40-100.degree. C.) with the more extreme conditions used to
remove the ethylene glycol. [Alternatively, once the low boiling
solvent is gone, the remaining solution can be partitioned between
ethyl acetate and water. The product will be in the organic phase.]
The residue was purified by column chromatography (2 kg silica gel,
ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate
fractions were combined, stripped and dried to product as a white
crisp foam (84g, 50%), contaminated starting material (17.4 g) and
pure reusable starting material 20 g. The yield based on starting
material less pure recovered starting material was 58%. TLC and NMR
were consistent with 99% pure product.
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylailyl-5-methyluridine
[0153]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g,
44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was
then dried over P.sub.2O.sub.5 under high vacuum for two days at
40.degree. C. The reaction mixture was flushed with argon and dry
THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear
solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added
dropwise to the reaction mixture. The rate of addition is
maintained such that resulting deep red coloration is just
discharged before adding the next drop. After the addition was
complete, the reaction was stirred for 4 hrs. By that time TLC
showed the completion of the reaction (ethylacetate:hexane, 60:40).
The solvent was evaporated in vacuum. Residue obtained was placed
on a flash column and eluted with ethyl acetate:hexane (60:40), to
get
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
as white foam (21.819 g, 86%).
5'-O-tert-butyldiphenyloilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methylurid-
ine
[0154]
2'-O-([2-phthalimidoxy)ethyl)-5'-t-butyldiphenylsilyl-5-methyluridi-
ne (3.1 g, 4.5 mmol) was dissolved in dry CH.sub.2Cl.sub.2 (4.5 mL)
and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at
-10.degree. C. to 0.degree. C. After 1 h the mixture was filtered,
the filtrate was washed with ice cold CH.sub.2Cl.sub.2 and the
combined organic phase was washed with water, brine and dried over
anhydrous Na.sub.2SO.sub.4. The solution was concentrated to get
2'-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH
(67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1
eq.) was added and the resulting mixture was strirred for 1 h.
Solvent was removed under vacuum; residue chromatographed to get
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)
ethyl]-5-methyluridine as white foam (1.95 g, 78%).
5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methylurid-
ine
[0155]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M
pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium
cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at
10.degree. C. under inert atmosphere. The reaction mixture was
stirred for 10 minutes at 10.degree. C. After that the reaction
vessel was removed from the ice bath and stirred at room
temperature for 2 h, the reaction monitored by TLC (5% MeOH in
CH.sub.2Cl.sub.2). Aqueous NaHCO.sub.3 solution (5%, 10 mL) was
added and extracted with ethyl acetate (2.times.20 mL). Ethyl
acetate phase was dried over anhydrous Na.sub.2SO.sub.4, evaporated
to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH
(30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and
the reaction mixture was stirred at room temperature for 10
minutes. Reaction mixture cooled to 10.degree. C. in an ice bath,
sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction
mixture stirred at 10.degree. C. for 10 minutes. After 10 minutes,
the reaction mixture was removed from the ice bath and stirred at
room temperature for 2 hrs. To the reaction mixture 5% NaHCO.sub.3
(25 mL) solution was added and extracted with ethyl acetate
(2.times.25 mL). Ethyl acetate layer was dried over anhydrous
Na.sub.2SO.sub.4 and evaporated to dryness. The residue obtained
was purified by flash column chromatography and eluted with 5% MeOH
in CH.sub.2Cl.sub.2 to get
5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluri-
dine as a white foam (14.6 g, 80%).
2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0156] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was
dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept
over KOH). This mixture of triethylamine-2HF was then added to
5'-O-tert-butyldiphenylsil-
yl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4
mmol) and stirred at room temperature for 24 hrs. Reaction was
monitored by TLC (5% MeOH in CH.sub.2Cl.sub.2). Solvent was removed
under vacuum and the residue placed on a flash column and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 to get
2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0157] 2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17
mmol) was dried over P.sub.2O.sub.5 under high vacuum overnight at
40.degree. C. It was then co-evaporated with anhydrous pyridine (20
mL). The residue obtained was dissolved in pyridine (11 mL) under
argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol),
4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the
mixture and the reaction mixture was stirred at room temperature
until all of the starting material disappeared. Pyridine was
removed under vacuum and the residue chromatographed and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 (containing a few drops of
pyridine) to get 5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5--
methyluridine (1.13 g, 80%).
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoet-
hyl)-N,N-diisopropylphosphoramidite]
[0158] 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08
g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the
residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was
added and dried over P2O.sub.5 under high vacuum overnight at
40.degree. C. Then the reaction mixture was dissolved in anhydrous
acetonitrile (8.4 mL) and
2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoramidite (2.12 mL, 6.08
mmol) was added. The reaction mixture was stirred at ambient
temperature for 4 hrs under inert atmosphere. The progress of the
reaction was monitored by TLC (hexane:ethyl acetate 1:1). The
solvent was evaporated, then the residue was dissolved in ethyl
acetate (70 mL) and washed with 5% aqueous NaHCO.sub.3 (40 mL).
Ethyl acetate layer was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. Residue obtained was chromatographed (ethyl acetate
as eluent) to get 5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyeth-
yl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]
as a foam (1.04 g, 74.9%).
2'-(Aminooxyethoxy) nucleoside amidites
[0159] 2'-(Aminooxyethoxy) nucleoside amidites (also known in the
art as 2'-O-(aminooxyethyl) nucleoside amidites] are prepared as
described in the following paragraphs. Adenosine, cytidine and
thymidine nucleoside amidites are prepared similarly.
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimeth-
oxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]
[0160] The 2'-O-aminooxyethyl guanosine analog may be obtained by
selective 2'-O-alkylation of diaminopurine riboside. Multigram
quantities of diaminopurine riboside may be purchased from Schering
AG (Berlin) to provide 2'-O-(2-ethylacetyl) diaminopurine riboside
along with a minor amount of the 3'-O-isomer. 2'-O-(2-ethylacetyl)
diaminopurine riboside may be resolved and converted to
2'-O-(2-ethylacetyl)guanosine by treatment with adenosine
deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO
94/02501 A1 940203.) Standard protection procedures should afford
2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine and
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'--
dimethoxytrityl)guanosine which may be reduced to provide
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-hydroxyethyl)-5'-O-(4,4'-dim-
ethoxytrityl)guanosine. As before the hydroxyl group may be
displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the
protected nucleoside may phosphitylated as usual to yield
2-N-isobutyryl-6-O-diphen-
ylcarbamoyl-2'-O-([2-phthalmidoxylethyl)-5'-O-(4,4'-dimethoxytrityl)guanos-
ine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites
[0161] 2'-dimethylaminoethoxyethoxy nucleoside amidites (also known
in the art as 2'-O-dimethylaminoethoxyethyl, i.e.,
2'-O--CH.sub.2--O--CH.sub.2--- N(CH.sub.2).sub.22 or 2'-DMAEOE
nucleoside amidites) are prepared as follows. Other nucleoside
amidites are prepared similarly.
2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine
[0162] 2[2-(Dimethylamino)ethoxylethanol (Aldrich, 6.66 g, 50 mmol)
is slowly added to a solution of borane in tetrahydrofuran (1 M, 10
mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves
as the solid dissolves. O.sup.2-,2'-anhydro-5-methyluridine (1.2 g,
5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is
sealed, placed in an oil bath and heated to 155.degree. C. for 26
hours. The bomb is cooled to room temperature and opened. The crude
solution is concentrated and the residue partitioned between water
(200 mL) and hexanes (200 mL). The excess phenol is extracted into
the hexane layer. The aqueous layer is extracted with ethyl acetate
(3.times.200 mL) and the combined organic layers are washed once
with water, dried over anhydrous sodium sulfate and concentrated.
The residue is columned on silica gel using methanol/methylene
chloride 1:20 (which has 2% triethylamine) as the eluent. As the
column fractions are concentrated a colorless solid forms which is
collected to give the title compound as a white solid.
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)
ethyl)]-5-methyl uridine
[0163] To 0.5 g (1.3 mmol) of
2'-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5- -methyl uridine in
anhydrous pyridine (8 mL), triethylamine (0.36 mL) and
dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and
stirred for 1 hour. The reaction mixture is poured into water (200
mL) and extracted with CH.sub.2Cl.sub.2 (2.times.200 mL). The
combined CH.sub.2Cl.sub.2 layers are washed with saturated
NaHCO.sub.3 solution, followed by saturated NaCl solution and dried
over anhydrous sodium sulfate. Evaporation of the solvent followed
by silica gel chromatography using MeOH:CH.sub.2Cl.sub.2:Et.sub.3N
(20:1, v/v, with 1% triethylamine) gives the title compound.
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite
[0164] Diisopropylaminotetrazolide (0.6 g) and
2-cyanoethoxy-N,N-diisoprop- yl phosphoramidite (1.1 mL, 2 eq.) are
added to a solution of
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methylu-
ridine (2.17 g, 3 mmol) dissolved in CH.sub.2Cl.sub.2 (20 mL) under
an atmosphere of argon. The reaction mixture is stirred overnight
and the solvent evaporated. The resulting residue is purified by
silica gel flash column chromatography with ethyl acetate as the
eluent to give the title compound.
Example 2
Oligonucleotide Synthesis
[0165] Unsubstituted and substituted phosphodiester (P.dbd.O)
oligo-nucleotides are synthesized on an automated DNA synthesizer
(Applied Biosystems model 380B) using standard phosphoramidite
chemistry with oxidation by iodine.
[0166] Phosphorothioates (P.dbd.S) are synthesized as for the
phosphodiester oligonucleotides except the standard oxidation
bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one
1,1-dioxide in acetonitrile for the stepwise thiation of the
phosphite linkages. The thiation wait step was increased to 68 sec
and was followed by the capping step. After cleavage from the CPG
column and deblocking in concentrated ammonium hydroxide at
55.degree. C. (18 h), the oligonucleotides were purified by
precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl
solution. Phosphinate oligonucleotides are prepared as described in
U.S. Pat. No. 5,508,270, herein incorporated by reference.
