U.S. patent application number 10/415198 was filed with the patent office on 2005-11-24 for antisense modulation of cellular apoptosis susceptibity gene expression.
Invention is credited to Cowsert, Lex M, Freier, Susan M.
Application Number | 20050260578 10/415198 |
Document ID | / |
Family ID | 24832854 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050260578 |
Kind Code |
A1 |
Cowsert, Lex M ; et
al. |
November 24, 2005 |
Antisense modulation of cellular apoptosis susceptibity gene
expression
Abstract
Antisense compounds, compositions and methods are provided for
modulating the expression of cellular apoptosis susceptibility
gene. The compositions comprise antisense compounds, particularly
antisense oligonucleotides, targeted to nucleic acids encoding
cellular apoptosis susceptibility gene. Methods of using these
compounds for modulation of cellular apoptosis susceptibility gene
expression and for treatment of diseases associated with expression
of cellular apoptosis susceptibility gene are provided.
Inventors: |
Cowsert, Lex M; (Pittsburgh,
PA) ; Freier, Susan M; (San Diego, CA) |
Correspondence
Address: |
ISIS PHARMACEUTICALS INC
1896 RUTHERFORD RD.
CARLSBAD
CA
92008
US
|
Family ID: |
24832854 |
Appl. No.: |
10/415198 |
Filed: |
November 10, 2003 |
PCT Filed: |
October 29, 2001 |
PCT NO: |
PCT/US01/51048 |
Current U.S.
Class: |
435/6.14 ;
435/6.16; 514/44A; 536/23.2 |
Current CPC
Class: |
C12N 2310/321 20130101;
C12N 2310/3525 20130101; C12N 2310/3341 20130101; C12N 2310/341
20130101; C12N 2310/346 20130101; A61K 38/00 20130101; C12N 15/113
20130101; C12N 2310/315 20130101; Y02P 20/582 20151101; C12N
2310/321 20130101 |
Class at
Publication: |
435/006 ;
514/044; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 1, 2000 |
US |
09/705,299 |
Claims
What is claimed is:
1. A compound 8 to 50 nucleobases in length targeted to a nucleic
acid molecule encoding cellular apoptosis susceptibility gene,
wherein said compound specifically hybridizes with and inhibits the
expression of cellular apoptosis susceptibility gene.
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: 17, 18, 19, 21, 22, 26, 27,
28, 29, 31, 33, 34, 35, 36, 37, 39, 41, 42, 43, 44, 45, 46, 47, 49,
50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 63, 65, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 or 84.
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 cellular apoptosis
susceptibility gene.
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 cellular apoptosis
susceptibility gene in cells or tissues comprising contacting said
cells or tissues with the compound of claim 1 so that expression of
cellular apoptosis susceptibility gene is inhibited.
16. A method of treating an animal having a disease or condition
associated with cellular apoptosis susceptibility gene comprising
administering to said animal a therapeutically or prophylactically
effective amount of the compound of claim 1 so that expression of
cellular apoptosis susceptibility gene is inhibited.
17. The method of claim 16 wherein the disease or condition is a
hyperproliferative disorder.
18. The method of claim 17 wherein the disease or condition is is
cancer.
19. The method of claim 18 wherein the cancer is breast cancer,
lung cancer, prostate cancer, melanoma, colon cancer, a lymphoid
cancer or a hematopoietic cancer.
20. The method of claim 19 wherein the cancer is breast cancer or
colon cancer.
Description
FIELD OF THE INVENTION
[0001] The present invention provides compositions and methods for
modulating the expression of cellular apoptosis susceptibility
gene. In particular, this invention relates to compounds,
particularly oligonucleotides, specifically hybridizable with
nucleic acids encoding cellular apoptosis susceptibility gene. Such
compounds have been shown to modulate the expression of cellular
apoptosis susceptibility gene.
BACKGROUND OF THE INVENTION
[0002] 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).
[0003] 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.
[0004] Cellular apoptosis susceptibility gene (also known as CAS,
CSE1 and CSP) is the human homolog of the yeast chromosome
segregation gene, CSE1, and has been simultaneously implicated in
the regulation of mitosis, apoptosis and cellular proliferation
(Brinkmann et al., Biochemistry, 1996, 35, 6891-6899; Scherf et
al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 2670-2674).
[0005] CAS was first identified in a screen for genes that affect
the sensitivity of breast cancer cells toward toxins used in
experimental cancer therapy. In these screens cellular apoptosis
susceptibility gene was isolated as antisense cDNA fragments that
rendered MCF-7 breast cancer cells resistent to cell death induced
by exotoxins, exotoxin-derived immunotoxins, diptheria toxin and
tumor necrosis factor (Brinkmann et al., Proc. Natl. Acad. Sci.
U.S.A., 1995, 92, 10427-10431; Ogryzko et al., Biochemistry, 1997,
36, 9493-9500). Characterization of the protein has since revealed
that the cellular apoptosis susceptibility gene is highly expressed
in other cancer cells including lymphoid neoplasms (Wellmann et
al., American Journal of Pathology, 1997, 150, 25-30), benign and
malignant cutaneous melanocytic lesions (Boni et al., The American
Journal of Dermatopathology, 1999, 21, 125-128), and colon cancer
cell lines (Brinkmann, Am. J. Hum. Genet., 1998, 62, 509-513;
Brinkmann et al., Genome Research, 1996, 6, 187-194).
[0006] It has been determined that cellular apoptosis
susceptibility gene undergoes alterntive splicing in a tissue- and
development-specific manner (Brinkmann et al., Genomics, 1999, 58,
41-49). Northern blot analyses have shown that the predominant
transcript in proliferating tissues is a 3147 nucleotide
transcript, while a larger transcripts can be detected in fetal and
adult brain and smaller transcripts can be detected in trachea,
liver and some cancers (Brinkmann et al., Genomics, 1999, 58,
41-49). The protein and nucleic acid sequences of the human
cellular apoptosis susceptibility gene are disclosed in U.S. Pat.
No. 5,759,782 and its corresponding PCT publication WO 96/40713 and
U.S. Pat. No. 6,072,031, respectively (Pastan and Brinkmann, 2000;
Pastan and Brinkmann, 1996; Pastan and Brinkmann, 1998). Also
disclosed are antibodies to the protein, protein fragments and an
isolated single-stranded antisense DNA sequence consisting of
nucleotides 2100-2536 of the human cellular apoptosis
susceptibility gene (Pastan and Brinkmann, 1996; Pastan and
Brinkmann, 1998).
[0007] The human cellular apoptosis susceptibility gene is located
on chromosome 20q13 and is amplified in BT474 breast cancer cells
(Brinkmann et al., Genome Research, 1996, 6, 187-194). This
chromosomal location harbors a remarkable degree of instability in
various tumors and amplification in this region is observed
frequently in aggressive types of breast cancers (Brinkmann et al.,
Genome Research, 1996, 6, 187-194).
[0008] Cellular apoptosis susceptibility gene has also been shown
to mediate export of importin-.alpha. from the nucleus (Kutay et
al., Cell, 1997, 90, 1061-1071). Importin-.alpha. is the nuclear
import receptor for nuclear localization signal-containing
proteins. This interaction, which is regulated by phosphorylation
events, requires the presence of RanGTP to form a ternary complex;
and it has been suggested that deregulation of importin transport
can cause cell cycle defects (Kutay et al., Cell, 1997, 90,
1061-1071; Scherf et al., Biochemical and Biophysical Research
Communications, 1998, 250, 623-628).
