U.S. patent application number 10/271887 was filed with the patent office on 2003-05-08 for antisense modulation of caspase 9 expression.
Invention is credited to Watt, Andrew T., Zhang, Hong.
Application Number | 20030087871 10/271887 |
Document ID | / |
Family ID | 24647062 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087871 |
Kind Code |
A1 |
Zhang, Hong ; et
al. |
May 8, 2003 |
Antisense modulation of caspase 9 expression
Abstract
Antisense compounds, compositions and methods are provided for
modulating the expression of caspase 9. The compositions comprise
antisense compounds, particularly antisense oligonucleotides,
targeted to nucleic acids encoding caspase 9. Methods of using
these compounds for modulation of caspase 9 expression and for
treatment of diseases associated with expression of caspase 9 are
provided.
Inventors: |
Zhang, Hong; (Carlsbad,
CA) ; Watt, Andrew T.; (Vista, CA) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
24647062 |
Appl. No.: |
10/271887 |
Filed: |
October 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10271887 |
Oct 15, 2002 |
|
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09659845 |
Sep 11, 2000 |
|
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6492170 |
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Current U.S.
Class: |
514/44A ;
435/375; 536/23.2 |
Current CPC
Class: |
C12N 2310/346 20130101;
C12N 2310/3525 20130101; A61K 48/00 20130101; C12Y 304/22062
20130101; A61K 38/00 20130101; C12N 2310/321 20130101; C12N 15/1137
20130101; C12N 2310/341 20130101; C12N 2310/321 20130101; C12N
2310/3341 20130101; Y02P 20/582 20151101; C12N 2310/315
20130101 |
Class at
Publication: |
514/44 ; 435/375;
536/23.2 |
International
Class: |
A61K 048/00; C07H
021/04 |
Claims
What is claimed is:
1. A compound 8 to 50 nucleobases in length targeted to a nucleic
acid molecule encoding caspase 9, wherein said compound
specifically hybridizes with and inhibits the expression of caspase
9.
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: 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68,
70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 83, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 101, 102, 103, 105, 106,
108, 109, 110, 111, 112, 113, 114, 116, 117, 118, 122, 124, 125,
126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 139, 140,
141, 143, 144, 145, 146, 147, 150, 152, 154, 155, 156, 157, 158,
159, 161, 162, 163, 164, 165, 166, 168, 169, 170, 171, 172, 173 or
174.
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 caspase 9.
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 caspase 9 in cells or
tissues comprising contacting said cells or tissues with the
compound of claim 1 so that expression of caspase 9 is
inhibited.
16. A method of treating an animal having a disease or condition
associated with caspase 9 comprising administering to said animal a
therapeutically or prophylactically effective amount of the
compound of claim 1 so that expression of caspase 9 is
inhibited.
17. The method of claim 16 wherein the disease or condition is
stroke, brain injury or a neurodegenerative disease.
18. The method of claim 16 wherein the disease or condition is a
hyperproliferative disorder.
19. The method of claim 16 wherein the disease or condition is a
haematopoetic disorder.
20. The method of claim 16 wherein the disease or condition is a
bone metabolism or cholesterol disorder.
Description
INTRODUCTION
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/659,845 filed Sep. 11, 2000.
FIELD OF THE INVENTION
[0002] The present invention provides compositions and methods for
modulating the expression of caspase 9. In particular, this
invention relates to compounds, particularly oligonucleotides,
specifically hybridizable with nucleic acids encoding caspase 9.
Such compounds have been shown to modulate the expression of
caspase 9.
BACKGROUND OF THE INVENTION
[0003] Apoptosis, or programmed cell death, is a naturally
occurring process that has been strongly conserved during evolution
to prevent uncontrolled cell proliferation. This form of cell
suicide plays a crucial role in ensuring the development and
maintenance of multicellular organisms by eliminating superfluous
or unwanted cells. However, if this process goes awry becoming
overstimulated, cell loss and degenerative disorders including
neurological disorders such as Alzheimers, Parkinsons, ALS,
retinitis pigmentosa and blood cell disorders can result. Stimuli
which can trigger apoptosis include growth factors such as tumor
necrosis factor (TNF), Fas and transforming growth factor beta
(TGF.beta.), neurotransmitters, growth factor withdrawal, loss of
extracellular matrix attachment and extreme fluctuations in
intracellular calcium levels (Afford and Randhawa, Mol. Pathol.,
2000, 53, 55-63).
[0004] Alternatively, insufficient apoptosis, triggered by growth
factors, extracellular matrix changes, CD40 ligand, viral gene
products neutral amino acids, zinc, estrogen and androgens, can
contribute to the development of cancer, autoimmune disorders and
viral infections (Afford and Randhawa, Mol. Pathol., 2000, 53,
55-63). Consequently, apoptosis is regulated under normal
circumstances by the interaction of gene products that either
induce or inhibit cell death and several gene products which
modulate the apoptotic process have now been identified.
[0005] The most well-characterized apoptotic signaling cascade to
date is that orchestrated by a family of cysteine proteases known
as caspases. These enzymes activate apoptosis through proteolytic
events triggered by one of several described mechanisms; including
ligand binding to the cell surface death receptors of either the
TNF or NGF receptor families, changes in mitochondrial integrity or
chemical induction (Thornberry, Br. Med. Bull., 1997, 53,
478-490).
[0006] Caspases have been classified into two groups, initiator
caspases and effector caspases, based upon their position in the
apoptotic signaling pathway.
[0007] Initiator caspases include caspase 1, 2, 4, 5, 8, 9, 10 and
14 and these enzymes have the largest prodomains of all the caspase
zymogens. These prodomains allow the initiator caspases to interact
with other downstream substrates including other caspases.
Initiator caspases are further divided into two groups based on
their protein binding domains. Caspases 8 and 10 contain the DED
(death effector domain) while caspases 1, 2, 4 and 9 contain the
CARD (caspase recruitment domain) (Bratton et al., Exp. Cell. Res.,
2000, 256, 27-33; Garcia-Calvo et al., Cell. Death Differ., 1999,
6, 362-369).
[0008] Effector caspases are activated by initiator caspases and
include caspase 3, 6, 7, 11 and 13 and these contain a shorter
prodomain. Once activated, effector caspases then cleave a number
of structural and regulatory proteins within the cell (Bratton et
al., Exp. Cell. Res., 2000, 256, 27-33; Garcia-Calvo et al., Cell.
Death Differ., 1999, 6, 362-369).
[0009] Caspase 9 (also known as CASP9, MCH6, APAF3, ICE-LAP6 or
ICE9) is a ubiquitously expressed initiator caspase which has been
shown to be the most upstream caspase in the apoptotic cascade and
to induce apoptosis in breast carcinoma cells when overexpressed
(Duan et al., J. Biol. Chem., 1996, 271, 16720-16724; Kuida, Int.
J. Biochem. Cell Biol., 2000, 32, 121-124; Li et al., Cell, 1997,
91, 479-489). Caspase 9 exists as two isoforms in both humans and
mice and the shorter of the isoforms has been shown to act as a
dominant-negative of the longer form in vitro by blocking
protein-protein interactions with the caspase 9 adaptor molecule,
Apaf-1 (Fujita et al., Biochem. Biophys. Res. Commun., 1999, 264,
550-555; Seol and Billiar, J. Biol. Chem., 1999, 274, 2072-2076;
Srinivasula et al., Cancer Res., 1999, 59, 999-1002).
[0010] Activation of the caspase 9 zymogen occurs upon
mitochondrial release of cytochrome c subsequent to triggers of
cell death followed by complex formation with Apaf-1 forming the
apoptosome. Proteolytic cleavage of the caspase 9 zymogen then
results in release of the mature caspase 9 from the apoptosome and
initiates the apoptotic cascade (Saleh et al., J. Biol. Chem.,
1999, 274, 17941-17945).
[0011] Disclosed in U.S. Pat. No. 6,010,878 are the polypeptide and
polynucleotide sequences of human caspase 9 as well as an
expression vector and host cells for the expression of said vector
(Dixit et al., 2000).
[0012] Caspase 9 is required for normal brain development and
mediates apoptosis induced by chemotherapeutic agents (Hakem et
al., Cell, 1998, 94, 339-352; Kuida, Int. J. Biochem. Cell Biol.,
2000, 32, 121-124) and oncogenic transformation (Fearnhead et al.,
Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 13664-13669; Kuwahara et
al., Cancer Lett., 2000, 148, 65-71; Soengas et al., Science, 1999,
284, 156-159; Wang et al., Eur. J. Cancer, 1999, 35, 1517-1525;
Zhuang and Cohen, Toxicol. Lett., 1998, 102-103, 121-129).
[0013] Caspase 9 also plays a role in ischemic recovery (Krajewski
et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 5752-5757) and
thymocyte apoptosis induced by sepsis (Tinsley et al., Shock, 2000,
13, 1-7). These data suggest that modulation of caspase 9 would
render opportunity to treat patients with disorders such as cancer,
stroke, brain injury or neurodegenerative diseases.
[0014] It is currently believed that modulation of caspase
expression represents a potential therapeutic target in a variety
of deregulated apoptotic pathologic conditions. Several types of
broad-spectrum caspase inhbitors have been identified for the
treatment of deregulated bone metabolism (Harada et al., 2000;
Reszka, 1999), as immunomodulatory agents (Gunasekera et al., 2000)
and as combination therapies for the regulation of blood
cholesterol (Reszka, 1999).
[0015] These non-specific caspase inhibitors fall into three main
classes; peptide-based molecules that mimic caspase substrates,
small molecules and naturally-occurring caspase inhibitors or
decoys (Deveraux et al., Embo J., 1998, 17, 2215-2223; Dong et al.,
Biochem. J., 2000, 347 Pt 3, 669-677; Gunasekera et al., 2000;
Harada et al., 2000; Reszka, 1999; Reszka, 1999; Robidoux et al.,
2000; Spruce et al., 1999).
[0016] Currently, however, there are no known therapeutic agents
which effectively inhibit the synthesis of caspase 9 and to date,
strategies aimed at modulating caspase 9 function have involved the
use of antibodies and molecules that block upstream entities such
as the death receptors, broad-spectrum caspase inhibitors or
targeted gene knockouts in mice. Mice lacking caspase 9 die
perinatally with a markedly enlarged and malformed cerebrum caused
by reduced apoptosis in the brain and embryonic stem cells lacking
caspase 9 show resistance to several apoptotic stimuli (Hakem et
al., Cell, 1998, 94, 339-352; Kuida et al., Cell, 1998, 94,
325-337).
[0017] There exists, therefore, a need to identify methods of
modulating apoptosis for the therapeutic treatment of human
diseases.
[0018] Antisense technology is emerging as an effective means for
reducing the expression of specific gene products and may therefore
prove to be uniquely useful in a number of therapeutic, diagnostic,
and research applications for the modulation of gene expression and
cellular processes.
[0019] The present invention satisfies this need and provides
compositions and methods for modulating caspase 9 expression,
including modulation of aberrant forms of caspase 9, including
mutated and alternatively spliced forms.
SUMMARY OF THE INVENTION
[0020] The present invention is directed to compounds, particularly
antisense oligonucleotides, which are targeted to a nucleic acid
encoding caspase 9, and which modulate the expression of caspase 9.