[0167] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0168] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050,
herein incorporated by reference.
[0169] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein
incorporated by reference.
[0170] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications PCT/US94/00902 and
PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively), herein incorporated by reference.
[0171] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0172] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0173] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated
by reference.
Example 3
Oligonucleoside Synthesis
[0174] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides,
methylenedimethyl-hydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligo-nucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289, all of which are herein
incorporated by reference.
[0175] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564,
herein incorporated by reference.
[0176] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Patent 5,223,618, herein incorporated by
reference.
Example 4
PNA Synthesis
[0177] Peptide nucleic acids (PNAs) are prepared in accordance with
any of the various procedures referred to in Peptide Nucleic Acids
(PNA): Synthesis, Properties and Potential Applications, Bioorganic
& Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared
in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and
5,719,262, herein incorporated by reference.
Example 5
Synthesis of Chimeric Oligonucleotides
[0178] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0179] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 380B, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
increasing the wait step after the delivery of tetrazole and base
to 600 s repeated four times for RNA and twice for 2'-O-methyl. The
fully protected oligonucleotide is cleaved from the support and the
phosphate group is deprotected in 3:1 ammonia/ethanol at room
temperature overnight then lyophilized to dryness. Treatment in
methanolic ammonia for 24 hrs at room temperature is then done to
deprotect all bases and sample was again lyophilized to dryness.
The pellet is resuspended in 1M TBAF in THF for 24 hrs at room
temperature to deprotect the 2' positions. The reaction is then
quenched with 1M TEAA and the sample is then reduced to 1/2 volume
by rotovac before being desalted on a G25 size exclusion column.
The oligo recovered is then analyzed spectrophotometrically for
yield and for purity by capillary electrophoresis and by mass
spectrometry.
[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric
Phosphorothioate Oligonucleotides
[0180] [2'-O-(2-methoxyethyl)]-[2'-deoxy]-[-2'-O-(methoxyethyl)]
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
[2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy
Phosphoro-thioate]-[2'-O-(- 2-Methoxyethyl) Phosphodiester]
Chimeric Oligonucleotides
[0181] [2'-O-(2-methoxyethyl phosphodiester]-[2'-deoxy
phosphoro-thioatel-[2'-O-(methoxyethyl) phosphodiester] chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidization with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0182] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 6
Oligonucleotide Isolation
[0183] 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 or
oligonucleosides 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, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7
Oligonucleotide Synthesis--96 Well Plate Format
[0184] Oligonucleotides were synthesized 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-cyanoethyldiisopropyl
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
known literature or patented methods. They are utilized as base
protected beta-cyanoethyldiisopropyl phosphoramidites.
[0185] 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.
Example 8
Oligonucleotide Analysis--96 Well Plate Format
[0186] The concentration of oligonucleotide in each well was
assessed by dilution of samples and UV absorption spectroscopy. The
full-length integrity of the individual products was evaluated by
capillary electrophoresis (CE) in either the 96 well format
(Beckman P/ACE.TM. MDQ) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
Base and backbone composition was confirmed by mass analysis of the
compounds utilizing electrospray-mass spectroscopy. All assay test
plates were diluted from the master plate using single and
multi-channel robotic pipettors. Plates were judged to be
acceptable if at least 85% of the compounds on the plate were at
least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
[0187] The effect of antisense compounds on target nucleic acid
expression can be tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. The following 5 cell types are provided for
illustrative purposes, but other cell types can be routinely used,
provided that the target is expressed in the cell type chosen. This
can be readily determined by methods routine in the art, for
example Northern blot analysis, Ribonuclease protection assays, or
RT-PCR.
T-24 Cells
[0188] The human transitional cell bladder carcinoma cell line T-24
was obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). T-24 cells were routinely cultured in complete
McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.)
supplemented with 10% fetal calf serum (Gibco/Life Technologies,
Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin
100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 7000 cells/well for use in
RT-PCR analysis.
[0189] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0190] A549 Cells
[0191] The human lung carcinoma cell line A549 was obtained from
the American Type Culture Collection (ATCC) (Manassas, Va.). A549
cells were routinely cultured in DMEM basal media (Gibco/Life
Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf
serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin lo
units per mL, and streptomycin 100 micrograms per mL (Gibco/Life
Technologies, Gaithersburg, Md.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
NHDF Cells
[0192] Human neonatal dermal fibroblast (NHDF) were obtained from
the Clonetics Corporation (Walkersville Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville Md.) supplemented as recommended by the supplier.
Cells were maintained for up to 10 passages as recommended by the
supplier.
HEK Cells
[0193] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville Md.). HEKs were routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville Md.) formulated as recommended by the supplier. Cells
were routinely maintained for up to 10 passages as recommended by
the supplier.
3T3-L1 Cells
[0194] The mouse embryonic adipocyte-like cell line 3T3-L1 was
obtained from the American Type Culure Collection (Manassas, Va.).
3T3-L1 cells were routinely cultured in DMEM, high glucose
(Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%
fetal calf serum (Gibco/Life Technologies, Gaithersburg,. Md.).
Cells were routinely passaged by trypsinization and dilution when
they reached 80% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 4000 cells/well for use in
RT-PCR analysis.
[0195] For Northern blotting or other analyses, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
Treatment with Antisense Compounds
[0196] When cells reached 80% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 200 .mu.L OPTI-MEM.TM.-1 reduced-serum medium
(Gibco BRL) and then treated with 130 .mu.L of OPTI-MEM.TM.-1
containing 3.75 .mu.g/mL LIPOFECTIN.TM. (Gibco BRL) and the desired
concentration of oligonucleotide. After 4-7 hours of treatment, the
medium was replaced with fresh medium. Cells were harvested 16-24
hours after oligonucleotide treatment.
[0197] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1,
a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in bold) with
a phosphorothioate backbone which is targeted to human H-ras. For
mouse or rat cells the positive control oligonucleotide is ISIS
15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2'-O-methoxyethyl
gapmer (2'-O-methoxyethyls shown in bold) with a phosphorothioate
backbone which is targeted to both mouse and rat c-raf. The
concentration of positive control oligonucleotide that results in
80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS
15770) mRNA is then utilized as the screening concentration for new
oligonucleotides in subsequent experiments for that cell line. If
80% inhibition is not achieved, the lowest concentration of
positive control oligonucleotide that results in 60% inhibition of
H-ras or c-raf mRNA is then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% inhibition is not achieved, that particular cell line
is deemed as unsuitable for oligonucleotide transfection
experiments.
Example 10
Analysis of Oligonucleotide Inhibition of src-c Expression
[0198] Antisense modulation of src-c expression can be assayed in a
variety of ways known in the art. For example, src-c mRNA levels
can be quantitated by, e.g., Northern blot analysis, competitive
polymerase chain reaction (PCR), or real-time PCR (RT-PCR).
Real-time quantitative PCR is presently preferred. RNA analysis can
be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA
isolation are taught in, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9
and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot
analysis is routine in the art and is taught in, for example,
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.
Real-time quantitative (PCR) can be conveniently accomplished using
the commercially available ABI PRISM.TM. 7700 Sequence Detection
System, available from PE-Applied Biosystems, Foster City, Calif.
and used according to manufacturer's instructions.
[0199] Protein levels of src-c can be quantitated in a variety of
ways well known in the art, such as immunoprecipitation, Western
blot analysis (immunoblotting), ELISA or fluorescence-activated
cell sorting (FACS). Antibodies directed to src-c can be identified
and obtained from a variety of sources, such as the MSRS catalog of
antibodies (Aerie Corporation, Birmingham, Mich.), or can be
prepared via conventional antibody generation methods. Methods for
preparation of polyclonal antisera are taught in, for example,
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997.
Preparation of monoclonal antibodies is taught in, for example,
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc.,
1997.
[0200] Immunoprecipitation methods are standard in the art and can
be found at, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley
& Sons, Inc., 1998. Western blot (immunoblot) analysis is
standard in the art and can be found at, for example, Ausubel, F.
M. et al., Current Protocols in Molecular Biology, Volume 2, pp.
10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked
immunosorbent assays (ELISA) are standard in the art and can be
found at, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley &
Sons, Inc., 1991.
Example 11
Poly(A)+ mRNA Isolation
[0201] Poly(A)+ mRNA was isolated according to Miura et al., Clin.
Chem., 1996, 42, 1758-1764. 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 .mu.L cold PBS. 60 .mu.L 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 .mu.L 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 .mu.L
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
.mu.L 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. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0202] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
Total RNA Isolation
[0203] Total RNA was isolated using an RNEASY 96.TM. 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 pipetting three times up and down. The
samples were then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. 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.TM. plate and the vacuum again applied for 15 seconds. 1 mL of
Buffer RPE was then added to each well of the RNEASY 96.TM. 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.TM. manifold
and blotted dry on paper towels. The plate was then re-attached to
the QIAVAC.TM. 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.
[0204] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 13
Real-Time Quantitative PCR Analysis of src-c mRNA Levels
[0205] Quantitation of src-c mRNA levels was determined by
real-time quantitative PCR using the ABI PRISM.TM. 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, FAM, or VIC, 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 intervals by
laser optics built into the ABI PRISM.TM. 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.
[0206] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0207] 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.TM. 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.TM., and 12.5 Units
MuLV reverse transcriptase) to 96 well plates containing 25 .mu.L
total RNA 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.TM., 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).
[0208] Gene target quantities obtained by real time RT-PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
from Molecular Probes. Methods of RNA quantification by
RiboGreen.TM. are taught in Jones, L. J., et al, Analytical
Biochemistry, 1998, 265, 368-374.
[0209] In this assay, 175 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 25 uL purified,
cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied
Biosystems) with excitation at 480 nm and emission at 520 nm.