[0009] Collectively, these data suggest that modulation of cellular
apoptosis susceptibility gene would render opportunity to treat
patients with various cancers and deregulated apoptotic pathologic
conditions.
[0010] Strategies aimed at modulating cellular apoptosis
susceptibility gene function have involved the use of antibodies
and antisense expression vectors but currently, there are no known
therapeutic agents which effectively inhibit the synthesis of
cellular apoptosis susceptibility gene. Consequently, there remains
a long felt need for agents capable of effectively inhibiting
cellular apoptosis susceptibility gene function.
[0011] 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 cellular apoptosis
susceptibility gene expression.
[0012] Currently there exists a need to identify methods of
modulating apoptosis for the therapeutic treatment of human
diseases and it is believed that cellular apoptosis susceptibility
gene modulators will be integral to these methods. The present
invention, therefore, provides compositions and methods for
modulating cellular apoptosis susceptibility gene expression,
including modulation of alternatively spliced forms of cellular
apoptosis susceptibility gene.
SUMMARY OF THE INVENTION
[0013] The present invention is directed to compounds, particularly
antisense oligonucleotides, which are targeted to a nucleic acid
encoding cellular apoptosis susceptibility gene, and which modulate
the expression of cellular apoptosis susceptibility gene.
Pharmaceutical and other compositions comprising the compounds of
the invention are also provided. Further provided are methods of
modulating the expression of cellular apoptosis susceptibility gene
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 cellular
apoptosis susceptibility gene 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
[0014] The present invention employs oligomeric compounds,
particularly antisense oligonucleotides, for use in modulating the
function of nucleic acid molecules encoding cellular apoptosis
susceptibility gene, ultimately modulating the amount of cellular
apoptosis susceptibility gene produced. This is accomplished by
providing antisense compounds which specifically hybridize with one
or more nucleic acids encoding cellular apoptosis susceptibility
gene. As used herein, the terms "target nucleic acid" and "nucleic
acid encoding cellular apoptosis susceptibility gene" encompass DNA
encoding cellular apoptosis susceptibility gene, 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 cellular apoptosis susceptibility gene. 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.
[0015] 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 cellular apoptosis susceptibility gene. 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 cellular apoptosis susceptibility gene, regardless of
the sequence(s) of such codons.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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 a basic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0029] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555;
5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are
commonly owned with this application, and each of which is herein
incorporated by reference.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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'-dimethylamino-ethoxyethoxy (also known in the art as
2'-O-dimethylamino-ethoxyethyl 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.
[0035] 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.
[0036] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy (2'-OCH.sub.2
CH.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.su- b.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.
[0037] 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]benzoxaz- in-2 (3H)-one), phenothiazine
cytidine (1H-pyrimido[5,4-b][1,4]benzothiazi- n-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-aminopropyl-adenine, 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.
[0038] 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.
[0039] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. The
compounds of the invention can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugates groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve oligomer
uptake, enhance oligomer resistance to degradation, and/or
strengthen sequence-specific hybridization with RNA. Groups that
enhance the pharmacokinetic properties, in the context of this
invention, include groups that improve oligomer uptake,
distribution, metabolism or excretion. Representative conjugate
groups are disclosed in International Patent Application
PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which
is incorporated herein by reference. Conjugate moieties include but
are not limited to lipid moieties such as a cholesterol moiety
(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86,
6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.,
1994, 4, 1053-1060), a thioether, e.g., hexyl-5-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,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) which is incorporated herein by reference in its
entirety.
[0040] 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.
[0041] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the
oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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 cellular apoptosis susceptibility gene
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.
[0052] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding cellular apoptosis susceptibility gene,
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 cellular apoptosis
susceptibility gene 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 cellular apoptosis susceptibility gene in a sample may
also be prepared.
[0053] 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.
[0054] 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.
[0055] 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. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673
(filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999),
Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298
(filed May 20, 1999) each of which is incorporated herein by
reference in their entirety.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] Emulsions
[0062] 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.
[0063] 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).
[0064] 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).
[0065] 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.
[0066] 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).
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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).
[0071] 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.
[0072] 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 (DA0750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized
C.sub.8-C.sub.10 glycerides, vegetable oils and silicone oil.
[0073] 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.
[0074] 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.
[0075] Liposomes
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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).
[0084] 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).
[0085] 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.
[0086] 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).
[0087] 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).
[0088] 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.).
[0089] 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. No. 5,540,935
(Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.)
describe PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0090] 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.
[0091] 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.
[0092] 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).
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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).
[0098] Penetration Enhancers
[0099] 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.
[0100] 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.
[0101] Surfactants: In connection with the present invention,
surfactants (br "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).
[0102] 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).
[0103] 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).
[0104] 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).
[0105] 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).
[0106] 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.
[0107] 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.
[0108] Carriers
[0109] 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).
[0110] Excipients
[0111] 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.).
[0112] 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.
[0113] 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.
[0114] 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.
[0115] Other Components
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] While the present invention has been described with
specificity in accordance with certain of its preferred
embodiments, the following examples serve only to illustrate the
invention and are not intended to limit the same.
EXAMPLES
Example 1
Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and
2'-alkoxy amidites
[0122] 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.
[0123] Oligonucleotides containing 5-methyl-2'-deoxycytidine
(5-Me-C) nucleotides were synthesized according to published
methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21,
3197-3203] using commercially available phosphoramidites (Glen
Research, Sterling Va. or ChemGenes, Needham Mass.).
2'-Fluoro amidites
2'-Fluorodeoxyadenosine amidites
[0124] 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.
2'-Fluorodeoxyguanosine
[0125] The synthesis of 2.sup.1-deoxy-2'-fluoroguanosine was
accomplished using tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosy- lguanine as starting material, and
conversion to the intermediate
diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS
group was followed by protection of the hydroxyl group with THP to
give diisobutyryl di-THP protected arabinofuranosylguanine.
Selective O-deacylation and triflation was followed by treatment of
the crude product with fluoride, then deprotection of the THP
groups. Standard methodologies were used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidi- tes.
2'-Fluorouridine
[0126] Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by
the modification of a literature procedure in which
2,2'-anhydro-1-beta-D-ara- binofuranosyluracil was treated with 70%
hydrogen fluoride-pyridine. Standard procedures were used to obtain
the 5'-DMT and 5'-DMT-3'phosphoramidites.
2'-Fluorodeoxycytidine
[0127] 2'-deoxy-2'-fluorocytidine was synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were
used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.
2'-O-(2-Methoxyethyl) modified amidites
[0128] 2'-O-Methoxyethyl-substituted nucleoside amidites are
prepared as follows, or alternatively, as per the methods of
Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.
2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]
[0129] 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.).
2'-O-Methoxyethyl-5-methyluridine
[0130] 2,2'-Anhydro-5-methyluridine (195 g, 0.81 M),
tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol
(1.2 L) were added to a 2 L stainless steel pressure vessel and
placed in a pre-heated oil bath at 160.degree. C. After heating for
48 hours at 155-160.degree. C., the vessel was opened and the
solution evaporated to dryness and triturated with MeOH (200 mL).
The residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 mL) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2Cl.sub.2/acetone/MeOH (20:5:3) containing 0.5% Et.sub.3NH.
The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL) and adsorbed
onto silica (150 g) prior to loading onto the column. The product
was eluted with the packing solvent to give 160 g (63%) of product.
Additional material was obtained by reworking impure fractions.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5 methyluridine
[0131] 2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was
co-evaporated with pyridine (250 mL) and the dried residue
dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the mixture stirred at
room temperature for one hour. A second aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the reaction stirred for
an additional one hour. Methanol (170 mL) was then added to stop
the reaction. HPLC showed the presence of approximately 70%
product. The solvent was evaporated and triturated with CH.sub.3CN
(200 mL). The residue was dissolved in CHCl.sub.3 (1.5 L) and
extracted with 2.times.500 mL of saturated NaHCO.sub.3 and
2.times.500 mL of saturated NaCl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered and evaporated. 275 g of residue was
obtained. The residue was purified on a 3.5 kg silica gel column,
packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5%
Et.sub.3NH. The pure fractions were evaporated to give 164 g of
product. Approximately 20 g additional was obtained from the impure
fractions to give a total yield of 183 g (57%).
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0132] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106
g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from
562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38
mL, 0.258 M) were combined and stirred at room temperature for 24
hours. The reaction was monitored by TLC by first quenching the TLC
sample with the addition of MeOH. Upon completion of the reaction,
as judged by TLC, MeOH (50 mL) was added and the mixture evaporated
at 35.degree. C. The residue was dissolved in CHCl.sub.3 (800 mL)
and extracted with 2.times.200 mL of saturated sodium bicarbonate
and 2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approx. 90% product). The residue was purified on a 3.5 kg silica
gel column and eluted using EtOAc/hexane (4:1). Pure product
fractions were evaporated to yield 96 g (84%). An additional 1.5 g
was recovered from later fractions.
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleurid-
ine
[0133] A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methox-
yethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in
CH.sub.3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M)
was added to a solution of triazole (90 g, 1.3 M) in CH.sub.3CN (1
L), cooled to -5.degree. C. and stirred for 0.5 h using an overhead
stirrer. POCl.sub.3 was added dropwise, over a 30 minute period, to
the stirred solution maintained at 0-10.degree. C., and the
resulting mixture stirred for an additional 2 hours. The first
solution was added dropwise, over a 45 minute period, to the latter
solution. The resulting reaction mixture was stored overnight in a
cold room. Salts were filtered from the reaction mixture and the
solution was evaporated. The residue was dissolved in EtOAc (1 L)
and the insoluble solids were removed by filtration. The filtrate
was washed with 1.times.300 mL of NaHCO.sub.3 and 2.times.300 mL of
saturated NaCl, dried over sodium sulfate and evaporated. The
residue was triturated with EtOAc to give the title compound.
2'-O-Methoxyethyl-51-O-dimethoxytrityl-5-methylcytidine
[0134] A solution of
3'-O-acetyl-21-O-methoxyethyl-5'-O-dimethoxytrityl-5--
methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and
NH.sub.4OH (30 mL) was stirred at room temperature for 2 hours. The
dioxane solution was evaporated and the residue azeotroped with
MeOH (2.times.200 mL). The residue was dissolved in MeOH (300 mL)
and transferred to a 2 liter stainless steel pressure vessel. MeOH
(400 mL) saturated with NH.sub.3 gas was added and the vessel
heated to 100.degree. C. for 2 hours (TLC showed complete
conversion). The vessel contents were evaporated to dryness and the
residue was dissolved in EtOAc (500 mL) and washed once with
saturated NaCl (200 mL). The organics were dried over sodium
sulfate and the solvent was evaporated to give 85 g (95%) of the
title compound.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0135] 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.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amid-
ite
[0136]
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.
2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites
2'-(Dimethylaminooxyethoxy) nucleoside amidites
[0137] 2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known
in the art as 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are
prepared as described in the following paragraphs. Adenosine,
cytidine and guanosine nucleoside amidites are prepared similarly
to the thymidine (5-methyluridine) except the exocyclic amines are
protected with a benzoyl moiety in the case of adenosine and
cytidine and with isobutyryl in the case of guanosine.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
[0138] 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.
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
[0139] 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.
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
[0140]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g,
44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was
then dried over P.sub.2O.sub.5 under high vacuum for two days at
40.degree. C. The reaction mixture was flushed with argon and dry
THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear
solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added
dropwise to the reaction mixture. The rate of addition is
maintained such that resulting deep red coloration is just
discharged before adding the next drop. After the addition was
complete, the reaction was stirred for 4 hrs. By that time TLC
showed the completion of the reaction (ethylacetate:hexane, 60:40).
The solvent was evaporated in vacuum. Residue obtained was placed
on a flash column and eluted with ethyl acetate:hexane (60:40), to
get
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
as white foam (21.819 g, 86%).
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methylurid-
ine
[0141]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridi-
ne (3.1 g, 4.5 mmol) was dissolved in dry CH.sub.2Cl.sub.2 (4.5 mL)
and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at
-10.degree. C. to 0.degree. C. After 1 h the mixture was filtered,
the filtrate was washed with ice cold CH.sub.2Cl.sub.2 and the
combined organic phase was washed with water, brine and dried over
anhydrous Na.sub.2SO.sub.4. The solution was concentrated to get
2'-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH
(67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1
eq.) was added and the resulting mixture was strirred for 1 h.
Solvent was removed under vacuum; residue chromatographed to get
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)
ethyl]-5-methyluridine as white foam (1.95 g, 78%).
5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methylurid-
ine
[0142]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M
pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium
cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at
10.degree. C. under inert atmosphere. The reaction mixture was
stirred for 10 minutes at 10.degree. C. After that the reaction
vessel was removed from the ice bath and stirred at room
temperature for 2 h, the reaction monitored by TLC (5% MeOH in
CH.sub.2Cl.sub.2). Aqueous NaHCO.sub.3 solution (5%, 10 mL) was
added and extracted with ethyl acetate (2.times.20 mL). Ethyl
acetate phase was dried over anhydrous Na.sub.2SO.sub.4, evaporated
to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH
(30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and
the reaction mixture was stirred at room temperature for 10
minutes. Reaction mixture cooled to 10.degree. C. in an ice bath,
sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction
mixture stirred at 10.degree. C. for 10 minutes. After 10 minutes,
the reaction mixture was removed from the ice bath and stirred at
room temperature for 2 hrs. To the reaction mixture 5% NaHCO.sub.3
(25 mL) solution was added and extracted with ethyl acetate
(2.times.25 mL). Ethyl acetate layer was dried over anhydrous
Na.sub.2SO.sub.4 and evaporated to dryness The residue obtained was
purified by flash column chromatography and eluted with 5% MeOH in
CH.sub.2Cl.sub.2 to get
5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluri-
dine as a white foam (14.6 g, 80%).