Pharmaceutical and other compositions comprising the compounds of
the invention are also provided. Further provided are methods of
modulating the expression of caspase 9 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 caspase 9 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
[0021] The present invention employs oligomeric compounds,
particularly antisense oligonucleotides, for use in modulating the
function of nucleic acid molecules encoding caspase 9, ultimately
modulating the amount of caspase 9 produced. This is accomplished
by providing antisense compounds which specifically hybridize with
one or more nucleic acids encoding caspase 9. As used herein, the
terms "target nucleic acid" and "nucleic acid encoding caspase 9"
encompass DNA encoding caspase 9, 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 caspase 9. 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.
[0022] 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 caspase 9. 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
caspase 9, regardless of the sequence(s) of such codons.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,
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 abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0036] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S.: 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.sub.1 to C.sub.10 alkyl or C.sub.2
to C.sub.10 alkenyl and alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.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.
[0042] A further prefered 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 3' or 4' carbon atom
wherein n is 1 or 2. LNAs and preparation thereof are described in
WO 98/39352 and WO 99/14226.
[0043] 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.dbd.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.
[0044] 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]benzoxazi- n-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.
[0045] 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.
[0046] 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 inter-calators,
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 triethylammonium 1,2-di-O-hexadecyl-rac-glyc-
ero-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,
indo-methicin, 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.
[0047] 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.
[0048] 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.
[0049] "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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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 Dec. 9, 1993 or in WO 94/26764 and U.S.
Pat. No. 5,770,713 to Imbach et al.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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 caspase 9 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.
[0060] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding caspase 9, 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 caspase 9 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 caspase 9 in a sample may also be
prepared.
[0061] 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.
[0062] 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.
[0063] 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. application
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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] Emulsions
[0070] 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.
[0071] 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).
[0072] 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).
[0073] 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.
[0074] 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).
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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).
[0079] 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.
[0080] 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 (SO750),
decaglycerol decaoleate (DAO750), 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 (C.sub.8-C.sub.12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized
C.sub.8-C.sub.10 glycerides, vegetable oils and silicone oil.
[0081] 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.
[0082] 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.
[0083] Liposomes
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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).
[0092] 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).
[0093] 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.
[0094] 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).
[0095] 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).
[0096] 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). 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
G.sub.M1, 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. No. 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-dimyristoylphosphatidylcholine are disclosed in WO 97/13499
(Lim et al.).
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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).
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0106] Penetration Enhancers
[0107] 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.
[0108] 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.
[0109] 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).
[0110] 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).
[0111] 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).
[0112] 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).
[0113] 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).
[0114] 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.
[0115] 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.
[0116] Carriers
[0117] 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).
[0118] Excipients
[0119] 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.).
[0120] 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.
[0121] 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.
[0122] 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.
[0123] Other Components
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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
[0130] Nucleoside Phosphoramidites for Oligonucleotide Synthesis
Deoxy and 2'-alkoxy Amidites
[0131] 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.
[0132] 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.).
[0133] 2'-Fluoro Amidites
[0134] 2'-Fluorodeoxyadenosine Amidites
[0135] 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
5'-DMT-3'-phosphoramidite intermediates.
[0136] 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.
[0138] 2'-Fluorouridine
[0139] 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.
[0140] 2'-Fluorodeoxycytidine
[0141] 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.
[0142] 2'-O-(2-Methoxyethyl) Modified Amidites
[0143] 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.
[0144]
2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]
[0145] 5-Methyluridine (ribosylthymine, commercially available
through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate
(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.).
[0146] 2'-O-Methoxyethyl-5-methyluridine
[0147] 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.
[0148] 2'-O-Methoxyethyl-51-O-dimethoxytrityl-5-methyluridine
[0149] 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%).
[0150]
3'-O-Acetyl-21-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0151] 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.
[0152]
31-O-Acetyl-21-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triaz-
oleuridine
[0153] 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.
[0154] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0155] A solution of
3.sup.1-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrit-
yl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL)
and NH.sub.4OH (30 mL) was stirred at room temperature for 2 hours.
The dioxane solution was evaporated and the residue azeotroped with
MeOH (2.times.200 mL). The residue was dissolved in MeOH (300 mL)
and transferred to a 2 liter stainless steel pressure vessel. MeOH
(400 mL) saturated with NH.sub.3 gas was added and the vessel
heated to 100.degree. C. for 2 hours (TLC showed complete
conversion). The vessel contents were evaporated to dryness and the
residue was dissolved in EtOAc (500 mL) and washed once with
saturated NaCl (200 mL). The organics were dried over sodium
sulfate and the solvent was evaporated to give 85 g (95%) of the
title compound.
[0156]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0157] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyl-cytidine (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.
[0158]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine--
3'-amidite
[0159]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
(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.
[0160] 2'-O-(Aminooxyethyl) Nucleoside Amidites and
2'-O-(dimethylaminooxyethyl)nucleoside Amidites
[0161] 2'-(Dimethylaminooxyethoxy) Nucleoside Amidites
[0162] 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.
[0163]
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
[0164] 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 -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.
[0165]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
[0166] 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 (84 g, 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.
[0167]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridi-
ne
[0168]
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%).
[0169]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine
[0170]
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-methyluri-
dine
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methy-
luridine (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.2c1.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-dimethylam-
inooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).
[0171] 2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0172] 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%).
[0173] 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0174] 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%).
[0175]
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2--
cyanoethyl)-N,N-diisopropylphosphoramidite]
[0176] 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 P.sub.2OS 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.sup.1,N.sup.1-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-dim-
ethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite] as a foam (1.04 g, 74.9%).
[0177] 2'-(Aminooxyethoxy) Nucleoside Amidites
[0178] 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.
[0179]
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-
-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidi-
te]
[0180] 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 Al 940203.) Standard protection procedures should afford
2.sup.1-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanos- ine
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-ethylacetyl)-5'-O-(4,4'-dime-
thoxytrityl)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-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[-
(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
[0181] 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) Nucleoside
Amidites
[0182] 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.2, or 2'-DMAEOE
nucleoside amidites) are prepared as follows. Other nucleoside
amidites are prepared similarly.
[0183] 2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl
Uridine
[0184] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol)
is slowly added to a solution of borane in tetra-hydrofuran (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.
[0185]
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-m-
ethyl Uridine
[0186] 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.2,Cl.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.
[0187]
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-me-
thyl uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite
[0188] 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-dimethylaminoethoxy)ethyl)]-5-methylur-
idine (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
[0189] Oligonucleotide Synthesis
[0190] Unsubstituted and substituted phosphodiester (P.dbd.O)
oligonucleotides are synthesized on an automated DNA synthesizer
(Applied Biosystems model 380B) using standard phosphoramidite
chemistry with oxidation by iodine.
[0191] 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.
[0192] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0197] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0198] 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
[0199] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides, methylenedimethylhydrazo
linked oligonucleosides, also identified as MDH linked
oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, 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.
[0200] 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.
[0201] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 4
[0202] PNA Synthesis
[0203] 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
[0204] Synthesis of Chimeric Oligonucleotides
[0205] 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".
[0206] [2'-O-Me]--[2'-deoxy]--[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0207] 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.
[0208] [2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)]
Chimeric Phosphorothioate Oligonucleotides
[0209] [2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxy-ethyl)]
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.
[0210] [2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy
Phosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0211] [2'-O-(2-methoxyethyl phosphodiester]--[2'-deoxy
phosphorothioate]--[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.
[0212] 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
[0213] Oligonucleotide Isolation
[0214] 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
[0215] Oligonucleotide Synthesis--96 Well Plate Format
[0216] 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.
[0217] 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
[0218] 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
[0219] Cell Culture and Oligonucleotide Treatment
[0220] 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.
[0221] T-24 Cells:
[0222] 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.
[0223] 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.
[0224] A549 Cells:
[0225] 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 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.
[0226] NHDF Cells:
[0227] 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.
[0228] HEK cells:
[0229] 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.
[0230] b.END Cells:
[0231] The mouse brain endothelial cell line b.END was obtained
from Dr. Werner Risau at the Max Plank Instititute (Bad Nauheim,
Germany). b.END 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 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 3000 cells/well for use in
RT-PCR analysis.
[0232] 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.
[0233] Treatment with Antisense Compounds:
[0234] 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.
[0235] 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
[0236] Analysis of Oligonucleotide Inhibition of Caspase 9
Expression
[0237] Antisense modulation of caspase 9 expression can be assayed
in a variety of ways known in the art. For example, caspase 9 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.
[0238] Protein levels of caspase 9 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 caspase 9 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.
[0239] 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
[0240] Poly(A)+ mRNA Isolation
[0241] 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 mL 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.
[0242] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
[0243] Total RNA Isolation
[0244] 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.
[0245] 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
[0246] Real-Time Quantitative PCR Analysis of Caspase 9 mRNA
Levels
[0247] Quantitation of caspase 9 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.
[0248] 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.
[0249] 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).
[0250] 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.
[0251] 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.
[0252] Probes and primers to human caspase 9 were designed to
hybridize to a human caspase 9 sequence, using published sequence
information (GenBank accession number U60521, incorporated herein
as SEQ ID NO:3). For human caspase 9 the PCR primers were:
[0253] forward primer: ATTGTGGGATGTTCAGCACTGT (SEQ ID NO: 4)
[0254] reverse primer: TGTTTGGCACCACTCAGGAA (SEQ ID NO: 5) and the
PCR probe was: FAM-CCTTGCCTCAATGCCAGTAACGCG-TAMRA
[0255] (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:
[0256] forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 7)
[0257] reverse primer: GGCAACAATATCCACTTTACCAGAGT (SEQ ID NO: 8)
and the PCR probe was: 5' JOE-CGCCTGGTCACCAGGGCTGCT-TAMPA 3' (SEQ
ID NO: 9) 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.
[0258] Probes and primers to mouse caspase 9 were designed to
hybridize to a mouse caspase 9 sequence, using published sequence
information (GenBank accession number AB019600, incorporated herein
as SEQ ID NO:10). For mouse caspase 9 the PCR primers were:
[0259] forward primer: CCAACTTGGACCGTGACAAA (SEQ ID NO:11)
[0260] reverse primer: CGTTCTTCACCTCCACCATGA (SEQ ID NO: 12) and
the PCR probe was: FAM-TTGAGCACCGATTCCGCTGGCT-TAMRA (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:
[0261] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)
[0262] reverse primer: GGGTCTCGCTCCTGGAAGCT (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
[0263] Northern Blot Analysis of Caspase 9 mRNA Levels
[0264] 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.
[0265] To detect human caspase 9, a human caspase 9 specific probe
was prepared by PCR using the forward primer ATTGTGGGATGTTCAGCACTGT
(SEQ ID NO: 4) and the reverse primer TGTTTGGCACCACTCAGGAA (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.).
[0266] To detect mouse caspase 9, a mouse caspase 9 specific probe
was prepared by PCR using the forward primer CCAACTTGGACCGTGACAAA
(SEQ ID NO:11) and the reverse primer CGTTCTTCACCTCCACCATGA (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.).