[0210] Probes and primers to human src-c were designed to hybridize
to a human src-c sequence, using published sequence information
(GenBank accession number NM.sub.--005417, incorporated herein as
SEQ ID NO:3). For human src-c the PCR primers were:
[0211] forward primer: AGCACAGGACAGACAGGCTACA (SEQ ID NO: 4)
[0212] reverse primer: CACTCCTCAGCCTGGATGGA (SEQ ID NO: 5) and the
PCR probe was: FAM-AGCAACTACGTGGCGCCCTCCG-TAMRA
[0213] (SEQ ID NO: 6) 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.
For human GAPDH the PCR primers were:
[0214] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)
[0215] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the
PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO:
9)
[0216] 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.
[0217] Probes and primers to mouse src-c were designed to hybridize
to a mouse src-c sequence, using published sequence information
(GenBank accession number M17031, incorporated herein as SEQ ID
NO:10). For mouse src-c the PCR primers were:
[0218] forward primer: ACCTCCCGCACCCAGTTC (SEQ ID NO:11)
[0219] reverse primer: GGCCATCAGCATGTTTGGA (SEQ ID NO: 12) and the
PCR probe was: FAM-AGCCTGCAGCAGCTCGTGGCTTA-TAMRA
[0220] (SEQ ID NO: 13) 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.
For mouse GAPDH the PCR primers were:
[0221] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)
[0222] reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 15) and the
PCR probe was: 5' JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3' (SEQ ID
NO: 16) 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 14
Northern Blot Analysis of src-c mRNA Levels
[0223] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM.
(TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared
following manufacturer's recommended protocols. Twenty micrograms
of total RNA was fractionated by electrophoresis through 1.2%
agarose gels containing 1.1% formaldehyde using a MOPS buffer
system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the
gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.TM.
UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then
robed using QUICKHYB.TM. hybridization solution (Stratagene, La
Jolla, Calif.) using manufacturer's recommendations for stringent
conditions.
[0224] To detect human src-c, a human src-c specific probe was
prepared by PCR using the forward primer AGCACAGGACAGACAGGCTACA
(SEQ ID NO: 4) and the reverse primer CACTCCTCAGCCTGGATGGA (SEQ ID
NO: 5). To normalize for variations in loading and transfer
efficiency membranes were stripped and probed for human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0225] To detect mouse src-c, a mouse src-c specific probe was
prepared by PCR using the forward primer ACCTCCCGCACCCAGTTC (SEQ ID
NO:11) and the reverse primer GGCCATCAGCATGTTTGGA (SEQ ID NO: 12).
To normalize for variations in loading and transfer efficiency
membranes were stripped and probed for mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0226] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software V3.3 (Molecular
Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels
in untreated controls.
Example 15
Antisense Inhibition of Human src-c Expression by Chimeric
Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy
Gap
[0227] In accordance with the present invention, a series of
oligonucleotides were designed to target different regions of the
human src-c RNA, using published sequences (GenBank accession
number NM.sub.--005417, incorporated herein as SEQ ID NO: 3,
GenBank accession number K03212, incorporated herein as SEQ ID NO:
17, GenBank accession number K03215, incorporated herein as SEQ ID
NO: 18, GenBank accession number K03218, incorporated herein as SEQ
ID NO: 19, GenBank accession number M16237, incorporated herein as
SEQ ID NO: 20, GenBank accession number M16245, incorporated herein
as SEQ ID NO: 21, GenBank accession number X03995, incorporated
herein as SEQ ID NO: 22, GenBank accession number X03996,
incorporated herein as SEQ ID NO: 23, GenBank accession number
X03998, incorporated herein as SEQ ID NO: 24, GenBank accession
number X03999, incorporated herein as SEQ ID NO: 25, GenBank
accession number AI798073, representing a 5'UTR variant of GenBank
accession number NM.sub.--005417, the complement of which is
incorporated herein as SEQ ID NO: 26, GenBank accession number
AI951646, representing a 5'UTR variant which diverges at position
66 of GenBank accession number NM.sub.--005417, the complement of
which is incorporated herein as SEQ ID NO: 27, and GenBank
accession number AI282193, representing a 5'UTR variant of GenBank
accession number NM.sub.--005417 which aligns with but is missing
220 nucleotides from GenBank accession number AI951646, the
complement of which is incorporated herein as SEQ ID NO: 28). The
oligonucleotides are shown in Table 1. "Target site" indicates the
first (5'-most) nucleotide number on the particular target sequence
to which the oligonucleotide binds. All compounds in Table 1 are
chimeric oligonucleotides ("gapmers") 20 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 five-nucleotide "wings". The wings are composed of
2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone)
linkages are phosphrothioate (P.dbd.S) throughout the
oligonucleotide. All cytidine residues are 5-methylcytidines. The
compounds were analyzed for their effect on human src-c mRNA levels
by quantitative real-time PCR as described in other examples
herein. Data are averages from two experiments. If present, "N.D."
indicates "no data".
1TABLE 1 Inhibition of human src-c mRNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap TARGET SEQ ID TARGET % SEQ ID ISIS # REGION NO SITE SEQUENCE
INHIB NO 143507 Exon 2 20 12 ttgctcttgttgctacccat 79 29 143508
Coding 3 4 ggcttgctcttgttgctacc 59 30 143509 Exon 26 6
ggttggaactgaccagtgtg 0 31 143510 Exon 28 7 atcgagagagaaagggagga 5
32 143511 Exon 28 9 agatcgagagagaaagggag 0 33 143512 Exon 26 10
ggaaggttggaactgaccag 0 34 143513 Coding 3 19 tggctggcatccttgggctt
77 35 143514 Coding 3 22 cgctggctggcatccttggg 48 36 143515 Exon 27
26 gggctcaccggcagacggac 31 37 143516 Exon 28 48
cggctcccaggccggaatgg 20 38 143517 Exon 2 20 73 agcgccgtgcacgttctcgg
38 39 143518 Exon 26 87 gcaagttgcttcacttctct 37 40 143519 Exon 2 20
133 gtggccgtcggccgaggctg 27 41 143520 Exon 28 155
aggcaggggctgggccggcg 22 42 143521 Coding 3 179 gcctccgaacagcttgggct
3 43 143522 Exon 2 20 193 gaagcctccgaacagcttgg 78 44 143523 Coding
3 191 cgaggagttgaagcctccga 64 45 143524 Coding 3 196
gtgtccgaggagttgaagcc 69 46 143525 Coding 3 200 gacggtgtccgaggagttga
43 47 143526 Exon 12 19 207 gcctgtgcctagaggttctc 59 48 143527 Exon
26 230 caccaggtgtggagtcaggg 5 49 143528 Exon 26 238
ggcacaaacaccaggtgtgg 8 50 143529 Coding 3 249 caaaggtggtcactccaccg
37 51 143530 Coding 3 258 agagggccacaaaggtggtc 48 52 143531 Coding
3 276 tcctagactcatagtcatag 75 53 143532 Coding 3 317
ctggagccgctcgcctttct 68 54 143533 Coding 3 349 agccaccagtctccctctgt
0 55 143534 Coding 3 366 tgctgagcgagtgggccagc 65 56 143535 Coding 3
378 ctgtctgtcctgtgctgagc 88 57 143536 Coding 3 427
tcagcctggatggagtcgga 80 58 143538 Coding 3 469 cgctctgactcccgtctggt
0 59 143539 Coding 3 476 cagtaaccgctctgactccc 83 60 143540 Coding 3
499 cctctcgggttctctgcatt 71 61 143541 Coding 3 505
aaggtccctctcgggttctc 77 62 143542 Coding 3 507 ggaaggtccctctcgggttc
69 63 143543 Exon 21 78 ctttctcgcacgaggaaggt 67 64 143544 Exon 17
23 tgtcgaagtcagacactgag 63 65 143545 Coding 3 589
ttcacgttgaggcccttggc 44 66 143546 Coding 3 658 aggctgttgaactgggtgcg
58 67 143547 Coding 3 660 gcaggctgttgaactgggtg 9 68 143548 Coding 3
666 gctgctgcaggctgttgaac 56 69 143549 Coding 3 671
caccagctgctgcaggctgt 66 70 143550 Coding 3 687 gtttggagtagtaggccacc
70 71 143551 Coding 3 711 ggcggtggcacaggccatcg 73 72 143552 Coding
3 747 gagtctgcggcttggacgtg 56 73 143553 Coding 3 748
tgagtctgcggcttggacgt 6 74 143554 Exon 7 22 49 gccctgagtctgcggcttgg
48 75 143555 Exon 7 22 89 gcagcgactcccgagggatc 65 76 143556 Coding
3 809 cagcttgacctccagccgca 80 77 143557 Coding 3 815
ctggcccagcttgacctcca 46 78 143558 Coding 3 857 ggtaccgttccaggtcccca
62 79 143559 Coding 3 871 atggccaccctggtggtacc 63 80 143560 Coding
3 873 tgatggccaccctggtggta 60 81 143561 Exon 8 23 26
gcttcagggttttgatggcc 34 82 143562 Exon 8 23 134
aaatgggctcctctgaaacc 78 83 143563 Exon 9 18 10 gagaaagtccagcaaactcc
64 84 143564 Coding 3 1050 tctcccccttgagaaagtcc 71 85 143565 Coding
3 1057 ttgcctgtctcccccttgag 78 86 143566 Coding 3 1072
ggcagccgcaggtacttgcc 68 87 143567 Exon 10 24 24
ggacgtagttcatccgctcc 74 88 143568 Coding 3 1184
caggttctctcccaccagga 75 89 143569 Exon 10 24 69
ccaggttctctcccaccagg 79 90 143570 Coding 3 1196
cactttgcacaccaggttct 68 91 143571 Coding 3 1202
gtcggccactttgcacacca 76 92 143572 Coding 3 1208
cccaaagtcggccactttgc 76 93 143573 Exon 11 25 34
gcggccatagagggcagctt 27 94 143574 Exon 11 25 79
agtcagcaggatcccgaagg 61 95 143575 Coding 3 1372
acccgtccctttgtggtgag 70 96 143576 Exon 12 19 19
ctggtccagcacctcgcggt 60 97 143577 Coding 3 1486
cagcactggcacatgaggtc 80 98 143578 Coding 3 1488
gccagcactggcacatgagg 77 99 143579 Coding 3 1492
ttccgccagcactggcacat 75 100 143580 Coding 3 1498
ggctccttccgccagcactg 85 101 143581 Coding 3 1510
ggccgctcctcaggctcctt 77 102 143582 Coding 3 1511
gggccgctcctcaggctcct 72 103 143583 Exon 12 19 128
actcgaaggtgggccgctcc 65 104 143584 Stop 3 1592 ctagaggttctccccgggct
57 105 Codon
[0228] As shown in Table 1, SEQ ID NOs 29, 30, 35, 44, 45, 46, 48,
53, 54, 56, 57, 58, 60, 61, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73,
76, 77, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95,
96, 97, 98, 99, 100, 101, 102, 103, 104 and 105 demonstrated at
least 50% inhibition of human src-c expression in this assay and
are therefore preferred. The target sites to which these preferred
sequences are complementary are herein referred to as "active
sites" and are therefore preferred sites for targeting by compounds
of the present invention.