2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0143] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was
dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept
over KOH). This mixture of triethylamine-2HF was then added to
5'-O-tert-butyldiphenylsil-
yl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4
mmol) and stirred at room temperature for 24 hrs. Reaction was
monitored by TLC (5% MeOH in CH.sub.2Cl.sub.2). Solvent was removed
under vacuum and the residue placed on a flash column and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 to get
2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0144] 2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17
mmol) was dried over P.sub.2O.sub.5 under high vacuum overnight at
40.degree. C. It was then co-evaporated with anhydrous pyridine (20
mL). The residue obtained was dissolved in pyridine (11 mL) under
argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol),
4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the
mixture and the reaction mixture was stirred at room temperature
until all of the starting material disappeared. Pyridine was
removed under vacuum and the residue chromatographed and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 (containing a few drops of
pyridine) to get 5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5--
methyluridine (1.13 g, 80%).
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoet-
hyl)-N,N-diisopropylphosphoramidite]
[0145] 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.2O.sub.5 under high vacuum overnight at
40.degree. C. Then the reaction mixture was dissolved in anhydrous
acetonitrile (8.4 mL) and
2-cyanoethyl-N,N,N.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%).
2'-(Aminooxyethoxy) nucleoside amidites
[0146] 2'-(Aminooxyethoxy) nucleoside amidites [also known in the
art as 2'-O-(aminooxyethyl) nucleoside amidites] are prepared as
described in the following paragraphs. Adenosine, cytidine and
thymidine nucleoside amidites are prepared similarly.
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimeth-
oxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]
[0147] The 2'-O-aminooxyethyl guanosine analog may be obtained by
selective 2'-O-alkylation of diaminopurine riboside. Multigram
quantities of diaminopurine riboside may be purchased from Schering
AG (Berlin) to provide 2'-O-(2-ethylacetyl)diaminopurine riboside
along with a minor amount of the 3'-O-isomer. 2'-O-(2-ethylacetyl)
diaminopurine riboside may be resolved and converted to
2'-O-(2-ethylacetyl)guanosine by treatment with adenosine
deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO
94/02501 A1 940203.) Standard protection procedures should afford
2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine and
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'--
dimethoxytrityl)guanosine which may be reduced to provide
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-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].
2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites
[0148] 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.
2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine
[0149] 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.
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)
ethyl)]-5-methyl uridine
[0150] To 0.5 g (1.3 mmol) of
2'-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5- -methyl uridine in
anhydrous pyridine (8 mL), triethylamine (0.36 mL) and
dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and
stirred for 1 hour. The reaction mixture is poured into water (200
mL) and extracted with CH.sub.2Cl.sub.2 (2.times.200 mL). The
combined CH.sub.2Cl.sub.2 layers are washed with saturated
NaHCO.sub.3 solution, followed by saturated NaCl solution and dried
over anhydrous sodium sulfate. Evaporation of the solvent followed
by silica gel chromatography using MeOH:CH.sub.2Cl.sub.2:Et.sub.3N
(20:1, v/v, with 1% triethylamine) gives the title compound.
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite
[0151] 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
[0152] Oligonucleotide Synthesis
[0153] 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.
[0154] 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.
[0155] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0156] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050,
herein incorporated by reference.
[0157] Phosphoramidite oligonucleotides are prepared as described
in U.S. Patent, 5,256,775 or U.S. Pat. No. 5,366,878, herein
incorporated by reference.
[0158] 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.
[0159] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0160] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0161] 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
[0162] Oligonucleoside Synthesis
[0163] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides,
methylenedi-methylhydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligo-nucleosides, also identified as amide-4 linked
oligo-nucleosides, 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.
[0164] 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.
[0165] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 4
[0166] PNA Synthesis
[0167] 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
[0168] Synthesis of Chimeric Oligonucleotides
[0169] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[2'-O-Me]--[2'-deoxy]--[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0170] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligo-nucleotide 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-phosphor-amidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
increasing the wait step after the delivery of tetrazole and base
to 600 s repeated four times for RNA and twice for 2'-O-methyl. The
fully protected oligonucleotide is cleaved from the support and the
phosphate group is deprotected in 3:1 ammonia/ethanol at room
temperature overnight then lyophilized to dryness. Treatment in
methanolic ammonia for 24 hrs at room temperature is then done to
deprotect all bases and sample was again lyophilized to dryness.
The pellet is resuspended in 1M TBAF in THF for 24 hrs at room
temperature to deprotect the 2' positions. The reaction is then
quenched with 1M TEAA and the sample is then reduced to 1/2 volume
by rotovac before being desalted on a G25 size exclusion column.
The oligo recovered is then analyzed spectrophotometrically for
yield and for purity by capillary electrophoresis and by mass
spectrometry.
[2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)] Chimeric
Phosphorothioate Oligonucleotides
[0171] [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.
[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy
Phosphorothioate]--[2'-O-- (2-Methoxyethyl) Phosphodiester]
Chimeric Oligonucleotides
[0172] [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.
[0173] Other chimeric oligonucleotides, chimeric oligo-nucleosides
and mixed chimeric oligonucleotides/oligo-nucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 6
[0174] Oligonucleotide Isolation
[0175] 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
[0176] Oligonucleotide Synthesis--96 Well Plate Format
[0177] 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.
[0178] 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
[0179] Oligonucleotide Analysis--96 Well Plate Format
[0180] 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
[0181] Cell Culture and Oligonucleotide Treatment
[0182] 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 4 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.
[0183] T-24 Cells:
[0184] 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.
[0185] 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.
[0186] A549 Cells:
[0187] 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.
[0188] NHDF Cells:
[0189] 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.
[0190] HEK Cells:
[0191] 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.
[0192] Treatment with Antisense Compounds:
[0193] 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.
[0194] 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
[0195] Analysis of Oligonucleotide Inhibition of Cellular Apoptosis
Susceptibility Gene Expression
[0196] Antisense modulation of cellular apoptosis susceptibility
gene expression can be assayed in a variety of ways known in the
art. For example, cellular apoptosis susceptibility gene 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.
[0197] Protein levels of cellular apoptosis susceptibility gene 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 cellular apoptosis susceptibility gene 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.
[0198] 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
[0199] Poly(A)+ mRNA Isolation
[0200] Poly(A)+ mRNA was isolated according to Miura et al., Clin.
Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA
isolation are taught in, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3,
John Wiley & Sons, Inc., 1993. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C. was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0201] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
[0202] Total RNA Isolation
[0203] Total RNA was isolated using an RNEASY 96.TM. kit and
buffers purchased from Qiagen Inc. (Valencia Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 100 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 100 .mu.L of 70% ethanol was then added to each well and
the contents mixed by pipetting three times up and down. The
samples were then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum was applied for 15
seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY
96.TM. plate and the vacuum again applied for 15 seconds. 1 mL of
Buffer RPE was then added to each well of the RNEASY 96.TM. plate
and the vacuum applied for a period of 15 seconds. The Buffer RPE
wash was then repeated and the vacuum was applied for an additional
10 minutes. The plate was then removed from the QIAVAC.TM. manifold
and blotted dry on paper towels. The plate was then re-attached to
the QIAVAC.TM. manifold fitted with a collection tube rack
containing 1.2 mL collection tubes. RNA was then eluted by
pipetting 60 .mu.L water into each well, incubating 1 minute, and
then applying the vacuum for 30 seconds. The elution step was
repeated with an additional 60 .mu.L water.
[0204] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 13
[0205] Real-Time Quantitative PCR Analysis of Cellular Apoptosis
Susceptibility Gene mRNA Levels
[0206] Quantitation of cellular apoptosis susceptibility gene mRNA
levels was determined by real-time quantitative PCR using the ABI
PRISMS 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 51 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.