[0267] 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
[0268] Antisense Inhibition of Human Caspase 9 Expression by
Chimeric Phosphorothioate Oligonucleotides having 2'-MOE Wings and
a Deoxy Gap
[0269] In accordance with the present invention, a series of
oligonucleotides were designed to target different regions of the
human caspase 9 RNA, using published sequences (GenBank accession
number U60521, incorporated herein as SEQ ID NO: 3, GenBank
accession number AL046716, incorporated herein as SEQ ID NO: 17,
GenBank accession number AB015653, incorporated herein as SEQ ID
NO: 18, GenBank accession number AB019198, incorporated herein as
SEQ ID NO: 19, GenBank accession number AB019199, incorporated
herein as SEQ ID NO: 20, GenBank accession number AB019200,
incorporated herein as SEQ ID NO: 21, GenBank accession number
AB019201, incorporated herein as SEQ ID NO: 22, GenBank accession
number AB019202, incorporated herein as SEQ ID NO: 23, GenBank
accession number AB019203, incorporated herein as SEQ ID NO: 24,
and GenBank accession number AB019204, incorporated herein as SEQ
ID NO: 25). 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 phosphorothioate (P.dbd.S) throughout the
oligonucleotide. All cytidine residues are 5-methylcytidines. The
compounds were analyzed for their effect on human caspase 9 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 caspase 9 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 135222 5' UTR 3 21 gtagccaactaagactccag 74 26 135223 Start
3 37 gcttcgtccatggcgagtag 79 27 Codon 135224 Coding 3 169
gagcctgcccgctggatgtc 96 28 135225 Coding 3 193 ctggcctgatcccgccgaga
63 29 135226 Coding 3 199 agctgcctggcctgatcccg 78 30 135227 Coding
3 208 tctatgatcagctgcctggc 78 31 135228 Coding 3 264
tgtcctctaagcaggagatg 95 32 135229 Coding 3 270 ggcctgtgtcctctaagcag
85 33 135230 Coding 3 283 gccagcatgtcctggcctgt 91 34 135231 Coding
3 308 ttgcctgttagttcgcagaa 91 35 135232 Coding 3 391
ctgagaacctctggtttgcg 96 36 135233 Coding 3 442 acatcaccaaatcctccaga
36 37 135234 Coding 3 455 ctcaagagcaccgacatcac 59 38 135235 Coding
3 472 gcatttcccctcaaactctc 88 39 135236 Coding 3 490
aggatgtaagccaaatctgc 51 40 135237 Coding 3 500 ctccatgctcaggatgtaag
94 41 135238 Coding 3 520 atgaggcagtggccacaggg 43 42 135239 Coding
3 541 cagaagttcacattgttgat 78 43 135240 Coding 3 599
ccgcaacttctcacagtcga 90 44 135241 Coding 3 652 gcagtcaggtcgcccttcac
76 45 135242 Coding 3 658 ttcttggcagtcaggtcgcc 81 46 135243 Coding
3 662 cattttcttggcagtcaggt 90 47 135244 Coding 3 666
gcaccattttcttggcagtc 62 48 135245 Coding 3 707 cagagcaccgtggtcctgct
83 49 135246 Coding 3 724 accaccacgcagcagtccag 84 50 135247 Coding
3 745 tgacagccgtgagagagaat 79 51 135248 Coding 3 753
ggctggcctgacagccgtga 76 52 135249 Coding 3 756 ggtggctggcctgacagccg
44 53 135250 Coding 3 782 gccgtagacagcccctggga 77 54 135251 Coding
3 791 tccatctgtgccgtagacag 86 55 135252 Coding 3 836
ggtcccattgaagatgttca 61 56 135253 Coding 3 884 ggcctggatgaaaaagagct
52 57 135254 Coding 3 1158 cagagtgagcccactgctca 61 58 135255 Coding
3 1174 agggactgcaggtcttcaga 29 59 135256 Coding 3 1194
cattagcgaccctaagcagg 87 60 135257 Coding 3 1228
ggcatctgtttataaatccc 93 61 135258 Stop 3 1286 gccctggccttatgatgttt
64 62 Codon 135259 3' UTR 3 1358 aagtccaggcctcagcctct 82 63 135260
3' UTR 3 1362 aggaaagtccaggcctcagc 75 64 135261 3' UTR 3 1386
ccggctgcaaagtccttgag 93 65 135262 3' UTR 3 1394
accctgtgccggctgcaaag 82 66 135263 3' UTR 3 1438
ggaagctgctaagagcctgt 87 67 135264 3' UTR 3 1468
cctccactgttcagcacttg 96 68 135265 3' UTR 3 1484
ttcatctgtccctcttcctc 40 69 135266 3' UTR 3 1503
ccacgtgcaatccacggcat 95 70 135267 3' UTR 3 1513
gctcaagaggccacgtgcaa 83 71 135268 3' UTR 3 1552
gatcatgggacacaagtcac 81 72 135269 3' UTR 3 1692
aggaagacgcgttactggca 97 73 135270 3' UTR 3 1715
aacctttttgtttggcacca 95 74 135271 3' UTR 3 1828
gtttgctgatctatgagcga 84 75 135272 3' UTR 3 1831
ccggtttgctgatctatgag 79 76 135273 3' UTR 3 1943
catatttgcctcagatttat 84 77 135274 3' UTR 3 1944
ccatatttgcctcagattta 85 78 135275 Exon 17 183 ggacgcatctccaaggcctc
1 79 135276 Exon 17 189 gcctccggacgcatctccaa 60 80 135277 Exon 17
210 ggtcagtcttcgctccccac 45 81 135278 Exon 17 215
ccccgggtcagtcttcgctc 31 82 135279 Exon 17 219 acggccccgggtcagtcttc
48 83 135280 Coding 18 429 ctttctgctcgacatcacca 0 84 135281 Intron
19 847 ctatctcaaaggcaggaaaa 74 85 135282 Intron 20 361
tcaagagcacctgaagaggc 91 86 135283 Exon 20 396 ggagactcaccaaatctgca
89 87 135284 Intron 20 425 agagcccgagctactggccc 81 88 135285 Intron
21 349 acagggacccacgtaaaccc 73 89 135286 Intron 21 367
ggatgtaagcctgccagcac 75 90 135287 Intron 21 544
gtgagtgtaccttggcagtc 82 91 135288 Intron 21 728
gccgagagagccagtcctca 81 92 135289 Intron 22 252
gcaccattttctacaagaga 81 93 135290 Exon 22 342 ggcttcctacctgacagccg
73 94 135291 Intron 23 232 ctgttcagaggttttgtggg 57 95 135292 Intron
23 250 gagtaggaatccaacagcct 86 96 135293 Intron 23 300
ggtggctggcctagaagacc 60 97 135294 Exon 23 450 ggagctgcttacccccacca
89 98 135295 Intron 24 253 acctcttgtcattgaaatac 32 99 135296 Intron
24 284 tctttctgctctgcaggaag 63 100 135297 Intron 25 196
gaccttgactcatacataca 85 101 135298 Intron 25 464
aggaggacacgctgcccctc 80 102 135299 Intron 25 688
gcactgaggaccagtccatc 63 103
[0270] As shown in Table 1, SEQ ID NOs 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68,
70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 83, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 101, 102 and 103
demonstrated at least 40% inhibition of human caspase 9 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
[0271] Antisense Inhibition of Mouse Caspase 9 Expression by
Chimeric Phosphorothioate Oligonucleotides having 2'-MOE Wings and
a Deoxy Gap.
[0272] In accordance with the present invention, a second series of
oligonucleotides were designed to target different regions of the
mouse caspase 9 RNA, using published sequences (GenBank accession
number AB019600, incorporated herein as SEQ ID NO: 10, and GenBank
accession number AB019601, incorporated herein as SEQ ID NO: 104).
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 caspase 9 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 caspase 9 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 135225 Coding 10 148 ctggcctgatcccgccgaga 87 29 135226
Coding 10 154 agctgcctggcctgatcccg 0 30 135228 Coding 10 219
tgtcctctaagcaggagatg 70 32 135229 Coding 10 225
ggcctgtgtcctctaagcag 10 33 135238 Coding 10 589
atgaggcagtggccacaggg 75 42 135239 Coding 10 610
cagaagttcacattgttgat 33 43 135243 Coding 10 731
cattttcttggcagtcaggt 54 47 135255 Coding 10 1243
agggactgcaggtcttcaga 0 59 135309 Coding 10 22 accctgcatcgccgcaggag
56 105 135310 Coding 10 24 gcaccctgcatcgccgcagg 33 106 135311
Coding 10 32 cactaggcgcaccctgcatc 24 107 135312 Coding 10 112
tgaatatcctcgatcatgtc 63 108 135313 Coding 10 113
ctgaatatcctcgatcatgt 79 109 135314 Coding 10 117
cctgctgaatatcctcgatc 52 110 135315 Coding 10 136
cgccgagacccagatcctgc 86 111 135316 Coding 10 168
caaggtctgtgaccagctgc 69 112 135317 Coding 10 171
tctcaaggtctgtgaccagc 71 113 135318 Coding 10 181
ctccctcgggtctcaaggtc 55 114 135319 Coding 10 200
gaagagaggaagggcctgcc 7 115 135320 Coding 10 240
aagccagggtgccttggcct 86 116 135321 Coding 10 251
ttgcaagagtgaagccaggg 84 117 135322 Coding 10 262
tgccgaccgctttgcaagag 73 118 135323 Coding 10 314
aggcaccaggtggtctaggg 17 119 135324 Coding 10 350
ctcctttgctgtgagtccca 29 120 135325 Coding 10 353
ctgctcctttgctgtgagtc 0 121 135326 Coding 10 375
acggctccagcttcactact 47 122 135327 Coding 10 379
tgtgacggctccagcttcac 22 123 135328 Coding 10 383
aggctgtgacggctccagct 70 124 135329 Coding 10 388
acggcaggctgtgacggctc 36 125 135330 Coding 10 395
gtttcccacggcaggctgtg 68 126 135331 Coding 10 459
tgagaacctctggcttgagc 74 127 135332 Coding 10 461
tctgagaacctctggcttga 49 128 135333 Coding 10 464
tggtctgagaacctctggct 84 129 135334 Coding 10 490
ccaatgtccaccggcctggg 13 130 135335 Coding 10 534