Example 16
Antisense Inhibition of Mouse src-c Expression by Chimeric
Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy
Gap
[0229] In accordance with the present invention, a second series of
oligonucleotides were designed to target different regions of the
mouse src-c RNA, using published sequences (GenBank accession
number M17031, incorporated herein as SEQ ID NO: 10, and GenBank
accession number AW213019, representing an mRNA varient which skips
exon 35, incorporated herein as SEQ ID NO: 106). The
oligonucleotides are shown in Table 2. "Target site" indicates the
first (5'-most) nucleotide number on the particular target sequence
to which the oligonucleotide binds. All compounds in Table 2 are
chimeric oligonucleotides ("gapmers") 20 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 five-nucleotide "wings". The wings are composed of
2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone)
linkages are phosphorothioate (P.dbd.S) throughout the
oligonucleotide. All cytidine residues are 5-methylcytidines. The
compounds were analyzed for their effect on mouse src-c mRNA levels
by quantitative real-time PCR as described in other examples
herein. Data are averages from two experiments. If present, "N.D."
indicates "no data".
2TABLE 2 Inhibition of mouse src-c mRNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap TARGET TARGET % SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE
INHIB NO 143523 Coding 10 188 cgaggagttgaagcctccga 76 45 143524
Coding 10 193 gtgtccgaggagttgaagcc 77 46 143530 Coding 10 255
agagggccacaaaggtggtc 55 52 143536 Coding 10 442
tcagcctggatggagtcgga 43 58 143541 Coding 10 520
aaggtccctctcgggttctc 70 62 143542 Coding 10 522
ggaaggtccctctcgggttc 9 63 143547 Coding 10 675 gcaggctgttgaactgggtg
84 68 143548 Coding 10 681 gctgctgcaggctgttgaac 37 69 143556 Coding
10 824 cagcttgacctccagccgca 64 77 143557 Coding 10 830
ctggcccagcttgacctcca 57 78 143570 Coding 10 1211
cactttgcacaccaggttct 56 91 143571 Coding 10 1217
gtcggccactttgcacacca 73 92 143572 Coding 10 1223
cccaaagtcggccactttgc 46 93 143579 Coding 10 1507
ttccgccagcactggcacat 6 100 143580 Coding 10 1513
ggctccttccgccagcactg 6 101 143607 Start 10 1 ttgctcttgttgctgcccat
13 107 Codon 143608 Coding 10 46 tccgagggctccaggctgcg 56 108 143609
Coding 10 173 tccgaagagcttgggctcgg 24 109 143610 Coding 10 177
agcctccgaagagcttgggc 65 110 143611 Coding 10 182
gttgaagcctccgaagagct 37 111 143612 Coding 10 184
gagttgaagcctccgaagag 37 112 143613 Coding 10 190
tccgaggagttgaagcctcc 65 113 143614 Coding 10 281
agtctctgtccgtgactcat 20 114 143615 Coding 10 291
aggacaggtcagtctctgtc 17 115 143616 Coding 10 292
aaggacaggtcagtctctgt 19 116 143617 Coding 10 307
cgctcccctttcttgaagga 1 117 143618 Coding 10 311
cagccgctcccctttcttga 41 118 143619 Coding 10 322
ttgacaatctgcagccgctc 16 119 143620 Coding 10 329
cgtgttattgacaatctgca 86 120 143621 Coding 10 343
acatccaccttcctcgtgtt 78 121 143622 Coding 10 373
gagtgtgccagccaccagtc 25 122 143623 Coding 10 380
gctcagcgagtgtgccagcc 17 123 143624 Coding 10 381
tgctcagcgagtgtgccagc 92 124 143625 Coding 10 383
cgtgctcagcgagtgtgcca 85 125 143626 Coding 10 386
tcccgtgctcagcgagtgtg 12 126 143627 Coding 10 393
cggtctgtcccgtgctcagc 29 127 143628 Coding 10 398
gtaaccggtctgtcccgtgc 69 128 143629 Coding 10 401
gatgtaaccggtctgtcccg 35 129 143630 Coding 10 473
ccgtctagtgatcttgccaa 82 130 143631 Coding 10 482
ctctgattcccgtctagtga 58 131 143632 Coding 10 487
agccgctctgattcccgtct 45 132 143633 Coding 10 530
cctcacgaggaaggtccctc 65 133 143634 Coding 10 542
ggtctcactctccctcacga 59 134 143635 Coding 10 570
atacagagaggcagtaggca 50 135 143636 Coding 10 575
gtcggatacagagaggcagt 49 136 143637 Coding 10 583
ttgtcgaagtcggatacaga 70 137 143638 Coding 10 598
tttaggcccttggcattgtc 76 138 143639 Coding 10 599
atttaggcccttggcattgt 79 139 143640 Coding 10 608
gtgtttcacatttaggccct 16 140 143641 Coding 10 651
aggtgatgtagaaaccgccg 3 141 143642 Coding 10 688
gccacgagctgctgcaggct 35 142 143643 Coding 10 704
atgtttggagtagtaagcca 71 143 143644 Coding 10 715
aggccatcagcatgtttgga 15 144 143645 Coding 10 724
cggtgacacaggccatcagc 76 145 143646 Coding 10 757
tgaggcttggatgtgggaca 72 146 143647 Coding 10 766
ccctgggtctgaggcttgga 65 147 143648 Coding 10 817
acctccagccgcagggactc 73 148 143649 Coding 10 836
gcaaccctggcccagcttga 14 149 143650 Coding 10 847
acctctccgaagcaaccctg 32 150 143651 Coding 10 875
cgtggtgccgttccaggtcc 66 151 143652 Coding 10 886
atggcaaccctcgtggtgcc 0 152 143653 Coding 10 888
tgatggcaaccctcgtggtg 13 153 143654 Coding 10 957
tcagtttcttcatgacttgg 30 154 143655 Coding 10 1035
ccttgttcatgtactctgtc 42 155 143656 Coding 10 1044
gcagactccccttgttcatg 24 156 143657 Coding 10 1046
cagcagactccccttgttca 80 157 143658 Coding 10 1067
cgtttcccccttgagaaagt 43 158 143659 Coding 10 1075
tatttgcccgtttccccctt 5 159 143660 Coding 10 1146
tcatccgctccacataggcc 35 160 143661 Coding 10 1160
ccggtgcacatagttcatcc 31 161 143662 Coding 10 1194
tctcccctactaggatattg 1 162 143663 Coding 10 1229
ggccaacccaaagtcggcca 50 163 143664 Coding 10 1265
ttgccgggctgtgtattcgt 66 164 143665 Coding 10 1455
gacaaggcatccggtagccc 0 165 143666 Coding 10 1496
ctggcacataaggtcatgca 0 166 143667 Coding 10 1510
tccttccgccagcactggca 83 167 143668 Stop 10 1607
ctataggttctccccgggct 62 168 Codon 143669 Exon 106 339
tctccctctgtgttattgac 57 169
[0230] As shown in Table 2, SEQ ID NOs 45, 46, 52, 58, 62, 68, 77,
78, 91, 92, 93, 108, 110, 113, 118, 120, 121, 124, 125, 128, 130,
131, 132, 133, 134, 135, 136, 137, 138, 139-, 143, 145, 146, 147,
148, 151, 155, 157, 158, 163, 164, 167, 168 and 169 demonstrated at
least 40% inhibition of mouse src-c expression in this experiment
and are therefore preferred. The target sites to which these
preferred sequences are complementary are herein referred to as
"active sites" and are therefore preferred sites for targeting by
compounds of the present invention.
Example 17
Western Blot Analysis of src-c Protein Levels
[0231] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a
16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to src-c is used, with a radiolabelled or
fluorescently labeled secondary antibody directed against the
primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale Calif.).