[0207] 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.
[0208] 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).
[0209] 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.
[0210] 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.
[0211] Probes and primers to human cellular apoptosis
susceptibility gene were designed to hybridize to a human cellular
apoptosis susceptibility gene sequence, using published sequence
information (GenBank accession number AF053641, incorporated herein
as SEQ ID NO:3). For human cellular apoptosis susceptibility gene
the PCR primers were:
[0212] forward primer: GGAGAATTGTTGAAGATGAACCAA (SEQ ID NO: 4)
[0213] reverse primer: CTGGGCTGCTAAGCATCAAGT (SEQ ID NO: 5) and the
PCR probe was: FAM-TTTGTGAAGCCGATCGAGTGGCC-TAMRA (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.
[0214] For human GAPDH the PCR primers were:
[0215] forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 7)
[0216] reverse primer: GGCAACAATATCCACTTTACCAGAGT (SEQ ID NO: 8)
and the PCR probe was: 5' JOE-CGCCTGGTCACCAGGGCTGCT-TAMRA 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.
Example 14
[0217] Northern Blot Analysis of Cellular Apoptosis Susceptibility
Gene mRNA Levels
[0218] 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.
[0219] To detect human cellular apoptosis susceptibility gene, a
human cellular apoptosis susceptibility gene specific probe was
prepared by PCR using the forward primer GGAGAATTGTTGAAGATGAACCAA
(SEQ ID NO: 4) and the reverse primer CTGGGCTGCTAAGCATCAAGT (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.).
[0220] 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
[0221] Antisense Inhibition of Human Cellular Apoptosis
Susceptibility Gene Expression by Chimeric Phosphorothioate
Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap
[0222] In accordance with the present invention, a series of
oligonucleotides were designed to target different regions of the
human cellular apoptosis susceptibility gene RNA, using published
sequences (GenBank accession number AF053641, incorporated herein
as SEQ ID NO: 3, GenBank accession number AF053643, incorporated
herein as SEQ ID NO: 10, GenBank accession number AF053644,
incorporated herein as SEQ ID NO: 11, GenBank accession number
AF053645, incorporated herein as SEQ ID NO: 12, GenBank accession
number AF053650, incorporated herein as SEQ ID NO: 13, GenBank
accession number AF053640, incorporated herein as SEQ ID NO: 14,
and GenBank accession number AF053642, incorporated herein as SEQ
ID NO: 15). 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 cellular apoptosis susceptibility gene 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 cellular apoptosis susceptibility gene
mRNA levels by chimeric phosphorothioate oligonucleotides having
2'-MOE wings and a deoxy gap TARGET ISIS SEQ ID TARGET % SEQ ID #
REGION NO SITE SEQUENCE INHIB NO 128205 Introl 10 1654
ggaagaataatacttgtgca 1 16 128206 Intron 11 1015
tcccagcactttgggaggct 17 17 128207 Intron 12 2982
cgaggtatgaagctggacaa 14 18 128208 Intron 12 3382
atgtctccaaatatactgcc 17 19 128209 Intron 12 5636
gcgacattacctgcttctga 3 20 128210 Intron 12 6013
tgaaataaacatcctagttg 21 21 128211 Intron 12 9585
cagggagatcctacagaata 12 22 128212 Intron 13 2161
ggaataaattctgttgataa 8 23 128213 5'UTR 3 3 aaccccggcaaaatggcgcg 10
24 128214 5'UTR 3 65 aaccccagccgcggaccgta 9 25 128215 Start Codon 3
115 ctgagttccattgctatagg 15 26 128216 Coding 3 202
agaaatttctcagctggacg 18 27 128217 Coding 3 227 attctgatttccttcaacag
14 28 128218 Coding 3 249 atgtcaaaagcaacagtgga 13 29 128219 Coding
3 268 tcctgggacttctccagtaa 0 30 128220 Coding 3 380
aatggccactcgatcggctt 33 31 128221 Coding 3 418 tctgggctgctaagcatcaa
0 32 128222 Coding 3 444 catcacttaactgcttctga 13 33 128223 Coding 3
479 ctgtggaaaatcttctctgc 22 34 128224 Coding 3 491
gtcaggccatttctgtggaa 14 35 128225 Coding 3 500 tgtcagcaagtcaggccatt
32 36 128226 Coding 3 567 aatgtgctgtacggaggact 22 37 128227 Coding
3 590 atgacggtatcttttaaata 8 38 128228 Coding 3 679
atagtggccttaaaaagatt 16 39 128229 Coding 3 688 cagagttcaatagtggcctt
5 40 128230 Coding 3 816 aagtttccatattaccttcc 25 41 128231 Coding 3
819 tccaagtttccatattacct 15 42 128232 Coding 3 830
gaaattattcatccaagttt 12 43 128233 Coding 3 900 gctccaataagccggcttcc
15 44 128234 Coding 3 921 cacaaatctgggattttaag 21 45 128235 Coding
3 1069 gccagaaattgaattgcatt 14 46 128236 Coding 3 1118
gttctggtcctcaaatagat 33 47 128237 Coding 3 1173
cagctctaaattccatgtta 5 48 128238 Coding 3 1315 attcctgtcacaggtccctc
16 49 128239 Coding 3 1351 tattcctgcagcatggaatt 31 50 128240 Coding
3 1405 actaggtagatggctgcatc 26 51 128241 Coding 3 1558
ataccgtcagctttaaggac 23 52 128242 Coding 3 1639
tgaagatgattaatcaagag 27 53 128243 Coding 3 1785
gagctttgaaaaggtttgtt 23 54 128244 Coding 3 1837
ctcatgatagctttcataat 28 55 128245 Coding 3 1926
tcttactaacagctaatagc 36 56 128246 Coding 3 1994
gcaagttattcttatggata 13 57 128247 Coding 3 2015