ccctgatcttccctggaaca 70 131 135336 Coding 10 549
ccatatctgcatgtcccctg 32 132 135337 Coding 10 560
cagggtgtatgccatatctg 33 133 135338 Coding 10 564
aatccagggtgtatgccata 49 134 135339 Coding 10 566
cgaatccagggtgtatgcca 71 135 135340 Coding 10 599
attgttgatgatgaggcagt 68 136 135341 Coding 10 601
acattgttgatgatgaggca 78 137 135342 Coding 10 659
gtcacggtccaagttggagc 7 138 135343 Coding 10 670
tgctcaagtttgtcacggtc 84 139 135344 Coding 10 681
agcggaatcggtgctcaagt 89 140 135345 Coding 10 707
cttcacctccaccatgaagc 41 141 135346 Coding 10 708
tcttcacctccaccatgaag 10 142 135347 Coding 10 726
tcttggcagtcaggtcgttc 69 143 135348 Coding 10 772
gcacggtggttccggtgtgc 61 144 135349 Coding 10 783
agcagtccagggcacggtgg 81 145 135350 Coding 10 789
ccacaaagcagtccagggca 69 146 135351 Coding 10 800
gaggatgaccaccacaaagc 44 147 135352 Coding 10 817
gcctggcagccatgagagag 10 148 135353 Coding 10 824
gtggctggcctggcagccat 0 149 135354 Coding 10 845
gacagcacccgggaactgga 62 150 135355 Coding 10 890
attcacaattttctcaatgg 6 151 135356 Coding 10 984
caaagccatggtctttctgc 61 152 135357 Coding 10 993
aggccacctcaaagccatgg 3 153 135358 Coding 10 1015
gtcctgccttgagaggaagt 66 154 135359 Coding 10 1040
atctggctcagagtcactgt 82 155 135360 Coding 10 1087
gcatccagctggtccaaggg 39 156 135361 Coding 10 1095
ttgacacagcatccagctgg 71 157 135362 Coding 10 1104
taggcaaacttgacacagca 72 158 135363 Coding 10 1106
ggtaggcaaacttgacacag 76 159 135364 Coding 10 1137
aggtggagtaggacacaagg 27 160 135365 Coding 10 1146
aacctgggaaggtggagtag 45 161 135366 Coding 10 1169
tttcttgtccctccaggaga 62 162 135367 Coding 10 1177
gagccacttttcttgtccct 82 163 135368 Coding 10 1183
taccaggagccacttttctt 66 164 135369 Coding 10 1187
gatgtaccaggagccacttt 67 165 135370 Coding 10 1198
tccaaggtctcgatgtacca 70 166 135371 Coding 10 1207
agaatgccatccaaggtctc 23 167 135372 Coding 10 1215
actgctccagaatgccatcc 59 168 135373 Coding 10 1255
accctgagaaggagggactg 66 169 135374 Coding 10 1284
aagtccctttctcagaaaca 44 170 135375 Coding 10 1303
cagccaggaatctgcttgta 90 171 135376 Coding 10 1323
ttttccggaggaagttaaaa 77 172 135377 Coding 10 1328
cagctttttccggaggaagt 93 173 135378 Coding 104 1154
gcattggcgacctgggaagg 53 174
[0273] As shown in Table 2, SEQ ID NOs 29, 32, 42, 43, 47, 105,
106, 108, 109, 110, 111, 112, 113, 114, 116, 117, 118, 122, 124,
125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 139,
140, 141, 143, 144, 145, 146, 147, 150, 152, 154, 155, 156, 157,
158, 159, 161, 162, 163, 164, 165, 166, 168, 169, 170, 171, 172,
173 and 174 demonstrated at least 30% inhibition of mouse caspase 9
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
[0274] Western Blot Analysis of Caspase 9 Protein Levels
[0275] 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 caspase 9 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
174 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1
tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense
Oligonucleotide 2 atgcattctg cccccaagga 20 3 1963 DNA Homo sapiens
CDS (46)...(1296) 3 cggaagcgga ctgaggcggc ctggagtctt agttggctac
tcgcc atg gac gaa gcg 57 Met Asp Glu Ala 1 gat cgg cgg ctc ctg cgg
cgg tgc cgg ctg cgg ctg gtg gaa gag ctg 105 Asp Arg Arg Leu Leu Arg
Arg Cys Arg Leu Arg Leu Val Glu Glu Leu 5 10 15 20 cag gtg gac cag
ctc tgg gac gcc ctg ctg agc agc gag ctg ttc agg 153 Gln Val Asp Gln
Leu Trp Asp Ala Leu Leu Ser Ser Glu Leu Phe Arg 25 30 35 ccc cat
atg atc gag gac atc cag cgg gca ggc tct gga tct cgg cgg 201 Pro His
Met Ile Glu Asp Ile Gln Arg Ala Gly Ser Gly Ser Arg Arg 40 45 50
gat cag gcc agg cag ctg atc ata gat ctg gag act cga ggg agt cag 249
Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr Arg Gly Ser Gln 55
60 65 gct ctt cct ttg ttc atc tcc tgc tta gag gac aca ggc cag gac
atg 297 Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr Gly Gln Asp
Met 70 75 80 ctg gct tcg ttt ctg cga act aac agg caa gca gca aag
ttg tcg aag 345 Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala Lys
Leu Ser Lys 85 90 95 100 cca acc cta gaa aac ctt acc cca gtg gtg
ctc aga cca gag att cgc 393 Pro Thr Leu Glu Asn Leu Thr Pro Val Val
Leu Arg Pro Glu Ile Arg 105 110 115 aaa cca gag gtt ctc aga ccg gaa
aca ccc aga cca gtg gac att ggt 441 Lys Pro Glu Val Leu Arg Pro Glu
Thr Pro Arg Pro Val Asp Ile Gly 120 125 130 tct gga gga ttt ggt gat
gtc ggt gct ctt gag agt ttg agg gga aat 489 Ser Gly Gly Phe Gly Asp
Val Gly Ala Leu Glu Ser Leu Arg Gly Asn 135 140 145 gca gat ttg gct
tac atc ctg agc atg gag ccc tgt ggc cac tgc ctc 537 Ala Asp Leu Ala
Tyr Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu 150 155 160 att atc
aac aat gtg aac ttc tgc cgt gag tcc ggg ctc cgc acc cgc 585 Ile Ile
Asn Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg 165 170 175
180 act ggc tcc aac atc gac tgt gag aag ttg cgg cgt cgc ttc tcc tcg
633 Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser
185 190 195 ccg cat ttc atg gtg gag gtg aag ggc gac ctg act gcc aag
aaa atg 681 Pro His Phe Met Val Glu Val Lys Gly Asp Leu Thr Ala Lys
Lys Met 200 205 210 gtg ctg gct ttg ctg gag ctg gcg cag cag gac cac
ggt gct ctg gac 729 Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His
Gly Ala Leu Asp 215 220 225 tgc tgc gtg gtg gtc att ctc tct cac ggc
tgt cag gcc agc cac ctg 777 Cys Cys Val Val Val Ile Leu Ser His Gly
Cys Gln Ala Ser His Leu 230 235 240 cag ttc cca ggg gct gtc tac ggc
aca gat gga tgc cct gtg tcg gtc 825 Gln Phe Pro Gly Ala Val Tyr Gly
Thr Asp Gly Cys Pro Val Ser Val 245 250 255 260 gag aag att gtg aac
atc ttc aat ggg acc agc tgc ccc agc ctg gga 873 Glu Lys Ile Val Asn
Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly 265 270 275 gga aag ccc
aag ctc ttt ttc atc cag gcc tgt ggt ggg gag cag aaa 921 Gly Lys Pro
Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys 280 285 290 gac
cat ggg ttt gag gtg gcc tcc act tcc cct gaa gac gag tcc cct 969 Asp
His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro 295 300
305 ggc agt aac ccc gag cca gat gcc acc ccg ttc cag gaa ggt ttg agg
1017 Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu
Arg 310 315 320 acc ttc gac cag ctg gac gcc ata tct agt ttg ccc aca
ccc agt gac 1065 Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro
Thr Pro Ser Asp 325 330 335 340 atc ttt gtg tcc tac tct act ttc cca
ggt ttt gtt tcc tgg agg gac 1113 Ile Phe Val Ser Tyr Ser Thr Phe
Pro Gly Phe Val Ser Trp Arg Asp 345 350 355 ccc aag agt ggc tcc tgg
tac gtt gag acc ctg gac gac atc ttt gag 1161 Pro Lys Ser Gly Ser
Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu 360 365 370 cag tgg gct
cac tct gaa gac ctg cag tcc ctc ctg ctt agg gtc gct 1209 Gln Trp
Ala His Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala 375 380 385
aat gct gtt tcg gtg aaa ggg att tat aaa cag atg cct ggt tgc ttt
1257 Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys
Phe 390 395 400 aat ttc ctc cgg aaa aaa ctt ttc ttt aaa aca tca taa
ggccagggcc 1306 Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser 405
410 415 cctcaccctg ccttatcttg caccccaaag ctttcctgcc ccaggcctga
aagaggctga 1366 ggcctggact ttcctgcaac tcaaggactt tgcagccggc
acagggtctg ctctttctct 1426 gccagtgaca gacaggctct tagcagcttc
cagattgacg acaagtgctg aacagtggag 1486 gaagagggac agatgaatgc
cgtggattgc acgtggcctc ttgagcagtg gctggtccag 1546 ggctagtgac
ttgtgtccca tgatccctgt gttgtctcta gagcagggat taacctctgc 1606
actactgaca tgtggggcca ggtcaccctt tgctgtgagg ctgtcctgta cattgtggga
1666 tgttcagcac tgtcccttgc ctcaatgcca gtaacgcgtc ttcctgagtg
gtgccaaaca 1726 aaaaggttct caggtgttgc caaatatgtc ctggggtata
aaactttcct cgcctgacaa 1786 ccactggtct gtagggattt ttggctacac
acaaaccagt atcgctcata gatcagcaaa 1846 ccggggccta ctagagtctg
aacagctgta atctatgaat tctaagtgaa attttaaaaa 1906 ttgttaattt
ttcctatatt gcattaattt taaaaaataa atctgaggca aatatgg 1963 4 22 DNA
Artificial Sequence PCR Primer 4 attgtgggat gttcagcact gt 22 5 20
DNA Artificial Sequence PCR Primer 5 tgtttggcac cactcaggaa 20 6 24