Sequence CWU 1
1
169 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1
tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense
Oligonucleotide 2 atgcattctg cccccaagga 20 3 1611 DNA Homo sapiens
CDS (1)...(1611) 3 atg ggt agc aac aag agc aag ccc aag gat gcc agc
cag cgg cgc cgc 48 Met Gly Ser Asn Lys Ser Lys Pro Lys Asp Ala Ser
Gln Arg Arg Arg 1 5 10 15 agc ctg gag ccc gcc gag aac gtg cac ggc
gct ggc ggg ggc gct ttc 96 Ser Leu Glu Pro Ala Glu Asn Val His Gly
Ala Gly Gly Gly Ala Phe 20 25 30 ccc gcc tcg cag acc ccc agc aag
cca gcc tcg gcc gac ggc cac cgc 144 Pro Ala Ser Gln Thr Pro Ser Lys
Pro Ala Ser Ala Asp Gly His Arg 35 40 45 ggc ccc agc gcg gcc ttc
gcc ccc gcg gcc gcc gag ccc aag ctg ttc 192 Gly Pro Ser Ala Ala Phe
Ala Pro Ala Ala Ala Glu Pro Lys Leu Phe 50 55 60 gga ggc ttc aac
tcc tcg gac acc gtc acc tcc ccg cag agg gcg ggc 240 Gly Gly Phe Asn
Ser Ser Asp Thr Val Thr Ser Pro Gln Arg Ala Gly 65 70 75 80 ccg ctg
gcc ggt gga gtg acc acc ttt gtg gcc ctc tat gac tat gag 288 Pro Leu
Ala Gly Gly Val Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu 85 90 95
tct agg acg gag aca gac ctg tcc ttc aag aaa ggc gag cgg ctc cag 336
Ser Arg Thr Glu Thr Asp Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln 100
105 110 att gtc aac aac aca gag gga gac tgg tgg ctg gcc cac tcg ctc
agc 384 Ile Val Asn Asn Thr Glu Gly Asp Trp Trp Leu Ala His Ser Leu
Ser 115 120 125 aca gga cag aca ggc tac atc ccc agc aac tac gtg gcg
ccc tcc gac 432 Thr Gly Gln Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala
Pro Ser Asp 130 135 140 tcc atc cag gct gag gag tgg tat ttt ggc aag
atc acc aga cgg gag 480 Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys
Ile Thr Arg Arg Glu 145 150 155 160 tca gag cgg tta ctg ctc aat gca
gag aac ccg aga ggg acc ttc ctc 528 Ser Glu Arg Leu Leu Leu Asn Ala
Glu Asn Pro Arg Gly Thr Phe Leu 165 170 175 gtg cga gaa agt gag acc
acg aaa ggt gcc tac tgc ctc tca gtg tct 576 Val Arg Glu Ser Glu Thr
Thr Lys Gly Ala Tyr Cys Leu Ser Val Ser 180 185 190 gac ttc gac aac
gcc aag ggc ctc aac gtg aag cac tac aag atc cgc 624 Asp Phe Asp Asn
Ala Lys Gly Leu Asn Val Lys His Tyr Lys Ile Arg 195 200 205 aag ctg
gac agc ggc ggc ttc tac atc acc tcc cgc acc cag ttc aac 672 Lys Leu
Asp Ser Gly Gly Phe Tyr Ile Thr Ser Arg Thr Gln Phe Asn 210 215 220
agc ctg cag cag ctg gtg gcc tac tac tcc aaa cac gcc gat ggc ctg 720
Ser Leu Gln Gln Leu Val Ala Tyr Tyr Ser Lys His Ala Asp Gly Leu 225
230 235 240 tgc cac cgc ctc acc acc gtg tgc ccc acg tcc aag ccg cag
act cag 768 Cys His Arg Leu Thr Thr Val Cys Pro Thr Ser Lys Pro Gln
Thr Gln 245 250 255 ggc ctg gcc aag gat gcc tgg gag atc cct cgg gag
tcg ctg cgg ctg 816 Gly Leu Ala Lys Asp Ala Trp Glu Ile Pro Arg Glu
Ser Leu Arg Leu 260 265 270 gag gtc aag ctg ggc cag ggc tgc ttt ggc
gag gtg tgg atg ggg acc 864 Glu Val Lys Leu Gly Gln Gly Cys Phe Gly
Glu Val Trp Met Gly Thr 275 280 285 tgg aac ggt acc acc agg gtg gcc
atc aaa acc ctg aag cct ggc acg 912 Trp Asn Gly Thr Thr Arg Val Ala
Ile Lys Thr Leu Lys Pro Gly Thr 290 295 300 atg tct cca gag gcc ttc
ctg cag gag gcc cag gtc atg aag aag ctg 960 Met Ser Pro Glu Ala Phe
Leu Gln Glu Ala Gln Val Met Lys Lys Leu 305 310 315 320 agg cat gag
aag ctg gtg cag ttg tat gct gtg gtt tca gag gag ccc 1008 Arg His
Glu Lys Leu Val Gln Leu Tyr Ala Val Val Ser Glu Glu Pro 325 330 335
att tac atc gtc acg gag tac atg agc aag ggg agt ttg ctg gac ttt
1056 Ile Tyr Ile Val Thr Glu Tyr Met Ser Lys Gly Ser Leu Leu Asp
Phe 340 345 350 ctc aag ggg gag aca ggc aag tac ctg cgg ctg cct cag
ctg gtg gac 1104 Leu Lys Gly Glu Thr Gly Lys Tyr Leu Arg Leu Pro
Gln Leu Val Asp 355 360 365 atg gct gct cag atc gcc tca ggc atg gcg
tac gtg gag cgg atg aac 1152 Met Ala Ala Gln Ile Ala Ser Gly Met
Ala Tyr Val Glu Arg Met Asn 370 375 380 tac gtc cac cgg gac ctt cgt
gca gcc aac atc ctg gtg gga gag aac 1200 Tyr Val His Arg Asp Leu
Arg Ala Ala Asn Ile Leu Val Gly Glu Asn 385 390 395 400 ctg gtg tgc
aaa gtg gcc gac ttt ggg ctg gct cgg ctc att gaa gac 1248 Leu Val
Cys Lys Val Ala Asp Phe Gly Leu Ala Arg Leu Ile Glu Asp 405 410 415
aat gag tac acg gcg cgg caa ggt gcc aaa ttc ccc atc aag tgg acg
1296 Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Phe Pro Ile Lys Trp
Thr 420 425 430 gct cca gaa gct gcc ctc tat ggc cgc ttc acc atc aag
tcg gac gtg 1344 Ala Pro Glu Ala Ala Leu Tyr Gly Arg Phe Thr Ile
Lys Ser Asp Val 435 440 445 tgg tcc ttc ggg atc ctg ctg act gag ctc
acc aca aag gga cgg gtg 1392 Trp Ser Phe Gly Ile Leu Leu Thr Glu
Leu Thr Thr Lys Gly Arg Val 450 455 460 ccc tac cct ggg atg gtg aac
cgc gag gtg ctg gac cag gtg gag cgg 1440 Pro Tyr Pro Gly Met Val
Asn Arg Glu Val Leu Asp Gln Val Glu Arg 465 470 475 480 ggc tac cgg
atg ccc tgc ccg ccg gag tgt ccc gag tcc ctg cac gac 1488 Gly Tyr
Arg Met Pro Cys Pro Pro Glu Cys Pro Glu Ser Leu His Asp 485 490 495
ctc atg tgc cag tgc tgg cgg aag gag cct gag gag cgg ccc acc ttc
1536 Leu Met Cys Gln Cys Trp Arg Lys Glu Pro Glu Glu Arg Pro Thr
Phe 500 505 510 gag tac ctg cag gcc ttc ctg gag gac tac ttc acg tcc
acc gag ccc 1584 Glu Tyr Leu Gln Ala Phe Leu Glu Asp Tyr Phe Thr
Ser Thr Glu Pro 515 520 525 cag tac cag ccc ggg gag aac ctc tag
1611 Gln Tyr Gln Pro Gly Glu Asn Leu 530 535 4 22 DNA Artificial
Sequence PCR Primer 4 agcacaggac agacaggcta ca 22 5 20 DNA
Artificial Sequence PCR Primer 5 cactcctcag cctggatgga 20 6 22 DNA
Artificial Sequence PCR Probe 6 agcaactacg tggcgccctc cg 22 7 19
DNA Artificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20
DNA Artificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20
DNA Artificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10
1626 DNA Mus musculus CDS (1)...(1626) 10 atg ggc agc aac aag agc
aag ccc aag gac gcc agc cag cgg cgc cgc 48 Met Gly Ser Asn Lys Ser
Lys Pro Lys Asp Ala Ser Gln Arg Arg Arg 1 5 10 15 agc ctg gag ccc
tcg gaa aac gtg cac ggg gca ggg ggc gcc ttc ccg 96 Ser Leu Glu Pro
Ser Glu Asn Val His Gly Ala Gly Gly Ala Phe Pro 20 25 30 gcc tca
cag aca ccg agc aag ccc gcc tcc gcc gac ggc cac cgc ggg 144 Ala Ser
Gln Thr Pro Ser Lys Pro Ala Ser Ala Asp Gly His Arg Gly 35 40 45
ccc agc gcc gcc ttc gtg ccg ccc gcg gcc gag ccc aag ctc ttc gga 192
Pro Ser Ala Ala Phe Val Pro Pro Ala Ala Glu Pro Lys Leu Phe Gly 50
55 60 ggc ttc aac tcc tcg gac acc gtc acc tcc ccg cag agg gcg ggc
gct 240 Gly Phe Asn Ser Ser Asp Thr Val Thr Ser Pro Gln Arg Ala Gly
Ala 65 70 75 80 ctg gca ggt ggg gtg acc acc ttt gtg gcc ctc tat gac
tat gag tca 288 Leu Ala Gly Gly Val Thr Thr Phe Val Ala Leu Tyr Asp
Tyr Glu Ser 85 90 95 cgg aca gag act gac ctg tcc ttc aag aaa ggg
gag cgg ctg cag att 336 Arg Thr Glu Thr Asp Leu Ser Phe Lys Lys Gly
Glu Arg Leu Gln Ile 100 105 110 gtc aat aac acg agg aag gtg gat gtc
aga gag gga gac tgg tgg ctg 384 Val Asn Asn Thr Arg Lys Val Asp Val
Arg Glu Gly Asp Trp Trp Leu 115 120 125 gca cac tcg ctg agc acg gga