aacagcagcagggttagctt 9 58 128255 Coding 3 2561 tccaaacatttttggttgta
3 59 128256 Coding 3 2653 agtaatttggttatgccaac 14 60 128257 Coding
3 2663 acattctgttagtaatttgg 13 61 128258 Coding 3 2795
tggtgtatcttctatgtcaa 5 62 128259 Coding 3 2905 ttgtgaagtgactgtgccag
13 63 128260 Coding 3 2913 tagacaacttgtgaagtgac 10 64 128261 Coding
3 2941 attgatggaacccttcctgg 36 65 128262 Coding 3 2976
actggagcgcttctgcattc 1 66 128263 Coding 3 2978 atactggagcgcttctgcat
25 67 128264 Stop Codon 3 3028 aatgcagtttaaagcagtgt 30 68 128265
3'UTR 3 3093 taatgcagctgtgctcagaa 36 69 128266 3'UTR 3 3160
agcaacatttaatatccttt 39 70 128267 3'UTR 3 3174 aaggttcaggttaaagcaac
18 71 128268 3'UTR 3 3196 acacaaaccaactaatttgc 23 72 128269 3'UTR 3
3222 gaagccacccacataactgt 30 73 128270 3'UTR 3 3224
tagaagccacccacataact 26 74 128271 3'UTR 3 3293 gtgcaaacgctcaacacaaa
50 75 128272 3'UTR 3 3319 cgtcaaaatttaagattatc 26 76 128273 3'UTR 3
3476 tgcactgcttggcaagtaac 18 77 128274 3'UTR 3 3496
agatttgaaactatgaaatg 36 78 128275 3'UTR 3 3504 ctgattacagatttgaaact
23 79 128276 3'UTR 3 3518 taggatttttattgctgatt 21 80 128277 Coding
14 655 gcattccataccttaaaaag 17 81 128278 3'UTR 14 1460
aagtccttactggttaatga 47 82 128279 3'UTR 14 1669
ggagatcttggtgcgccaat 17 83 128230 3'UTR 14 1714
ccattgatggaacctacagg 32 84 128281 Coding 15 624
atgccttcaaaaataggtcc 4 85 128282 3'UTR 15 787 gatggaacctacaagagatg
9 86
[0223] As shown in Table 1, SEQ ID NOs 17, 18, 19, 21, 22, 26, 27,
28, 29, 31, 33, 34, 35, 36, 37, 39, 41, 42, 43, 44, 45, 46, 47, 49,
50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 63, 65, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 and 84 demonstrated
at least 12% inhibition of human cellular apoptosis susceptibility
gene 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
[0224] Western Blot Analysis of Cellular Apoptosis Susceptibility
Gene Protein Levels
[0225] 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 cellular apoptosis susceptibility gene 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
86 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1
tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense
Oligonucleotide 2 atgcattctg cccccaagga 20 3 3569 DNA Homo sapiens
misc_feature 3356 n=a, c, g or t 3 gtcgcgccat tttgccgggg tttgaatgtg
aggcggagcg gcggcaggag cggatagtgc 60 cagctacggt ccgcggctgg
ggttccctcc tccgtttctg tatccccacg agatcctata 120 gca atg gaa ctc agc
gat gca aat ctg caa aca cta aca gaa tat tta 168 Met Glu Leu Ser Asp
Ala Asn Leu Gln Thr Leu Thr Glu Tyr Leu 1 5 10 15 aag aaa aca ctt
gat cct gat cct gcc atc cga cgt cca gct gag aaa 216 Lys Lys Thr Leu
Asp Pro Asp Pro Ala Ile Arg Arg Pro Ala Glu Lys 20 25 30 ttt ctt
gaa tct gtt gaa gga aat cag aat tat cca ctg ttg ctt ttg 264 Phe Leu
Glu Ser Val Glu Gly Asn Gln Asn Tyr Pro Leu Leu Leu Leu 35 40 45
aca tta ctg gag aag tcc cag gat aat gtt atc aaa gta tgt gct tca 312
Thr Leu Leu Glu Lys Ser Gln Asp Asn Val Ile Lys Val Cys Ala Ser 50
55 60 gta aca ttc aaa aac tat att aaa agg aac tgg aga att gtt gaa
gat 360 Val Thr Phe Lys Asn Tyr Ile Lys Arg Asn Trp Arg Ile Val Glu
Asp 65 70 75 gaa cca aac aaa att tgt gaa gcc gat cga gtg gcc att
aaa gcc aac 408 Glu Pro Asn Lys Ile Cys Glu Ala Asp Arg Val Ala Ile
Lys Ala Asn 80 85 90 95 ata gtg cac ttg atg ctt agc agc cca gag caa
att cag aag cag tta 456 Ile Val His Leu Met Leu Ser Ser Pro Glu Gln
Ile Gln Lys Gln Leu 100 105 110 agt gat gca att agc att att ggc aga
gaa gat ttt cca cag aaa tgg 504 Ser Asp Ala Ile Ser Ile Ile Gly Arg
Glu Asp Phe Pro Gln Lys Trp 115 120 125 cct gac ttg ctg aca gaa atg
gtg aat cgc ttt cag agt gga gat ttc 552 Pro Asp Leu Leu Thr Glu Met
Val Asn Arg Phe Gln Ser Gly Asp Phe 130 135 140 cat gtt att aat gga
gtc ctc cgt aca gca cat tca tta ttt aaa aga 600 His Val Ile Asn Gly
Val Leu Arg Thr Ala His Ser Leu Phe Lys Arg 145 150 155 tac cgt cat
gaa ttt aag tca aac gag tta tgg act gaa att aag ctt 648 Tyr Arg His
Glu Phe Lys Ser Asn Glu Leu Trp Thr Glu Ile Lys Leu 160 165 170 175
gtt ctg gat gcc ttt gct ttg cct ttg act aat ctt ttt aag gcc act 696
Val Leu Asp Ala Phe Ala Leu Pro Leu Thr Asn Leu Phe Lys Ala Thr 180
185 190 att gaa ctc tgc agt acc cat gca aat gat gcc tct gcc ctg agg
att 744 Ile Glu Leu Cys Ser Thr His Ala Asn Asp Ala Ser Ala Leu Arg
Ile 195 200 205 ctg ttt tct tcc ctg atc ctg atc tca aaa ttg ttc tat
agt tta aac 792 Leu Phe Ser Ser Leu Ile Leu Ile Ser Lys Leu Phe Tyr
Ser Leu Asn 210 215 220 ttt cag gat ctc cct gaa ttt tgg gaa ggt aat
atg gaa act tgg atg 840 Phe Gln Asp Leu Pro Glu Phe Trp Glu Gly Asn
Met Glu Thr Trp Met 225 230 235 aat aat ttc cat act ctc tta aca ttg
gat aat aag ctt tta caa act 888 Asn Asn Phe His Thr Leu Leu Thr Leu
Asp Asn Lys Leu Leu Gln Thr 240 245 250 255 gat gat gaa gag gaa gcc
ggc tta ttg gag ctc tta aaa tcc cag att 936 Asp Asp Glu Glu Glu Ala
Gly Leu Leu Glu Leu Leu Lys Ser Gln Ile 260 265 270 tgt gat aat gcc
gca ctc tat gca caa aag tac gat gaa gaa ttc cag 984 Cys Asp Asn Ala
Ala Leu Tyr Ala Gln Lys Tyr Asp Glu Glu Phe Gln 275 280 285 cga tac