DNA Artificial Sequence PCR Probe 6 ccttgcctca atgccagtaa cgcg 24 7
21 DNA Artificial Sequence PCR Primer 7 caacggattt ggtcgtattg g 21
8 26 DNA Artificial Sequence PCR Primer 8 ggcaacaata tccactttac
cagagt 26 9 21 DNA Artificial Sequence PCR Probe 9 cgcctggtca
ccagggctgc t 21 10 1365 DNA Mus musculus CDS (1)...(1365) 10 atg
gac gag gcg gac cgg cag ctc ctg cgg cga tgc agg gtg cgc cta 48 Met
Asp Glu Ala Asp Arg Gln Leu Leu Arg Arg Cys Arg Val Arg Leu 1 5 10
15 gtg agc gag ctg caa gtc gcg gag ctc tgg gac gct ctg ctg agt cga
96 Val Ser Glu Leu Gln Val Ala Glu Leu Trp Asp Ala Leu Leu Ser Arg
20 25 30 gag ctc ttc acg cgc gac atg atc gag gat att cag cag gca
gga tct 144 Glu Leu Phe Thr Arg Asp Met Ile Glu Asp Ile Gln Gln Ala
Gly Ser 35 40 45 ggg tct cgg cgg gat cag gcc agg cag ctg gtc aca
gac ctt gag acc 192 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Val Thr
Asp Leu Glu Thr 50 55 60 cga ggg agg cag gcc ctt cct ctc ttc atc
tcc tgc tta gag gac aca 240 Arg Gly Arg Gln Ala Leu Pro Leu Phe Ile
Ser Cys Leu Glu Asp Thr 65 70 75 80 ggc caa ggc acc ctg gct tca ctc
ttg caa agc ggt cgg caa gca gcc 288 Gly Gln Gly Thr Leu Ala Ser Leu
Leu Gln Ser Gly Arg Gln Ala Ala 85 90 95 aag cag gat cca gag gct
gtt aaa ccc cta gac cac ctg gtg cct gtg 336 Lys Gln Asp Pro Glu Ala
Val Lys Pro Leu Asp His Leu Val Pro Val 100 105 110 gtc ctg gga cca
atg gga ctc aca gca aag gag cag aga gta gtg aag 384 Val Leu Gly Pro
Met Gly Leu Thr Ala Lys Glu Gln Arg Val Val Lys 115 120 125 ctg gag
ccg tca cag cct gcc gtg gga aac ctc acc cca gtg gtg ctg 432 Leu Glu
Pro Ser Gln Pro Ala Val Gly Asn Leu Thr Pro Val Val Leu 130 135 140
ggg cca gaa gag ctc tgg cct gct cgg ctc aag cca gag gtt ctc aga 480
Gly Pro Glu Glu Leu Trp Pro Ala Arg Leu Lys Pro Glu Val Leu Arg 145
150 155 160 cca gaa aca ccc agg ccg gtg gac att ggt tct ggc gga gct
cat gat 528 Pro Glu Thr Pro Arg Pro Val Asp Ile Gly Ser Gly Gly Ala
His Asp 165 170 175 gtc tgt gtt cca ggg aag atc agg gga cat gca gat
atg gca tac acc 576 Val Cys Val Pro Gly Lys Ile Arg Gly His Ala Asp
Met Ala Tyr Thr 180 185 190 ctg gat tcg gat ccc tgt ggc cac tgc ctc
atc atc aac aat gtg aac 624 Leu Asp Ser Asp Pro Cys Gly His Cys Leu
Ile Ile Asn Asn Val Asn 195 200 205 ttc tgc cct tcc tcg ggg ctc ggc
aca cgc acg ggc tcc aac ttg gac 672 Phe Cys Pro Ser Ser Gly Leu Gly
Thr Arg Thr Gly Ser Asn Leu Asp 210 215 220 cgt gac aaa ctt gag cac
cga ttc cgc tgg ctg cgc ttc atg gtg gag 720 Arg Asp Lys Leu Glu His
Arg Phe Arg Trp Leu Arg Phe Met Val Glu 225 230 235 240 gtg aag aac
gac ctg act gcc aag aaa atg gtc acg gct ttg atg gag 768 Val Lys Asn
Asp Leu Thr Ala Lys Lys Met Val Thr Ala Leu Met Glu 245 250 255 atg
gca cac cgg aac cac cgt gcc ctg gac tgc ttt gtg gtg gtc atc 816 Met
Ala His Arg Asn His Arg Ala Leu Asp Cys Phe Val Val Val Ile 260 265
270 ctc tct cat ggc tgc cag gcc agc cac ctc cag ttc ccg ggt gct gtc
864 Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val
275 280 285 tat ggg aca gat gga tgc tcc gtg tcc att gag aaa att gtg
aat atc 912 Tyr Gly Thr Asp Gly Cys Ser Val Ser Ile Glu Lys Ile Val
Asn Ile 290 295 300 ttc aac ggg agc ggc tgc ccc agc ctg gga ggg aag
ccc aag ctc ttc 960 Phe Asn Gly Ser Gly Cys Pro Ser Leu Gly Gly Lys
Pro Lys Leu Phe 305 310 315 320 ttc atc cag gcc tgc ggt ggt gag cag
aaa gac cat ggc ttt gag gtg 1008 Phe Ile Gln Ala Cys Gly Gly Glu
Gln Lys Asp His Gly Phe Glu Val 325 330 335 gcc tgc act tcc tct caa
ggc agg acc ttg gac agt gac tct gag cca 1056 Ala Cys Thr Ser Ser
Gln Gly Arg Thr Leu Asp Ser Asp Ser Glu Pro 340 345 350 gat gct gtc
cca tat cag gaa ggc cca agg ccc ttg gac cag ctg gat 1104 Asp Ala
Val Pro Tyr Gln Glu Gly Pro Arg Pro Leu Asp Gln Leu Asp 355 360 365
gct gtg tca agt ttg cct acc ccc agt gac atc ctt gtg tcc tac tcc
1152 Ala Val Ser Ser Leu Pro Thr Pro Ser Asp Ile Leu Val Ser Tyr
Ser 370 375 380 acc ttc cca ggt ttt gtc tcc tgg agg gac aag aaa agt
ggc tcc tgg 1200 Thr Phe Pro Gly Phe Val Ser Trp Arg Asp Lys Lys
Ser Gly Ser Trp 385 390 395 400 tac atc gag acc ttg gat ggc att ctg
gag cag tgg gct cgc tct gaa 1248 Tyr Ile Glu Thr Leu Asp Gly Ile
Leu Glu Gln Trp Ala Arg Ser Glu 405 410 415 gac ctg cag tcc ctc ctt
ctc agg gtc gcc aat gct gtt tct gag aaa 1296 Asp Leu Gln Ser Leu
Leu Leu Arg Val Ala Asn Ala Val Ser Glu Lys 420 425 430 ggg act tac
aag cag att cct ggc tgt ttt aac ttc ctc cgg aaa aag 1344 Gly Thr
Tyr Lys Gln Ile Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys 435 440 445
ctg ttt ttt aaa act tca tga 1365 Leu Phe Phe Lys Thr Ser 450 11 20
DNA Artificial Sequence PCR Primer 11 ccaacttgga ccgtgacaaa 20 12
21 DNA Artificial Sequence PCR Primer 12 cgttcttcac ctccaccatg a 21
13 22 DNA Artificial Sequence PCR Probe 13 ttgagcaccg attccgctgg ct
22 14 20 DNA Artificial Sequence PCR Primer 14 ggcaaattca
acggcacagt 20 15 20 DNA Artificial Sequence PCR Primer 15
gggtctcgct cctggaagct 20 16 27 DNA Artificial Sequence PCR Probe 16
aaggccgaga atgggaagct tgtcatc 27 17 603 DNA Homo sapiens 17
gacgcagtgg tggattctgg agcggggcgg tgacgtgggg cgggttctgg ggcggggcgg
60 tgacgcgagc gtgttctggg gcggggcggt gatgggaggc ttggccctgg
gggcggggcg 120 aggcgcagag gtgcgtcctg agggcggggc ggtgacgcaa
gagcgactcc tgggggcggg 180 gcgaggcctt ggagatgcgt ccggaggcgg
tggggagcga agactgaccc ggggccgtga 240 cgcggggcag gccctggggc
gggggcgggt cctggggact ggggcgggcg gccgaggccc 300 ggaagcggac
tgaggcggcc tggagtctta gttggctact cgccatggac gaagcggatc 360
ggcggctcct gcggcggtgc cggctgcggc tggtggaaga gctgcaggtg gaccagctct
420 gggacgtcct gctgagtcgc gagctgttca ggccccatat gatcgaggac
atccagcggg 480 caggctctgg atctcggcgg gatcaggcca ggcagctgat
catagatctg gagactcgag 540 ggagtcaggc tcttcctttg ttcatctcct
gcttagagga cacaggccag gacatgctgg 600 ctt 603 18 1137 DNA Homo
sapiens CDS (22)...(822) 18 agtcttagtt ggctactcgc c atg gac gaa gcg
gat cgg cgg ctc ctg cgg 51 Met Asp Glu Ala Asp Arg Arg Leu Leu Arg
1 5 10 cgg tgc cgg ctg cgg ctg gtg gaa gag ctg cag gtg gac cag ctc
tgg 99 Arg Cys Arg Leu Arg Leu Val Glu Glu Leu Gln Val Asp Gln Leu
Trp 15 20 25 gac gcc ctg ctg agc cgc gag ctg ttc agg ccc cat atg
atc gag gac 147 Asp Ala Leu Leu Ser Arg Glu Leu Phe Arg Pro His Met
Ile Glu Asp 30 35 40 atc cag cgg gca ggc tct gga tct cgg cgg gat
cag gcc agg cag ctg 195 Ile Gln Arg Ala Gly Ser Gly Ser Arg Arg Asp
Gln Ala Arg Gln Leu 45 50 55 atc ata gat ctg gag act cga ggg agt
cag gct ctt cct ttg ttc atc 243 Ile Ile Asp Leu Glu Thr Arg Gly Ser
Gln Ala Leu Pro Leu Phe Ile 60 65 70 tcc tgc tta gag gac aca ggc
cag gac atg ctg gct tcg ttt ctg cga 291 Ser Cys Leu Glu Asp Thr Gly
Gln Asp Met Leu Ala Ser Phe Leu Arg 75 80 85 90 act aac agg caa gca
gca aag ttg tcg aag cca acc cta gaa aac ctt 339 Thr Asn Arg Gln Ala
Ala Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu 95 100 105 acc cca gtg
gtg ctc aga cca gag att cgc aaa cca gag gtt ctc aga 387 Thr Pro Val
Val Leu Arg Pro Glu Ile Arg Lys Pro Glu Val Leu Arg 110 115 120 ccg
gaa aca ccc aga cca gtg gac att ggt tct gga gga ttt ggt gat 435 Pro
Glu Thr Pro Arg Pro Val Asp Ile Gly Ser Gly Gly Phe Gly Asp 125 130
135 gtc gag cag aaa gac cat ggg ttt gag gtg gcc tcc act tcc cct gaa
483 Val Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu
140 145 150 gac gag tcc cct ggc agt aac ccc gag cca gat gcc acc ccg
ttc cag 531 Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro
Phe Gln 155 160 165 170 gaa ggt ttg agg acc ttc gac cag ctg gac gcc
ata tct agt ttg ccc 579 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala
Ile Ser Ser Leu Pro 175 180 185 aca ccc agt gac atc ttt gtg tcc tac
tct act ttc cca ggt ttt gtt 627 Thr Pro Ser Asp Ile Phe Val Ser Tyr
Ser Thr Phe Pro Gly Phe Val 190 195 200 tcc tgg agg gac ccc aag agt
ggc tcc tgg tac gtt gag acc ctg gac 675 Ser Trp Arg Asp Pro Lys Ser
Gly Ser Trp Tyr Val Glu Thr Leu Asp 205 210 215 gac atc ttt gag cag
tgg gct cac tct gaa gac ctg cag tcc ctc ctg 723 Asp Ile Phe Glu Gln
Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu 220 225 230 ctt agg gtc
gct aat gct gtt tcg gtg aaa ggg att tat aaa cag atg 771 Leu Arg Val
Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 235 240 245 250
cct ggt tgc ttt aat ttc ctc cgg aaa aaa ctt ttc ttt aaa aca tca 819
Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser 255
260 265 taa ggccagggcc cctcaccctg ccttatcttg caccccaaag ctttcctgcc
872 ccaggcctga aagaggctga ggcctggact ttcctgcaac tcaaggactt
tgcagccggc 932 acagggtctg ctctttctct gccagtgaca gacaggctct
tagcagcttc cagattgacg 992 acaagtgctg aacagtggag gaagagggac
agatgaatgc cgtggattgc acgtggcctc 1052 ttgagcagtg gctggtccag
ggctagtgac ttgtgtccca tgatccctgt gttgtctcta 1112 gagcagggat
taacctctgc actac 1137 19 908 DNA Homo sapiens 19 tccctctggg
cacttgtctc ttgttgtgtc attgctgatt tgtctgattg cattccccat 60
tagaaatttg ctccttaagg actgagactg tccatggcag cattttcagc acttagcata
120 gtgctgggca tcttttgccc tgcaaaataa tgaacagatc agcaaaggct
ggtaaatggc 180 acacagtgca cctgcagctc cattgctgcc cacgtgcctg
attgtttaga cttacagtag 240 ctgggaaatg gggagacaag gtgagagcca
tataaaagta cgtggataat tagtatcctg 300 cctttctttc caaacagcgg
gcaggctctg gatctcggcg ggatcaggcc
aggcagctga 360 tcatagatct ggagactcga gggagtcagg ctcttccttt
gttcatctcc tgcttagagg 420 acacaggcca ggacatgctg gcttcgtttc
tgcgaactaa caggcaagca gcaaagttgt 480 cgaagccaac cctagaaaac
cttaccccag tggtgctcag accagagatt cgcaaaccag 540 aggttctcag
accggaaaca cccagaccag tggacattgg ttctggagga tttggtgatg 600
tcggtaagta gcaagagagt gattggtggg tgggcatgac acgtagtcat ttgggactca
660 ctatgagtac agctgggtgg gctgagattc agaatttaat tttagcattg
tctttctgaa 720 tgttcatagg gatggcaaaa ttctgggagg ttttcaggag
gttgaatctt ggcagcccct 780 cctctgaaaa gcatgtgttg aggtctgttt
tggatgtccc aggttttttg tttttgtttt 840 tgtttttttt cctgcctttg
agatagcagg ccccaggttt tgatcaatgt ttccagagcc 900 atctattg 908 20 731
DNA Homo sapiens unsure 536 unknown 20 ctgtgtaatg ttctcttgtg
tggctgtatc acaatttgta tatctaccaa actgttgtta 60 gattggttgt
ttccatgttg gggcgtgatg aataaggcct cagggaacat tttggccaag 120
tcttttgtgc ccgtttgcac tccttgctct ttgatatata cctaagggtg gacttgctgt
180 cagagggtag gtgtgtgttt aactttgtta gaaacctcta aacagatctc
caaggtggct 240 ctcccaccag cagagtctga gggtctgagt tctcgctgct
ccgttcttgg ggggctgtcc 300 tcacagagcc ccttgcccct cctctgctgt
aacccaacat tcatgtttct ctctttctct 360 gcctcttcag gtgctcttga
gagtttgagg ggaaatgcag atttggtgag tctccatgaa 420 atttgggcca
gtagctcggg ctctgggccc ttccctcccc acccctcagg tccttccctg 480
caggtgtgtc aggagcccag ggaacccaac cctaggcagt gcagtggggg tctggnctca
540 gcctccctga ctccggccca gcgcctgctg gggcagattg ttcatctctg
cgcttcctca 600 tctgcaaagc tgacagcaga agcagggcag acctgatgag
gggcagtcat cgggatccag 660 accaagtcgc cactctctca ttcacaagta
tttattgtgt ggctcccatg ctctgtgctg 720 gggccacctg g 731 21 791 DNA
Homo sapiens 21 tgccttagag aattacaagg ggactgagcc tgggagaggg
cagccttggg tgcctccagg 60 tggcctgggc atggttctgc tgacagagtt
ctcaggtaca ccccggccta ggtgacccgt 120 gaggtccttt gggatgaatg
ctcatggctg ttggatttcc aggttaccag ctcccttccc 180 ttaataccaa
gatggggggg tcaccttcta gtgctagtct tccctgcagt tgcttctctt 240
ttgccctgga atgccgggtg gagaccctgc tactccttgt gcgagagggc agaccccgtt
300 ttcttcccgc ctctccagac tgtcctctag ggagcgtggg gagagcccgg
gtttacgtgg 360 gtccctgtgc tggcaggctt acatcctgag catggagccc
tgtggccact gcctcattat 420 caacaatgtg aacttctgcc gtgagtccgg
gctccgcacc cgcactggct ccaacatcga 480 ctgtgagaag ttgcggcgtc
gcttctcctc gccgcatttc atggtggagg tgaagggcga 540 cctgactgcc
aaggtacact cactatctgt ggagggagac agggtggggg gcagtgggtg 600
gggaagtatc ttttgaggga ctccaaaagc cagctgactc cccaggcgag cccttacatc
660 tggaggacca tgggaggtag gacggcccac ggctacaggt tcaagttcag
acgcagaagt 720 ccgggtttga ggactggctc tctcggccac ctgctgtgtg
acctcctgtg cctcagtttc 780 cttgtctgga a 791 22 604 DNA Homo sapiens
22 gggattacag gtgtgtgcca ccatgcctgg ctaaattttt ttttttgtat
ttttagtaga 60 gacggggttt caccatgttg gccaggctgg tctcgaactc
gtgacctcaa atgatccacc 120 tgccttggcc tcccaaagtg ctgggattac
aggcgtgaac caccatgcct ggccaggctg 180 ggccctttta ttgttaggaa
cggtccagtc tgcatctaga cctatccgtg cttctggctc 240 acctgcagcc
ctctcttgta gaaaatggtg ctggctttgc tggagctggc gcagcaggac 300
cacggtgctc tggactgctg cgtggtggtc attctctctc acggctgtca ggtaggaagc
360 ctcccactgt tccctgggca ggcattgggt actggccgtg ccaagaggct
gtgcaggggc 420 catgtccctt cttgtgtcca aaacaccctt ggctttgtgg
aaaagggctg tggggccctg 480 cccacctcct gttgttttct tgggagccat
gtggtcctct gaggagttgg ctgcaccgtc 540 ctgggcaggt cggtgttcct
gggagaagcc ctctgggaga gggagggcag agaccaggtc 600 tgct 604 23 810 DNA
Homo sapiens 23 ccaagcgata ttgatgccgc tgctgcaggg gccaaacttt
gagaaccact ggtctagagg 60 ccgcagccag cactctccag gcacactggc
ccctagaggc acatgagttt gacatgcaac 120 cccttgctgt caaggtgtct
ttccagatgc tgaccagggt ctttccagaa ccaacctgct 180 ctgtctcaat
cagcctcgtg cccctggctt tgaccttcca aatccttggc tcccacaaaa 240
cctctgaaca ggctgttgga ttcctactcc aacttcttgg ttttgtaacc agggttcttg
300 gtcttctagg ccagccacct gcagttccca ggggctgtct acggcacaga
tggatgccct 360 gtgtcggtcg agaagattgt gaacatcttc aatgggacca
gctgccccag cctgggagga 420 aagcccaagc tctttttcat ccaggcctgt
ggtgggggta agcagctcct cagcctccct 480 ctgggtgggt ctggtgggga
gggagccgcc acctgcttct ttctccagcc tgcccctcac 540 agggcccatg
aggtctctcc aggcagtcag agggtaccac acatggtccc tttttggcag 600
cacctctgtc tggctggagc aggccctgct ctgtctgctt tgtagaggtc agagtcttct
660 gtttcatggc acagtggcat catgggctca agtaactttg gcagcttcaa
ttccaaagac 720 ttggcaaaaa gaaagcagaa gagagaggag aaacctcttt
ttatttttct tgtcaccatt 780 ctcctgacct ccatcaacta gaccccgttt 810 24
675 DNA Homo sapiens 24 tttatcagac aactatcttg gagctcacca ggaatttcct
tgtgggcaaa atatgggaga 60 ggtgtgtagc ttttcatctt gtagccatct
tatttaggaa ccaaaacggg gaggcaggtt 120 tgcgtgaccc agttcccagc
ttgacttttc cgtttggctt aatgagtttg gggtcccaag 180 atttattttc
ctttcacact gttaatgtag tatattgcac cgactttcat atgttgaacc 240
atccttgtct gtgtatttca atgacaagag gtgtttgttt ctgcttcctg cagagcagaa
300 agaccatggg tttgaggtgg cctccacttc ccctgaagac gagtcccctg
gcagtaaccc 360 cgagccagat gccaccccgt tccaggaagg tttgaggacc
ttcgaccagc tggacgccat 420 atctagtttg cccacaccca gtgacatctt
tgtgtcctac tctactttcc caggtgagca 480 catcagaagg gctcgtcctc
gcagccagtg ggtcttcccg tctgccctag aggcagctgt 540 gtggtgagag
aaaagaccag ggtatgagtc ctggttctgt cttttgcccc tctgtgaccc 600
tgagcaagtt acttctctct gagcctcggc tcaggctgag cgggcaagga tgcgcgctgc
660 aaggaggcct gcgcc 675 25 718 DNA Homo sapiens unsure 710 unknown
25 ggattacagg tgtgagccac tgtgcctggc agtttccttc ctttcttatg
tcttggatga 60 gtcacttgac ttttccaagc cttggtttcc ttgctgggaa
aacaggaatt atagccggaa 120 tcaggggatg gtcatgaggg tgaaataaga
tcatgattag ggcagagcct caggggccag 180 gctgctcctg ttggatgtat
gtatgagtca aggtctggag ggcgcccgag ccagcgtgca 240 tggccagagg
ggtggtgggg agccggcgga ggggtggctc tccagcagtg ttcagccctc 300
ctccctccaa aggttttgtt tcctggaggg accccaagag tggctcctgg tacgttgaga
360 ccctggacga catctttgag cagtgggctc actctgaaga cctgcagtcc
ctcctgctta 420 gggtgagtgc tgccttcctc tgcaaaggag aggggaggct
gctgaggggc agcgtgtcct 480 cctggggctg gggatttggg gtgagcaggg
caggcccaaa ccaagggtaa aaggtagtag 540 actcctgcct ctgagccttg
gagtcggcac ttttgtgtct ctttatgaga ggcatcctgc 600 ctgtggtgac
gtttgccctt tattcaaaga gttgccttcc ctgtctcctc caaggtcccc 660
aggtttgaca cctcccgctc ctccactgat ggactggtcc tcagtgccan cgcagatg 718
26 20 DNA Artificial Sequence Antisense Oligonucleotide 26
gtagccaact aagactccag 20 27 20 DNA Artificial Sequence Antisense
Oligonucleotide 27 gcttcgtcca tggcgagtag 20 28 20 DNA Artificial
Sequence Antisense Oligonucleotide 28 gagcctgccc gctggatgtc 20 29
20 DNA Artificial Sequence Antisense Oligonucleotide 29 ctggcctgat
cccgccgaga 20 30 20 DNA Artificial Sequence Antisense
Oligonucleotide 30 agctgcctgg cctgatcccg 20 31 20 DNA Artificial
Sequence Antisense Oligonucleotide 31 tctatgatca gctgcctggc 20 32
20 DNA Artificial Sequence Antisense Oligonucleotide 32 tgtcctctaa
gcaggagatg 20 33 20 DNA Artificial Sequence Antisense
Oligonucleotide 33 ggcctgtgtc ctctaagcag 20 34 20 DNA Artificial
Sequence Antisense Oligonucleotide 34 gccagcatgt cctggcctgt 20 35