cag acc ggt tac atc ccc agc aac tat 432 Ala His Ser Leu Ser Thr Gly
Gln Thr Gly Tyr Ile Pro Ser Asn Tyr 130 135 140 gtg gcg ccc tcc gac
tcc atc cag gct gag gag tgg tac ttt ggc aag 480 Val Ala Pro Ser Asp
Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys 145 150 155 160 atc act
aga cgg gaa tca gag cgg ctg ctg ctc aac gcc gag aac ccg 528 Ile Thr
Arg Arg Glu Ser Glu Arg Leu Leu Leu Asn Ala Glu Asn Pro 165 170 175
aga ggg acc ttc ctc gtg agg gag agt gag acc aca aaa ggt gcc tac 576
Arg Gly Thr Phe Leu Val Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr 180
185 190 tgc ctc tct gta tcc gac ttc gac aat gcc aag ggc cta aat gtg
aaa 624 Cys Leu Ser Val Ser Asp Phe Asp Asn Ala Lys Gly Leu Asn Val
Lys 195 200 205 cac tac aag atc cgc aag ctg gac agc ggc ggt ttc tac
atc acc tcc 672 His Tyr Lys Ile Arg Lys Leu Asp Ser Gly Gly Phe Tyr
Ile Thr Ser 210 215 220 cgc acc cag ttc aac agc ctg cag cag ctc gtg
gct tac tac tcc aaa 720 Arg Thr Gln Phe Asn Ser Leu Gln Gln Leu Val
Ala Tyr Tyr Ser Lys 225 230 235 240 cat gct gat ggc ctg tgt cac cgc
ctc act acc gta tgt ccc aca tcc 768 His Ala Asp Gly Leu Cys His Arg
Leu Thr Thr Val Cys Pro Thr Ser 245 250 255 aag cct cag acc cag gga
ttg gcc aag gat gcg tgg gag atc ccc cgg 816 Lys Pro Gln Thr Gln Gly
Leu Ala Lys Asp Ala Trp Glu Ile Pro Arg 260 265 270 gag tcc ctg cgg
ctg gag gtc aag ctg ggc cag ggt tgc ttc gga gag 864 Glu Ser Leu Arg
Leu Glu Val Lys Leu Gly Gln Gly Cys Phe Gly Glu 275 280 285 gtg tgg
atg ggg acc tgg aac ggc acc acg agg gtt gcc atc aaa act 912 Val Trp
Met Gly Thr Trp Asn Gly Thr Thr Arg Val Ala Ile Lys Thr 290 295 300
ctg aag cca ggc acc atg tcc cca gag gcc ttc ctg cag gag gcc caa 960
Leu Lys Pro Gly Thr Met Ser Pro Glu Ala Phe Leu Gln Glu Ala Gln 305
310 315 320 gtc atg aag aaa ctg agg cac gag aaa ctg gtg cag ctg tat
gct gtg 1008 Val Met Lys Lys Leu Arg His Glu Lys Leu Val Gln Leu
Tyr Ala Val 325 330 335 gtg tcg gaa gaa ccc att tac att gtg aca gag
tac atg aac aag ggg 1056 Val Ser Glu Glu Pro Ile Tyr Ile Val Thr
Glu Tyr Met Asn Lys Gly 340 345 350 agt ctg ctg gac ttt ctc aag ggg
gaa acg ggc aaa tat ttg cgg cta 1104 Ser Leu Leu Asp Phe Leu Lys
Gly Glu Thr Gly Lys Tyr Leu Arg Leu 355 360 365 ccc cag ctg gtg gac
atg tct gct cag atc gct tca ggc atg gcc tat 1152 Pro Gln Leu Val
Asp Met Ser Ala Gln Ile Ala Ser Gly Met Ala Tyr 370 375 380 gtg gag
cgg atg aac tat gtg cac cgg gac ctt cga gcc gcc aat atc 1200 Val
Glu Arg Met Asn Tyr Val His Arg Asp Leu Arg Ala Ala Asn Ile 385 390
395 400 cta gta ggg gag aac ctg gtg tgc aaa gtg gcc gac ttt ggg ttg
gcc 1248 Leu Val Gly Glu Asn Leu Val Cys Lys Val Ala Asp Phe Gly
Leu Ala 405 410 415 cgg ctc ata gaa gac aac gaa tac aca gcc cgg caa
ggt gcc aaa ttc 1296 Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg
Gln Gly Ala Lys Phe 420 425 430 ccc atc aag tgg acc gcc cct gaa gct
gct ctg tac ggc agg ttc acc 1344 Pro Ile Lys Trp Thr Ala Pro Glu
Ala Ala Leu Tyr Gly Arg Phe Thr 435 440 445 atc aag tcg gat gtg tgg
tcc ttt ggg att ctg ctg acc gag ctc acc 1392 Ile Lys Ser Asp Val
Trp Ser Phe Gly Ile Leu Leu Thr Glu Leu Thr 450 455 460 act aag gga
aga gtg ccc tat cct ggg atg gtg aac cgt gag gtt ctg 1440 Thr Lys
Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu Val Leu 465 470 475
480 gac cag gtg gag cgg ggc tac cgg atg cct tgt ccc ccc gag tgc ccc
1488 Asp Gln Val Glu Arg Gly Tyr Arg Met Pro Cys Pro Pro Glu Cys
Pro 485 490 495 gag tcc ctg cat gac ctt atg tgc cag tgc tgg cgg aag
gag ccc gag 1536 Glu Ser Leu His Asp Leu Met Cys Gln Cys Trp Arg
Lys Glu Pro Glu 500 505 510 gag cgg ccc acc ttc gag tac ctg cag gcc
ttc ctg gaa gac tac ttt 1584 Glu Arg Pro Thr Phe Glu Tyr Leu Gln
Ala Phe Leu Glu Asp Tyr Phe 515 520 525 acg tcc act gag cca cag tac
cag ccc ggg gag aac cta tag 1626 Thr Ser Thr Glu Pro Gln Tyr Gln
Pro Gly Glu Asn Leu 530 535 540 11 18 DNA Artificial Sequence PCR
Primer 11 acctcccgca cccagttc 18 12 19 DNA Artificial Sequence PCR
Primer 12 ggccatcagc atgtttgga 19 13 23 DNA Artificial Sequence PCR
Probe 13 agcctgcagc agctcgtggc tta 23 14 20 DNA Artificial Sequence
PCR Primer 14 ggcaaattca acggcacagt 20 15 20 DNA Artificial
Sequence PCR Primer 15 gggtctcgct cctggaagat 20 16 27 DNA
Artificial Sequence PCR Probe 16 aaggccgaga atgggaagct tgtcatc 27
17 164 DNA Homo sapiens 17 gccccgcagg tgcctactgc ctctcagtgt
ctgacttcga caacgccaag ggcctcaacg 60 tgaagcacta caagatccgc
aagctggaca gcggcggctt ctacatcacc tcccgcaccc 120 agttcaacag
cctgcagcag ctggtggcct actactccag tgag 164 18 91 DNA Homo sapiens 18
tctgcccagg gagtttgctg gactttctca agggggagac aggcaagtac ctgcggctgc
60 ctcagctggt ggacatggct gctcaggtga g 91 19 255 DNA Homo sapiens 19
ctgccacagg gatggtgaac cgcgaggtgc tggaccaggt ggagcggggc taccggatgc
60 cctgcccgcc ggagtgtccc gagtccctgc acgacctcat gtgccagtgc
tggcggaagg 120 agcctgagga gcggcccacc ttcgagtacc tgcaggcctt
cctggaggac tacttcacgt 180 ccaccgagcc ccagtaccag cccggggaga
acctctaggc acaggcgggc ccagaccggc 240 ttctcggctt ggatc 255 20 271
DNA Homo sapiens 20 ctgccaggac catgggtagc aacaagagca agcccaagga
tgccagccag cggcgccgca 60 gcctggagcc cgccgagaac gtgcacggcg
ctggcggggg cgctttcccc gcctcgcaga 120 cccccagcaa gccagcctcg
gccgacggcc accgcggccc cagcgcggcc ttcgcccccg 180 cggccgccga
gcccaagctg ttcggaggct tcaactcctc ggacaccgtc acctccccgc 240
agagggcggg cccgctggcc ggtcagtgcg c 271 21 115 DNA Homo sapiens 21
cccccaggtg gtattttggc aagatcacca gacgggagtc agagcggtta ctgctcaatg
60 cagagaaccc gagagggacc ttcctcgtgc gagaaagtga gaccacgaaa ggtac 115
22 156 DNA Homo sapiens 22 aacacgccga tggcctgtgc caccgcctca
ccaccgtgtg ccccacgtcc aagccgcaga 60 ctcagggcct ggccaaggat
gcctgggaga tccctcggga gtcgctgcgg ctggaggtca 120 agctgggcca
gggctgcttt ggcgaggtgt ggatgg 156 23 180 DNA Homo sapiens 23
ggacctggaa cggtaccacc agggtggcca tcaaaaccct gaagcctggc acgatgtctc
60 cagaggcctt cctgcaggag gcccaggtca tgaagaagct gaggcatgag
aagctggtgc 120 agttgtatgc tgtggtttca gaggagccca tttacatcgt
cacggagtac atgagcaagg 180 24 154 DNA Homo sapiens 24 atcgcctcag
gcatggcgta cgtggagcgg atgaactacg tccaccggga ccttcgtgca 60
gccaacatcc tggtgggaga gaacctggtg tgcaaagtgg ccgactttgg gctggctcgg
120 ctcattgaag acaatgagta cacggcgcgg caag 154 25 132 DNA Homo
sapiens 25 gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc
cgcttcacca 60 tcaagtcgga cgtgtggtcc ttcgggatcc tgctgactga
gctcaccaca aagggacggg 120 tgccctaccc tg 132 26 464 DNA Homo sapiens
26 atcctcacac tggtcagttc caaccttccc aggaatcttc tgtggccatg
ttcactccgg 60 ttttacagaa cagagaacag aagctcagag aagtgaagca
acttgcccag ctatgagaga 120 cagagccagg atttgaaacc agatgaggac
gctgaggccc agagagggaa agccacttgc 180 ctagggacac acagcgggga
gaggtggagc agggcctcta tttcgagacc cctgactcca 240 cacctggtgt
ttgtgccaag accccaggct gcctcccagg tcctctggga cagcccctgc 300
cttctaccag gaccatgggt agcaacaaga gcaagcccaa ggatgccagc cagcggcgcc
360 gcagcctgga gcccgccgag aacgtgcacg gcgctggcgg gggcgctttc
cccgcctcgc 420 agacccccag caagccagcc tcggccgacg accaccgcgg cccc 464