ctg cct cgt ttt gtt aca gcc atc tgg aat tta cta gtt aca 1032 Arg
Tyr Leu Pro Arg Phe Val Thr Ala Ile Trp Asn Leu Leu Val Thr 290 295
300 acg ggt caa gag gtt aaa tat gat ttg ttg gta agt aat gca att caa
1080 Thr Gly Gln Glu Val Lys Tyr Asp Leu Leu Val Ser Asn Ala Ile
Gln 305 310 315 ttt ctg gct tca gtt tgt gag aga cct cat tat aag aat
cta ttt gag 1128 Phe Leu Ala Ser Val Cys Glu Arg Pro His Tyr Lys
Asn Leu Phe Glu 320 325 330 335 gac cag aac acg ctg aca agt atc tgt
gaa aag gtt att gtg cct aac 1176 Asp Gln Asn Thr Leu Thr Ser Ile
Cys Glu Lys Val Ile Val Pro Asn 340 345 350 atg gaa ttt aga gct gct
gat gaa gaa gca ttt gaa gat aat tct gag 1224 Met Glu Phe Arg Ala
Ala Asp Glu Glu Ala Phe Glu Asp Asn Ser Glu 355 360 365 gag tac ata
agg aga gat ttg gaa gga tct gat att gat act aga cgc 1272 Glu Tyr
Ile Arg Arg Asp Leu Glu Gly Ser Asp Ile Asp Thr Arg Arg 370 375 380
agg gct gct tgt gat ctg gta cga gga tta tgc aag ttt ttt gag gga
1320 Arg Ala Ala Cys Asp Leu Val Arg Gly Leu Cys Lys Phe Phe Glu
Gly 385 390 395 cct gtg aca gga atc ttc tct ggt tat gtt aat tcc atg
ctg cag gaa 1368 Pro Val Thr Gly Ile Phe Ser Gly Tyr Val Asn Ser
Met Leu Gln Glu 400 405 410 415 tac gca aaa aat cca tct gtc aac tgg
aaa cac aaa gat gca gcc atc 1416 Tyr Ala Lys Asn Pro Ser Val Asn
Trp Lys His Lys Asp Ala Ala Ile 420 425 430 tac cta gtg aca tct ttg
gca tca aaa gcc caa aca cag aag cat gga 1464 Tyr Leu Val Thr Ser
Leu Ala Ser Lys Ala Gln Thr Gln Lys His Gly 435 440 445 att aca caa
gca aat gaa ctt gta aac cta act gag ttc ttt gtg aat 1512 Ile Thr
Gln Ala Asn Glu Leu Val Asn Leu Thr Glu Phe Phe Val Asn 450 455 460
cac atc ctc cct gat tta aaa tca gct aat gtg aat gaa ttt cct gtc
1560 His Ile Leu Pro Asp Leu Lys Ser Ala Asn Val Asn Glu Phe Pro
Val 465 470 475 ctt aaa gct gac ggt atc aaa tat att atg att ttt aga
aat caa gtg 1608 Leu Lys Ala Asp Gly Ile Lys Tyr Ile Met Ile Phe
Arg Asn Gln Val 480 485 490 495 cca aaa gaa cat ctt tta gtc tcg att
cct ctc ttg att aat cat ctt 1656 Pro Lys Glu His Leu Leu Val Ser
Ile Pro Leu Leu Ile Asn His Leu 500 505 510 caa gct gga agt att gtt
gtt cat act tac gca gct cat gct ctt gaa 1704 Gln Ala Gly Ser Ile
Val Val His Thr Tyr Ala Ala His Ala Leu Glu 515 520 525 cgg ctc ttt
act atg cga ggg cct aac aat gcc act ctc ttt aca gct 1752 Arg Leu
Phe Thr Met Arg Gly Pro Asn Asn Ala Thr Leu Phe Thr Ala 530 535 540
gca gaa atc gca ccg ttt gtt gag att ctg cta aca aac ctt ttc aaa
1800 Ala Glu Ile Ala Pro Phe Val Glu Ile Leu Leu Thr Asn Leu Phe
Lys 545 550 555 gct ctc aca ctt cct ggc tct tca gaa aat gaa tat att
atg aaa gct 1848 Ala Leu Thr Leu Pro Gly Ser Ser Glu Asn Glu Tyr
Ile Met Lys Ala 560 565 570 575 atc atg aga agt ttt tct ctc cta caa
gaa gcc ata atc ccc tac atc 1896 Ile Met Arg Ser Phe Ser Leu Leu
Gln Glu Ala Ile Ile Pro Tyr Ile 580 585 590 cct act ctc atc act cag
ctt aca cag aag cta tta gct gtt agt aag 1944 Pro Thr Leu Ile Thr
Gln Leu Thr Gln Lys Leu Leu Ala Val Ser Lys 595 600 605 aac cca agc
aaa cct cac ttt aat cac tac atg ttt gaa gca ata tgt 1992 Asn Pro
Ser Lys Pro His Phe Asn His Tyr Met Phe Glu Ala Ile Cys 610 615 620
tta tcc ata aga ata act tgc aaa gct aac cct gct gct gtt gta aat
2040 Leu Ser Ile Arg Ile Thr Cys Lys Ala Asn Pro Ala Ala Val Val
Asn 625 630 635 ttt gag gag gct ttg ttt ttg gtg ttt act gaa atc tta
caa aat gat 2088 Phe Glu Glu Ala Leu Phe Leu Val Phe Thr Glu Ile
Leu Gln Asn Asp 640 645 650 655 gtg caa gaa ttt att cca tac gtc ttt
caa gtg atg tct ttg ctt ctg 2136 Val Gln Glu Phe Ile Pro Tyr Val
Phe Gln Val Met Ser Leu Leu Leu 660 665 670 gaa aca cac aaa aat gac
atc ccg tct tcc tat atg gcc tta ttt cct 2184 Glu Thr His Lys Asn
Asp Ile Pro Ser Ser Tyr Met Ala Leu Phe Pro 675 680 685 cat ctc ctt
cag cca gtg ctt tgg gaa aga aca gga aat att cct gct 2232 His Leu
Leu Gln Pro Val Leu Trp Glu Arg Thr Gly Asn Ile Pro Ala 690 695 700
cta gtg agg ctt ctt caa gca ttc tta gaa cgc ggt tca aac aca ata
2280 Leu Val Arg Leu Leu Gln Ala Phe Leu Glu Arg Gly Ser Asn Thr
Ile 705 710 715 gca agt gct gca gct gac aaa att cct ggg tta cta ggt
gtc ttt cag 2328 Ala Ser Ala Ala Ala Asp Lys Ile Pro Gly Leu Leu
Gly Val Phe Gln 720 725 730 735 aag ctg att gca tcc aaa gca aat gac
cac caa ggt ttt tat ctt cta 2376 Lys Leu Ile Ala Ser Lys Ala Asn
Asp His Gln Gly Phe Tyr Leu Leu 740 745 750 aac agt ata ata gag cac
atg cct cct gaa tca gtt gac caa tat agg 2424 Asn Ser Ile Ile Glu
His Met Pro Pro Glu Ser Val Asp Gln Tyr Arg 755 760 765 aaa caa atc
ttc att ctg cta ttc cag aga ctt cag aat tcc aaa aca 2472 Lys Gln
Ile Phe Ile Leu Leu Phe Gln Arg Leu Gln Asn Ser Lys Thr 770 775 780
acc aag ttt atc aag agt ttt tta gtc ttt att aat ttg tat tgc ata
2520 Thr Lys Phe Ile Lys Ser Phe Leu Val Phe Ile Asn Leu Tyr Cys
Ile 785 790 795 aaa tat ggg gca cta gca cta caa gaa ata ttt gat ggt
ata caa cca 2568 Lys Tyr Gly Ala Leu Ala Leu Gln Glu Ile Phe Asp
Gly Ile Gln Pro 800 805 810 815 aaa atg ttt gga atg gtt ttg gaa aaa
att att att cct gaa att cag 2616 Lys Met Phe Gly Met Val Leu Glu