20 DNA Artificial Sequence Antisense Oligonucleotide 35 ttgcctgtta
gttcgcagaa 20 36 20 DNA Artificial Sequence Antisense
Oligonucleotide 36 ctgagaacct ctggtttgcg 20 37 20 DNA Artificial
Sequence Antisense Oligonucleotide 37 acatcaccaa atcctccaga 20 38
20 DNA Artificial Sequence Antisense Oligonucleotide 38 ctcaagagca
ccgacatcac 20 39 20 DNA Artificial Sequence Antisense
Oligonucleotide 39 gcatttcccc tcaaactctc 20 40 20 DNA Artificial
Sequence Antisense Oligonucleotide 40 aggatgtaag ccaaatctgc 20 41
20 DNA Artificial Sequence Antisense Oligonucleotide 41 ctccatgctc
aggatgtaag 20 42 20 DNA Artificial Sequence Antisense
Oligonucleotide 42 atgaggcagt ggccacaggg 20 43 20 DNA Artificial
Sequence Antisense Oligonucleotide 43 cagaagttca cattgttgat 20 44
20 DNA Artificial Sequence Antisense Oligonucleotide 44 ccgcaacttc
tcacagtcga 20 45 20 DNA Artificial Sequence Antisense
Oligonucleotide 45 gcagtcaggt cgcccttcac 20 46 20 DNA Artificial
Sequence Antisense Oligonucleotide 46 ttcttggcag tcaggtcgcc 20 47
20 DNA Artificial Sequence Antisense Oligonucleotide 47 cattttcttg
gcagtcaggt 20 48 20 DNA Artificial Sequence Antisense
Oligonucleotide 48 gcaccatttt cttggcagtc 20 49 20 DNA Artificial
Sequence Antisense Oligonucleotide 49 cagagcaccg tggtcctgct 20 50
20 DNA Artificial Sequence Antisense Oligonucleotide 50 accaccacgc
agcagtccag 20 51 20 DNA Artificial Sequence Antisense
Oligonucleotide 51 tgacagccgt gagagagaat 20 52 20 DNA Artificial
Sequence Antisense Oligonucleotide 52 ggctggcctg acagccgtga 20 53
20 DNA Artificial Sequence Antisense Oligonucleotide 53 ggtggctggc
ctgacagccg 20 54 20 DNA Artificial Sequence Antisense
Oligonucleotide 54 gccgtagaca gcccctggga 20 55 20 DNA Artificial
Sequence Antisense Oligonucleotide 55 tccatctgtg ccgtagacag 20 56
20 DNA Artificial Sequence Antisense Oligonucleotide 56 ggtcccattg
aagatgttca 20 57 20 DNA Artificial Sequence Antisense
Oligonucleotide 57 ggcctggatg aaaaagagct 20 58 20 DNA Artificial
Sequence Antisense Oligonucleotide 58 cagagtgagc ccactgctca 20 59
20 DNA Artificial Sequence Antisense Oligonucleotide 59 agggactgca
ggtcttcaga 20 60 20 DNA Artificial Sequence Antisense
Oligonucleotide 60 cattagcgac cctaagcagg 20 61 20 DNA Artificial
Sequence Antisense Oligonucleotide 61 ggcatctgtt tataaatccc 20 62
20 DNA Artificial Sequence Antisense Oligonucleotide 62 gccctggcct
tatgatgttt 20 63 20 DNA Artificial Sequence Antisense
Oligonucleotide 63 aagtccaggc ctcagcctct 20 64 20 DNA Artificial
Sequence Antisense Oligonucleotide 64 aggaaagtcc aggcctcagc 20 65
20 DNA Artificial Sequence Antisense Oligonucleotide 65 ccggctgcaa
agtccttgag 20 66 20 DNA Artificial Sequence Antisense
Oligonucleotide 66 accctgtgcc ggctgcaaag 20 67 20 DNA Artificial
Sequence Antisense Oligonucleotide 67 ggaagctgct aagagcctgt 20 68
20 DNA Artificial Sequence Antisense Oligonucleotide 68 cctccactgt
tcagcacttg 20 69 20 DNA Artificial Sequence Antisense
Oligonucleotide 69 ttcatctgtc cctcttcctc 20 70 20 DNA Artificial
Sequence Antisense Oligonucleotide 70 ccacgtgcaa tccacggcat 20 71
20 DNA Artificial Sequence Antisense Oligonucleotide 71 gctcaagagg
ccacgtgcaa 20 72 20 DNA Artificial Sequence Antisense
Oligonucleotide 72 gatcatggga cacaagtcac 20 73 20 DNA Artificial
Sequence Antisense Oligonucleotide 73 aggaagacgc gttactggca 20 74
20 DNA Artificial Sequence Antisense Oligonucleotide 74 aacctttttg
tttggcacca 20 75 20 DNA Artificial Sequence Antisense
Oligonucleotide 75 gtttgctgat ctatgagcga 20 76 20 DNA Artificial
Sequence Antisense Oligonucleotide 76 ccggtttgct gatctatgag 20 77
20 DNA Artificial Sequence Antisense Oligonucleotide 77 catatttgcc
tcagatttat 20 78 20 DNA Artificial Sequence Antisense
Oligonucleotide 78 ccatatttgc ctcagattta 20 79 20 DNA Artificial
Sequence Antisense Oligonucleotide 79 ggacgcatct ccaaggcctc 20 80
20 DNA Artificial Sequence Antisense Oligonucleotide 80 gcctccggac
gcatctccaa 20 81 20 DNA Artificial Sequence Antisense
Oligonucleotide 81 ggtcagtctt cgctccccac 20 82 20 DNA Artificial
Sequence Antisense Oligonucleotide 82 ccccgggtca gtcttcgctc 20 83
20 DNA Artificial Sequence Antisense Oligonucleotide 83 acggccccgg
gtcagtcttc 20 84 20 DNA Artificial Sequence Antisense
Oligonucleotide 84 ctttctgctc gacatcacca 20 85 20 DNA Artificial
Sequence Antisense Oligonucleotide 85 ctatctcaaa ggcaggaaaa 20 86
20 DNA Artificial Sequence Antisense Oligonucleotide 86 tcaagagcac
ctgaagaggc 20 87 20 DNA Artificial Sequence Antisense
Oligonucleotide 87 ggagactcac caaatctgca 20 88 20 DNA Artificial
Sequence Antisense Oligonucleotide 88 agagcccgag ctactggccc 20 89
20 DNA Artificial Sequence Antisense Oligonucleotide 89 acagggaccc
acgtaaaccc 20 90 20 DNA Artificial Sequence Antisense
Oligonucleotide 90 ggatgtaagc ctgccagcac 20 91 20 DNA Artificial
Sequence Antisense Oligonucleotide 91 gtgagtgtac cttggcagtc 20 92
20 DNA Artificial Sequence Antisense Oligonucleotide 92 gccgagagag
ccagtcctca 20 93 20 DNA Artificial Sequence Antisense
Oligonucleotide 93 gcaccatttt ctacaagaga 20 94 20 DNA Artificial
Sequence Antisense Oligonucleotide 94 ggcttcctac ctgacagccg 20 95
20 DNA Artificial Sequence Antisense Oligonucleotide 95 ctgttcagag
gttttgtggg 20 96 20 DNA Artificial Sequence Antisense
Oligonucleotide 96 gagtaggaat ccaacagcct 20 97 20 DNA Artificial
Sequence Antisense Oligonucleotide 97 ggtggctggc ctagaagacc 20 98
20 DNA Artificial Sequence Antisense Oligonucleotide 98 ggagctgctt
acccccacca 20 99 20 DNA Artificial Sequence Antisense
Oligonucleotide 99 acctcttgtc attgaaatac 20 100 20 DNA Artificial
Sequence Antisense Oligonucleotide 100 tctttctgct ctgcaggaag 20 101
20 DNA Artificial Sequence Antisense Oligonucleotide 101 gaccttgact
catacataca 20 102 20 DNA Artificial Sequence Antisense
Oligonucleotide 102 aggaggacac gctgcccctc 20 103 20 DNA Artificial
Sequence Antisense Oligonucleotide 103 gcactgagga ccagtccatc 20 104
1255 DNA Mus musculus CDS (1)...(1182) 104 atg gac gag gcg gac cgg
cag ctc ctg cgg cga tgc agg gtg cgc cta 48 Met Asp Glu Ala Asp Arg
Gln Leu Leu Arg Arg Cys Arg Val Arg Leu 1 5 10 15 gtg agc gag ctg
caa gtc gcg gag ctc tgg gac gct ctg ctg agt cga 96 Val Ser Glu Leu
Gln Val Ala Glu Leu Trp Asp Ala Leu Leu Ser Arg 20 25
30 gag ctc ttc acg cgc gac atg atc gag gat att cag cag gca gga tct
144 Glu Leu Phe Thr Arg Asp Met Ile Glu Asp Ile Gln Gln Ala Gly Ser
35 40 45 ggg tct cgg cgg gat cag gcc agg cag ctg gtc aca gac ctt
gag acc 192 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Val Thr Asp Leu
Glu Thr 50 55 60 cga ggg agg cag gcc ctt cct ctc ttc atc tcc tgc
tta gag gac aca 240 Arg Gly Arg Gln Ala Leu Pro Leu Phe Ile Ser Cys
Leu Glu Asp Thr 65 70 75 80 ggc caa ggc acc ctg gct tca ctc ttg caa
agc ggt cgg caa gca gcc 288 Gly Gln Gly Thr Leu Ala Ser Leu Leu Gln
Ser Gly Arg Gln Ala Ala 85 90 95 aag cag gat cca gag gct gtt aaa
ccc cta gac cac ctg gtg cct gtg 336 Lys Gln Asp Pro Glu Ala Val Lys
Pro Leu Asp His Leu Val Pro Val 100 105 110 gtc ctg gga cca atg gga
ctc aca gca aag gag cag aga gta gtg aag 384 Val Leu Gly Pro Met Gly
Leu Thr Ala Lys Glu Gln Arg Val Val Lys 115 120 125 ctg gag ccg tca
cag cct gcc gtg gga aac ctc acc cca gtg gtg ctg 432 Leu Glu Pro Ser
Gln Pro Ala Val Gly Asn Leu Thr Pro Val Val Leu 130 135 140 ggg cca
gaa gag ctc tgg cct gct cgg ctc aag cca gag gtt ctc aga 480 Gly Pro
Glu Glu Leu Trp Pro Ala Arg Leu Lys Pro Glu Val Leu Arg 145 150 155
160 cca gaa aca ccc agg ccg gtg gac att ggt tct ggc gga gct cat gat
528 Pro Glu Thr Pro Arg Pro Val Asp Ile Gly Ser Gly Gly Ala His Asp
165 170 175 gtc tgt gtt cca ggg aag atc agg gga cat gca gat atg gca
tac acc 576 Val Cys Val Pro Gly Lys Ile Arg Gly His Ala Asp Met Ala
Tyr Thr 180 185 190 ctg gat tcg gat ccc tgt ggc cac tgc ctc atc atc
aac aat gtg aac 624 Leu Asp Ser Asp Pro Cys Gly His Cys Leu Ile Ile
Asn Asn Val Asn 195 200 205 ttc tgc cct tcc tcg ggg ctc ggc aca cgc