27 459 DNA Homo sapiens 27 cgtcccgcgg tccgccctcc cgtgcgtccg
tctgccggtg agcccgcccg cccgccggcc 60 cagaacagag aacagaagct
cagagaagtg aagcaacttg cccagctatg agagacagag 120 ccaggatttg
aaaccagatg aggacgctga ggcccagaga gggaaagcca cttgcctagg 180
gacacacagc ggggagaggt ggagcagggc ctctatttcg agacccctga ctccacacct
240 ggtgtttgtg ccaagacccc aggctgcctc ccaggtcctc tgggacagcc
cctgccttct 300 accaggacca tgggtagcaa caagagcaag cccaaggatg
ccagccagcg
gcgccgcagc 360 ctggagcccg ccgagaacgt gcacggcgct ggcgggggcg
ctttccccgc ctcgcagacc 420 cccagcaagc cagcctcgac cgacggccac
cgcggcccc 459 28 336 DNA Homo sapiens 28 tcctcctcct ccctttctct
ctcgatctgt ctctcccggc ccggaatcca ttccggcctg 60 ggagccggag
cggccaggcc gccgtctgcc cgtcccgctg gacgtcccgc ggtccgccct 120
cccgtgcgtc cgtctgccgg tgagcccgcc cgcccgccgg cccagcccct gccttctacc
180 aggaccatgg gtagcaacaa gagcaagccc aaggatgcca gccagcggcg
ccgcagcctg 240 gagcccgccg agaacgtgca cggcgctggc gggggcgctt
tccccgcctc gcagaccccc 300 agcaagccag cctcggccga cggccaccgc ggcccc
336 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29
ttgctcttgt tgctacccat 20 30 20 DNA Artificial Sequence Antisense
Oligonucleotide 30 ggcttgctct tgttgctacc 20 31 20 DNA Artificial
Sequence Antisense Oligonucleotide 31 ggttggaact gaccagtgtg 20 32
20 DNA Artificial Sequence Antisense Oligonucleotide 32 atcgagagag
aaagggagga 20 33 20 DNA Artificial Sequence Antisense
Oligonucleotide 33 agatcgagag agaaagggag 20 34 20 DNA Artificial
Sequence Antisense Oligonucleotide 34 ggaaggttgg aactgaccag 20 35
20 DNA Artificial Sequence Antisense Oligonucleotide 35 tggctggcat
ccttgggctt 20 36 20 DNA Artificial Sequence Antisense
Oligonucleotide 36 cgctggctgg catccttggg 20 37 20 DNA Artificial
Sequence Antisense Oligonucleotide 37 gggctcaccg gcagacggac 20 38
20 DNA Artificial Sequence Antisense Oligonucleotide 38 cggctcccag
gccggaatgg 20 39 20 DNA Artificial Sequence Antisense
Oligonucleotide 39 agcgccgtgc acgttctcgg 20 40 20 DNA Artificial
Sequence Antisense Oligonucleotide 40 gcaagttgct tcacttctct 20 41
20 DNA Artificial Sequence Antisense Oligonucleotide 41 gtggccgtcg
gccgaggctg 20 42 20 DNA Artificial Sequence Antisense
Oligonucleotide 42 aggcaggggc tgggccggcg 20 43 20 DNA Artificial
Sequence Antisense Oligonucleotide 43 gcctccgaac agcttgggct 20 44
20 DNA Artificial Sequence Antisense Oligonucleotide 44 gaagcctccg
aacagcttgg 20 45 20 DNA Artificial Sequence Antisense
Oligonucleotide 45 cgaggagttg aagcctccga 20 46 20 DNA Artificial
Sequence Antisense Oligonucleotide 46 gtgtccgagg agttgaagcc 20 47
20 DNA Artificial Sequence Antisense Oligonucleotide 47 gacggtgtcc
gaggagttga 20 48 20 DNA Artificial Sequence Antisense
Oligonucleotide 48 gcctgtgcct agaggttctc 20 49 20 DNA Artificial
Sequence Antisense Oligonucleotide 49 caccaggtgt ggagtcaggg 20 50
20 DNA Artificial Sequence Antisense Oligonucleotide 50 ggcacaaaca
ccaggtgtgg 20 51 20 DNA Artificial Sequence Antisense
Oligonucleotide 51 caaaggtggt cactccaccg 20 52 20 DNA Artificial
Sequence Antisense Oligonucleotide 52 agagggccac aaaggtggtc 20 53
20 DNA Artificial Sequence Antisense Oligonucleotide 53 tcctagactc
atagtcatag 20 54 20 DNA Artificial Sequence Antisense
Oligonucleotide 54 ctggagccgc tcgcctttct 20 55 20 DNA Artificial
Sequence Antisense Oligonucleotide 55 agccaccagt ctccctctgt 20 56
20 DNA Artificial Sequence Antisense Oligonucleotide 56 tgctgagcga
gtgggccagc 20 57 20 DNA Artificial Sequence Antisense
Oligonucleotide 57 ctgtctgtcc tgtgctgagc 20 58 20 DNA Artificial
Sequence Antisense Oligonucleotide 58 tcagcctgga tggagtcgga 20 59
20 DNA Artificial Sequence Antisense Oligonucleotide 59 cgctctgact
cccgtctggt 20 60 20 DNA Artificial Sequence Antisense
Oligonucleotide 60 cagtaaccgc tctgactccc 20 61 20 DNA Artificial
Sequence Antisense Oligonucleotide 61 cctctcgggt tctctgcatt 20 62
20 DNA Artificial Sequence Antisense Oligonucleotide 62 aaggtccctc
tcgggttctc 20 63 20 DNA Artificial Sequence Antisense
Oligonucleotide 63 ggaaggtccc tctcgggttc 20 64 20 DNA Artificial
Sequence Antisense Oligonucleotide 64 ctttctcgca cgaggaaggt 20 65
20 DNA Artificial Sequence Antisense Oligonucleotide 65 tgtcgaagtc
agacactgag 20 66 20 DNA Artificial Sequence Antisense
Oligonucleotide 66 ttcacgttga ggcccttggc 20 67 20 DNA Artificial
Sequence Antisense Oligonucleotide 67 aggctgttga actgggtgcg 20 68
20 DNA Artificial Sequence Antisense Oligonucleotide 68 gcaggctgtt
gaactgggtg 20 69 20 DNA Artificial Sequence Antisense
Oligonucleotide 69 gctgctgcag gctgttgaac 20 70 20 DNA Artificial
Sequence Antisense Oligonucleotide 70 caccagctgc tgcaggctgt 20 71
20 DNA Artificial Sequence Antisense Oligonucleotide 71 gtttggagta
gtaggccacc 20 72 20 DNA Artificial Sequence Antisense
Oligonucleotide 72 ggcggtggca caggccatcg 20 73 20 DNA Artificial
Sequence Antisense Oligonucleotide 73 gagtctgcgg cttggacgtg 20 74
20 DNA Artificial Sequence Antisense Oligonucleotide 74 tgagtctgcg
gcttggacgt 20 75 20 DNA Artificial Sequence Antisense
Oligonucleotide 75 gccctgagtc tgcggcttgg 20 76 20 DNA Artificial
Sequence Antisense Oligonucleotide 76 gcagcgactc ccgagggatc 20 77
20 DNA Artificial Sequence Antisense Oligonucleotide 77 cagcttgacc
tccagccgca 20 78 20 DNA Artificial Sequence Antisense
Oligonucleotide 78 ctggcccagc ttgacctcca 20 79 20 DNA Artificial
Sequence Antisense Oligonucleotide 79 ggtaccgttc caggtcccca 20 80
20 DNA Artificial Sequence Antisense Oligonucleotide 80 atggccaccc
tggtggtacc 20 81 20 DNA Artificial Sequence Antisense
Oligonucleotide 81 tgatggccac cctggtggta 20 82 20 DNA Artificial
Sequence Antisense Oligonucleotide 82 gcttcagggt tttgatggcc 20 83
20 DNA Artificial Sequence Antisense Oligonucleotide 83 aaatgggctc
ctctgaaacc 20 84 20 DNA Artificial Sequence Antisense
Oligonucleotide 84 gagaaagtcc agcaaactcc 20 85 20 DNA Artificial
Sequence Antisense Oligonucleotide 85 tctccccctt gagaaagtcc 20 86
20 DNA Artificial Sequence Antisense Oligonucleotide 86 ttgcctgtct
cccccttgag 20 87 20 DNA Artificial Sequence Antisense
Oligonucleotide 87 ggcagccgca ggtacttgcc 20 88 20 DNA Artificial
Sequence Antisense Oligonucleotide 88 ggacgtagtt catccgctcc 20 89
20 DNA Artificial Sequence Antisense Oligonucleotide 89 caggttctct
cccaccagga 20 90 20 DNA Artificial Sequence Antisense
Oligonucleotide 90 ccaggttctc tcccaccagg 20 91 20 DNA Artificial
Sequence Antisense Oligonucleotide 91 cactttgcac accaggttct 20 92
20 DNA Artificial Sequence Antisense Oligonucleotide 92 gtcggccact
ttgcacacca 20 93 20 DNA Artificial Sequence Antisense
Oligonucleotide 93 cccaaagtcg gccactttgc 20 94 20 DNA Artificial
Sequence Antisense Oligonucleotide 94 gcggccatag agggcagctt 20 95
20 DNA Artificial Sequence Antisense Oligonucleotide 95 agtcagcagg
atcccgaagg 20 96 20 DNA Artificial Sequence Antisense
Oligonucleotide 96 acccgtccct ttgtggtgag 20 97 20 DNA Artificial
Sequence Antisense Oligonucleotide 97 ctggtccagc acctcgcggt 20 98