acg ggc tcc aac ttg gac 672 Phe Cys Pro Ser Ser Gly Leu Gly Thr Arg
Thr Gly Ser Asn Leu Asp 210 215 220 cgt gac aaa ctt gag cac cga ttc
cgc tgg ctg cgc ttc atg gtg gag 720 Arg Asp Lys Leu Glu His Arg Phe
Arg Trp Leu Arg Phe Met Val Glu 225 230 235 240 gtg aag aac gac ctg
act gcc aag aaa atg gtc acg gct ttg atg gag 768 Val Lys Asn Asp Leu
Thr Ala Lys Lys Met Val Thr Ala Leu Met Glu 245 250 255 atg gca cac
cgg aac cac cgt gcc ctg gac tgc ttt gtg gtg gtc atc 816 Met Ala His
Arg Asn His Arg Ala Leu Asp Cys Phe Val Val Val Ile 260 265 270 ctc
tct cat ggc tgc cag gcc agc cac ctc cag ttc ccg ggt gct gtc 864 Leu
Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val 275 280
285 tat ggg aca gat gga tgc tcc gtg tcc att gag aaa att gtg aat atc
912 Tyr Gly Thr Asp Gly Cys Ser Val Ser Ile Glu Lys Ile Val Asn Ile
290 295 300 ttc aac ggg agc ggc tgc ccc agc ctg gga ggg aag ccc aag
ctc ttc 960 Phe Asn Gly Ser Gly Cys Pro Ser Leu Gly Gly Lys Pro Lys
Leu Phe 305 310 315 320 ttc atc cag gcc tgc ggt ggt gag cag aaa gac
cat ggc ttt gag gtg 1008 Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys
Asp His Gly Phe Glu Val 325 330 335 gcc tgc act tcc tct caa ggc agg
acc ttg gac agt gac tct gag cca 1056 Ala Cys Thr Ser Ser Gln Gly
Arg Thr Leu Asp Ser Asp Ser Glu Pro 340 345 350 gat gct gtc cca tat
cag gaa ggc cca agg ccc ttg gac cag ctg gat 1104 Asp Ala Val Pro
Tyr Gln Glu Gly Pro Arg Pro Leu Asp Gln Leu Asp 355 360 365 gct gtg
tca agt ttg cct acc ccc agt gac atc ctt gtg tcc tac tcc 1152 Ala
Val Ser Ser Leu Pro Thr Pro Ser Asp Ile Leu Val Ser Tyr Ser 370 375
380 acc ttc cca ggt cgc caa tgc tgt ttc tga gaaagggact tacaagcaga
1202 Thr Phe Pro Gly Arg Gln Cys Cys Phe 385 390 ttcctggctg
ttttaacttc ctccggaaaa agctgttttt taaaacttca tga 1255 105 20 DNA
Artificial Sequence Antisense Oligonucleotide 105 accctgcatc
gccgcaggag 20 106 20 DNA Artificial Sequence Antisense
Oligonucleotide 106 gcaccctgca tcgccgcagg 20 107 20 DNA Artificial
Sequence Antisense Oligonucleotide 107 cactaggcgc accctgcatc 20 108
20 DNA Artificial Sequence Antisense Oligonucleotide 108 tgaatatcct
cgatcatgtc 20 109 20 DNA Artificial Sequence Antisense
Oligonucleotide 109 ctgaatatcc tcgatcatgt 20 110 20 DNA Artificial
Sequence Antisense Oligonucleotide 110 cctgctgaat atcctcgatc 20 111
20 DNA Artificial Sequence Antisense Oligonucleotide 111 cgccgagacc
cagatcctgc 20 112 20 DNA Artificial Sequence Antisense
Oligonucleotide 112 caaggtctgt gaccagctgc 20 113 20 DNA Artificial
Sequence Antisense Oligonucleotide 113 tctcaaggtc tgtgaccagc 20 114
20 DNA Artificial Sequence Antisense Oligonucleotide 114 ctccctcggg
tctcaaggtc 20 115 20 DNA Artificial Sequence Antisense
Oligonucleotide 115 gaagagagga agggcctgcc 20 116 20 DNA Artificial
Sequence Antisense Oligonucleotide 116 aagccagggt gccttggcct 20 117
20 DNA Artificial Sequence Antisense Oligonucleotide 117 ttgcaagagt
gaagccaggg 20 118 20 DNA Artificial Sequence Antisense
Oligonucleotide 118 tgccgaccgc tttgcaagag 20 119 20 DNA Artificial
Sequence Antisense Oligonucleotide 119 aggcaccagg tggtctaggg 20 120
20 DNA Artificial Sequence Antisense Oligonucleotide 120 ctcctttgct
gtgagtccca 20 121 20 DNA Artificial Sequence Antisense
Oligonucleotide 121 ctgctccttt gctgtgagtc 20 122 20 DNA Artificial
Sequence Antisense Oligonucleotide 122 acggctccag cttcactact 20 123
20 DNA Artificial Sequence Antisense Oligonucleotide 123 tgtgacggct
ccagcttcac 20 124 20 DNA Artificial Sequence Antisense
Oligonucleotide 124 aggctgtgac ggctccagct 20 125 20 DNA Artificial
Sequence Antisense Oligonucleotide 125 acggcaggct gtgacggctc 20 126
20 DNA Artificial Sequence Antisense Oligonucleotide 126 gtttcccacg
gcaggctgtg 20 127 20 DNA Artificial Sequence Antisense
Oligonucleotide 127 tgagaacctc tggcttgagc 20 128 20 DNA Artificial
Sequence Antisense Oligonucleotide 128 tctgagaacc tctggcttga 20 129
20 DNA Artificial Sequence Antisense Oligonucleotide 129 tggtctgaga
acctctggct 20 130 20 DNA Artificial Sequence Antisense
Oligonucleotide 130 ccaatgtcca ccggcctggg 20 131 20 DNA Artificial
Sequence Antisense Oligonucleotide 131 ccctgatctt ccctggaaca 20 132
20 DNA Artificial Sequence Antisense Oligonucleotide 132 ccatatctgc
atgtcccctg 20 133 20 DNA Artificial Sequence Antisense
Oligonucleotide 133 cagggtgtat gccatatctg 20 134 20 DNA Artificial
Sequence Antisense Oligonucleotide 134 aatccagggt gtatgccata 20 135
20 DNA Artificial Sequence Antisense Oligonucleotide 135 cgaatccagg
gtgtatgcca 20 136 20 DNA Artificial Sequence Antisense
Oligonucleotide 136 attgttgatg atgaggcagt 20 137 20 DNA Artificial
Sequence Antisense Oligonucleotide 137 acattgttga tgatgaggca 20 138
20 DNA Artificial Sequence Antisense Oligonucleotide 138 gtcacggtcc
aagttggagc 20 139 20 DNA Artificial Sequence Antisense
Oligonucleotide 139 tgctcaagtt tgtcacggtc 20 140 20 DNA Artificial
Sequence Antisense Oligonucleotide 140 agcggaatcg gtgctcaagt 20 141
20 DNA Artificial Sequence Antisense Oligonucleotide 141 cttcacctcc
accatgaagc 20 142 20 DNA Artificial Sequence Antisense
Oligonucleotide 142 tcttcacctc caccatgaag 20 143 20 DNA Artificial
Sequence Antisense Oligonucleotide 143 tcttggcagt caggtcgttc 20 144
20 DNA Artificial Sequence Antisense Oligonucleotide 144 gcacggtggt
tccggtgtgc 20 145 20 DNA Artificial Sequence Antisense
Oligonucleotide 145 agcagtccag ggcacggtgg 20 146 20 DNA Artificial
Sequence Antisense Oligonucleotide 146 ccacaaagca gtccagggca 20 147
20 DNA Artificial Sequence Antisense Oligonucleotide 147 gaggatgacc
accacaaagc 20 148 20 DNA Artificial Sequence Antisense
Oligonucleotide 148 gcctggcagc catgagagag 20 149 20 DNA Artificial
Sequence Antisense Oligonucleotide 149 gtggctggcc tggcagccat 20 150
20 DNA Artificial Sequence Antisense Oligonucleotide 150 gacagcaccc
gggaactgga 20 151 20 DNA Artificial Sequence Antisense
Oligonucleotide 151 attcacaatt ttctcaatgg 20 152 20 DNA Artificial
Sequence Antisense Oligonucleotide 152 caaagccatg gtctttctgc 20 153
20 DNA Artificial Sequence Antisense Oligonucleotide 153 aggccacctc
aaagccatgg 20 154 20 DNA Artificial Sequence Antisense
Oligonucleotide 154 gtcctgcctt gagaggaagt 20 155 20 DNA Artificial
Sequence Antisense Oligonucleotide 155 atctggctca gagtcactgt 20 156
20 DNA Artificial Sequence Antisense Oligonucleotide 156 gcatccagct
ggtccaaggg 20 157 20 DNA Artificial Sequence Antisense
Oligonucleotide 157 ttgacacagc atccagctgg 20 158 20 DNA Artificial
Sequence Antisense Oligonucleotide 158 taggcaaact tgacacagca 20 159
20 DNA Artificial Sequence Antisense Oligonucleotide 159 ggtaggcaaa
cttgacacag 20 160 20 DNA Artificial Sequence Antisense
Oligonucleotide 160 aggtggagta ggacacaagg 20 161 20 DNA Artificial
Sequence Antisense Oligonucleotide 161 aacctgggaa ggtggagtag 20 162
20 DNA Artificial Sequence Antisense Oligonucleotide 162 tttcttgtcc
ctccaggaga 20 163 20 DNA Artificial Sequence Antisense
Oligonucleotide 163 gagccacttt tcttgtccct 20 164 20 DNA Artificial
Sequence Antisense Oligonucleotide 164 taccaggagc cacttttctt 20 165
20 DNA Artificial Sequence Antisense Oligonucleotide 165 gatgtaccag
gagccacttt 20 166 20 DNA Artificial Sequence Antisense
Oligonucleotide 166 tccaaggtct cgatgtacca 20 167 20 DNA Artificial
Sequence Antisense Oligonucleotide 167 agaatgccat ccaaggtctc 20 168
20 DNA Artificial Sequence Antisense Oligonucleotide 168 actgctccag
aatgccatcc 20 169 20 DNA Artificial Sequence Antisense
Oligonucleotide 169 accctgagaa ggagggactg 20 170 20 DNA Artificial
Sequence Antisense Oligonucleotide 170 aagtcccttt ctcagaaaca 20 171
20 DNA Artificial Sequence Antisense Oligonucleotide 171 cagccaggaa
tctgcttgta 20 172 20 DNA Artificial Sequence Antisense
Oligonucleotide 172 ttttccggag gaagttaaaa 20 173 20 DNA Artificial
Sequence Antisense Oligonucleotide 173 cagctttttc cggaggaagt 20 174
20 DNA Artificial Sequence Antisense Oligonucleotide 174 gcattggcga
cctgggaagg 20
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