20 DNA Artificial Sequence Antisense Oligonucleotide 98 cagcactggc
acatgaggtc 20 99 20 DNA Artificial Sequence Antisense
Oligonucleotide 99 gccagcactg gcacatgagg 20 100 20 DNA Artificial
Sequence Antisense Oligonucleotide 100 ttccgccagc actggcacat 20 101
20 DNA Artificial Sequence Antisense Oligonucleotide 101 ggctccttcc
gccagcactg 20 102 20 DNA Artificial Sequence Antisense
Oligonucleotide 102 ggccgctcct caggctcctt 20 103 20 DNA Artificial
Sequence Antisense Oligonucleotide 103 gggccgctcc tcaggctcct 20 104
20 DNA Artificial Sequence Antisense Oligonucleotide 104 actcgaaggt
gggccgctcc 20 105 20 DNA Artificial Sequence Antisense
Oligonucleotide 105 ctagaggttc tccccgggct 20 106 493 DNA Mus
musculus unsure 205 unknown 106 acgcgtccgg caacaagagc aagcccaagg
acgccagcca gcggcgccgc agcctggagc 60 cctcggaaaa cgtgcacggg
gcagggggcg ccttcccggc ctcacagaca ccgagcaagc 120 ccgcctccgc
cgacggccac cgcgggccca gcgccgcctt cgtgccgccc gcggccgagc 180
ccaagctctt cggaggcttc aactnctcgg acaccgtcac ctccccgcag agggcggggc
240 ctctggcagg tggggtgacc acctttgtgg ccctctatga ctatgagtca
cggacagaga 300 ctgacctgtc cttcaagaaa ggggagcggc tgcagattgt
caataacaca gagggagact 360 ggtggctggc acactcgctg agcacgggac
agaccggtta catccncagc aactatgtgg 420 cgccctccga ctccatccag
gctgaggagt ggtactttgg caggatcact agacgggaat 480 cagagcggct gct 493
107 20 DNA Artificial Sequence Antisense Oligonucleotide 107
ttgctcttgt tgctgcccat 20 108 20 DNA Artificial Sequence Antisense
Oligonucleotide 108 tccgagggct ccaggctgcg 20 109 20 DNA Artificial
Sequence Antisense Oligonucleotide 109 tccgaagagc ttgggctcgg 20 110
20 DNA Artificial Sequence Antisense Oligonucleotide 110 agcctccgaa
gagcttgggc 20 111 20 DNA Artificial Sequence Antisense
Oligonucleotide 111 gttgaagcct ccgaagagct 20 112 20 DNA Artificial
Sequence Antisense Oligonucleotide 112 gagttgaagc ctccgaagag 20 113
20 DNA Artificial Sequence Antisense Oligonucleotide 113 tccgaggagt
tgaagcctcc 20 114 20 DNA Artificial Sequence Antisense
Oligonucleotide 114 agtctctgtc cgtgactcat 20 115 20 DNA Artificial
Sequence Antisense Oligonucleotide 115 aggacaggtc agtctctgtc 20 116
20 DNA Artificial Sequence Antisense Oligonucleotide 116 aaggacaggt
cagtctctgt 20 117 20 DNA Artificial Sequence Antisense
Oligonucleotide 117 cgctcccctt tcttgaagga 20 118 20 DNA Artificial
Sequence Antisense Oligonucleotide 118 cagccgctcc cctttcttga 20 119
20 DNA Artificial Sequence Antisense Oligonucleotide 119 ttgacaatct
gcagccgctc 20 120 20 DNA Artificial Sequence Antisense
Oligonucleotide 120 cgtgttattg acaatctgca 20 121 20 DNA Artificial
Sequence Antisense Oligonucleotide 121 acatccacct tcctcgtgtt 20 122
20 DNA Artificial Sequence Antisense Oligonucleotide 122 gagtgtgcca
gccaccagtc 20 123 20 DNA Artificial Sequence Antisense
Oligonucleotide 123 gctcagcgag tgtgccagcc 20 124 20 DNA Artificial
Sequence Antisense Oligonucleotide 124 tgctcagcga gtgtgccagc 20 125
20 DNA Artificial Sequence Antisense Oligonucleotide 125 cgtgctcagc
gagtgtgcca 20 126 20 DNA Artificial Sequence Antisense
Oligonucleotide 126 tcccgtgctc agcgagtgtg 20 127 20 DNA Artificial
Sequence Antisense Oligonucleotide 127 cggtctgtcc cgtgctcagc 20 128
20 DNA Artificial Sequence Antisense Oligonucleotide 128 gtaaccggtc
tgtcccgtgc 20 129 20 DNA Artificial Sequence Antisense
Oligonucleotide 129 gatgtaaccg gtctgtcccg 20 130 20 DNA Artificial
Sequence Antisense Oligonucleotide 130 ccgtctagtg atcttgccaa 20 131
20 DNA Artificial Sequence Antisense Oligonucleotide 131 ctctgattcc
cgtctagtga 20 132 20 DNA Artificial Sequence Antisense
Oligonucleotide 132 agccgctctg attcccgtct 20 133 20 DNA Artificial
Sequence Antisense Oligonucleotide 133 cctcacgagg aaggtccctc 20 134
20 DNA Artificial Sequence Antisense Oligonucleotide 134 ggtctcactc
tccctcacga 20 135 20 DNA Artificial Sequence Antisense
Oligonucleotide 135 atacagagag gcagtaggca 20 136 20 DNA Artificial
Sequence Antisense Oligonucleotide 136 gtcggataca gagaggcagt 20 137
20 DNA Artificial Sequence Antisense Oligonucleotide 137 ttgtcgaagt
cggatacaga 20 138 20 DNA Artificial Sequence Antisense
Oligonucleotide 138 tttaggccct tggcattgtc 20 139 20 DNA Artificial
Sequence Antisense Oligonucleotide 139 atttaggccc ttggcattgt 20 140
20 DNA Artificial Sequence Antisense Oligonucleotide 140 gtgtttcaca
tttaggccct 20 141 20 DNA Artificial Sequence Antisense
Oligonucleotide 141 aggtgatgta gaaaccgccg 20 142 20 DNA Artificial
Sequence Antisense Oligonucleotide 142 gccacgagct gctgcaggct 20 143
20 DNA Artificial Sequence Antisense Oligonucleotide 143 atgtttggag
tagtaagcca 20 144 20 DNA Artificial Sequence Antisense
Oligonucleotide 144 aggccatcag catgtttgga 20 145 20 DNA Artificial
Sequence Antisense Oligonucleotide 145 cggtgacaca ggccatcagc 20 146
20 DNA Artificial Sequence Antisense Oligonucleotide 146 tgaggcttgg
atgtgggaca
20 147 20 DNA Artificial Sequence Antisense Oligonucleotide 147
ccctgggtct gaggcttgga 20 148 20 DNA Artificial Sequence Antisense
Oligonucleotide 148 acctccagcc gcagggactc 20 149 20 DNA Artificial
Sequence Antisense Oligonucleotide 149 gcaaccctgg cccagcttga 20 150
20 DNA Artificial Sequence Antisense Oligonucleotide 150 acctctccga
agcaaccctg 20 151 20 DNA Artificial Sequence Antisense
Oligonucleotide 151 cgtggtgccg ttccaggtcc 20 152 20 DNA Artificial
Sequence Antisense Oligonucleotide 152 atggcaaccc tcgtggtgcc 20 153
20 DNA Artificial Sequence Antisense Oligonucleotide 153 tgatggcaac
cctcgtggtg 20 154 20 DNA Artificial Sequence Antisense
Oligonucleotide 154 tcagtttctt catgacttgg 20 155 20 DNA Artificial
Sequence Antisense Oligonucleotide 155 ccttgttcat gtactctgtc 20 156
20 DNA Artificial Sequence Antisense Oligonucleotide 156 gcagactccc
cttgttcatg 20 157 20 DNA Artificial Sequence Antisense
Oligonucleotide 157 cagcagactc cccttgttca 20 158 20 DNA Artificial
Sequence Antisense Oligonucleotide 158 cgtttccccc ttgagaaagt 20 159
20 DNA Artificial Sequence Antisense Oligonucleotide 159 tatttgcccg
tttccccctt 20 160 20 DNA Artificial Sequence Antisense
Oligonucleotide 160 tcatccgctc cacataggcc 20 161 20 DNA Artificial
Sequence Antisense Oligonucleotide 161 ccggtgcaca tagttcatcc 20 162
20 DNA Artificial Sequence Antisense Oligonucleotide 162 tctcccctac
taggatattg 20 163 20 DNA Artificial Sequence Antisense
Oligonucleotide 163 ggccaaccca aagtcggcca 20 164 20 DNA Artificial
Sequence Antisense Oligonucleotide 164 ttgccgggct gtgtattcgt 20 165
20 DNA Artificial Sequence Antisense Oligonucleotide 165 gacaaggcat
ccggtagccc 20 166 20 DNA Artificial Sequence Antisense
Oligonucleotide 166 ctggcacata aggtcatgca 20 167 20 DNA Artificial
Sequence Antisense Oligonucleotide 167 tccttccgcc agcactggca 20 168
20 DNA Artificial Sequence Antisense Oligonucleotide 168 ctataggttc
tccccgggct 20 169 20 DNA Artificial Sequence Antisense
Oligonucleotide 169 tctccctctg tgttattgac 20
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