Lys Ile Ile Ile Pro Glu Ile Gln 820 825 830 aag gta tct gga aat gta
gag aaa aag atc tgt gcg gtt ggc ata acc 2664 Lys Val Ser Gly Asn
Val Glu Lys Lys Ile Cys Ala Val Gly Ile Thr 835 840 845 aaa tta cta
aca gaa tgt ccc cca atg atg gac act gag tat acc aaa 2712 Lys Leu
Leu Thr Glu Cys Pro Pro Met Met Asp Thr Glu Tyr Thr Lys 850 855 860
ctg tgg act cca tta tta cag tct ttg att ggt ctt ttt gag tta ccc
2760 Leu Trp Thr Pro Leu Leu Gln Ser Leu Ile Gly Leu Phe Glu Leu
Pro 865 870 875 gaa gat gat acc att cct gat gag gaa cat ttt att gac
ata gaa gat 2808 Glu Asp Asp Thr Ile Pro Asp Glu Glu His Phe Ile
Asp Ile Glu Asp 880 885 890 895 aca cca gra tat cag act gcc ttc tca
cag ttg gca ttt gct ggg aaa 2856 Thr Pro Xaa Tyr Gln Thr Ala Phe
Ser Gln Leu Ala Phe Ala Gly Lys 900 905 910 aaa gag cat gat cct gta
ggt caa atg gtg aat aac ccc aaa att cac 2904 Lys Glu His Asp Pro
Val Gly Gln Met Val Asn Asn Pro Lys Ile His 915 920 925 ctg gca cag
tca ctt cac aag ttg tct acc gcc tgt cca gga agg gtt 2952 Leu Ala
Gln Ser Leu His Lys Leu Ser Thr Ala Cys Pro Gly Arg Val 930 935 940
cca tca atg gtg agc acc agc ctg aat gca gaa gcg ctc cag tat ctc
3000 Pro Ser Met Val Ser Thr Ser Leu Asn Ala Glu Ala Leu Gln Tyr
Leu 945 950 955 caa ggg tac ctt cag gca gcc agt gtg aca ctg ctt taa
actgcatttt 3049 Gln Gly Tyr Leu Gln Ala Ala Ser Val Thr Leu Leu 960
965 970 tctaatgggc taaacccaga tggtttccta ggaaatcaca ggcttctgag
cacagctgca 3109 ttaaaacaaa ggaagttctc cttttgaact tgtcacgaat
tccatcttgt aaaggatatt 3169 aaatgttgct ttaacctgaa ccttgagcaa
attagttggt ttgtgtgatc atacagttat 3229 gtgggtggct tctagtttgc
aacttcaagg gacaagtatt aatagttcag tgtatggcgt 3289 tggtttgtgt
tgagcgtttg cacggtttgg ataatcttaa attttgacgg acactgtgga 3349
gactttnctg ttactaaatc cttttgtttt gaagctgttg ctatttgtat ttctcttgtc
3409 ctttatattt tttgtctgtt tatttacgct tttattggaa atgtgaataa
gtaaagaatt 3469 acttgtgtta cttgccaagc agtgcacatt tcatagtttc
aaatctgtaa tcagcaataa 3529 aaatcctaaa atatgtacct aaaaaaaaaa
aaaaaaaaaa 3569 4 24 DNA Artificial Sequence PCR Primer 4
ggagaattgt tgaagatgaa ccaa 24 5 21 DNA Artificial Sequence PCR
Primer 5 ctgggctgct aagcatcaag t 21 6 23 DNA Artificial Sequence
PCR Probe 6 tttgtgaagc cgatcgagtg gcc 23 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 2929 DNA Homo sapiens 10 gtcgcgccat tttgccgggg tttgaatgtg
aggcggagcg gcggcaggag cgggtagtgc 60 cagctacggt ccgcggctgg
ggttcctcct ccgtttctgt atccccacga ggtgaggcgc 120 ggggcgtgca
cggcctacca gagtggctct tggggcccag ctgaggaagg gatgaggcgc 180
tcccggtact aacgagcgct agggagtgag aaccgcgcct ctgggcgaga gcggaatgtg
240 ggcccggggt tcgggtggtg cgctcaggca aggtcttcgg cttccctagg
gagcgcttgc 300 cgcgcctcgc ggcatcctag gtctctggcc caggtgcggc
gaccccaggg cctgtgaggg 360 ctgagggcaa ctgaggcgcg gcctaacgcg
agcctgcgga ttgcccgccc tttcctctcc 420 atcacggcgc tgattggctg
cgccgccgcc tctccgctcg ggaaggccgc tccttattgg 480 tcgcgctcgc
atgtccattc tctgcgacgg tggctgctag ccgcgcgagc tgagtgtgcg 540
gcggcgtggc ctgcccgggc ggggtctgcg ggcgcgtgga gctgcggctg cctcgcgtcg
600 tcccttgccc taactctagt gaagcccccg tcgtgatctc actttgctga
attgtttgtg 660 agaattctag tcggtacacc cgggacatag aaatcccctg
taacgcgggt tgggggaggg 720 ttccctcgtt ttgggagaaa cgctgtgctg
tggtggttcg gagcatagac tttggaatta 780 gatcgcctgg gttcattcca
gacgtagcct ctcgttaggg gtgtaacttg aggctggtca 840 cctcatctca
ctgagcctcc atttccttag ctgcaaaatg gggttaataa cagaatctac 900
ctcggaaagt ggctttgagg atgcgctgag ttatttaccg tggtttacaa aggttccctg
960 ggagctgacg gttgtttatt catctctccc acccatcttt ctccttagcc
agtgtggctt 1020 tctgtttctt ccatattcca gggttattcc gatttaaaat
tatttgcaca tgccttttgt 1080 ttgctgggga agtaccaaca tttttgcaag
gcaggccgcc tctcccaagt ttccctcacc 1140 ttaggcctcc cctaaccaac
ctcttctttt gttttctttt tctttttctt ttctcttttc 1200 tttttttttt
tttttttttt tttttttttg agagagagag tctcgctctg cctcccaggc 1260
tggagtgcag tggcgcgatc tcgactcaat gcaacctccg cctcccggat tcaagcgatt
1320 ttcctgcctc agcctcccga atagctgaga ctacaggtct gagaatacac
gccaccacac 1380 ccggctaatt tttgtatttt tattagagac ggggtttcac
catgttgatc aggctgtcta 1440 atcaacctgt ttaggacctt tctcccatgg
ctctccatca acatcacctg gttttagtta 1500 cttcatagca tttactaaca
cttacttatt gcttacttgt tatctttctc aactgctaga 1560 atgtaagctg
tttgagagca aggaccttgt ctgtcttgct tacttctctc tccctggtgg 1620
gtgccatccc acctgtgctt gccacatggt agatgcacaa gtattattct tccttggagg
1680 cacagaaggg gaccctttag tcttggattc caccaattgc tctttttctc
accttgggcc 1740 cacatagtca tatctttggc ctttctctgt gtgtgtctct
ctcttttttt tttttttttt 1800 tgacatgctt tcagaggatc ctaatggacc
tcgtctctcc gctcctacaa ccctttatct 1860 ttcacttctt accatcagtg
caagggaagt ctctctcccc ttctgattac ttctgctggt 1920 tggggtctgg
gacaggattg gatggatgca agagcagtgc tctagttttg gaattaattt 1980
gtttctttat tcatttaata actttattga gcatccactt aatctctggg aatatagtga
2040 aaaaacaaaa atggacaagg tcccttccct tgaggaccat gtattctcgt
gggcgaaaga 2100 aaaaacatta gataatttca gatagtgaca gaattatgtg
acaggtgaag aaggcttttc 2160 tgaggaggtg acatttgggt agagaccagc
cattaaaagg tgaggggcag aacattccag 2220 gtagagggaa tagaaggctc
aaggctcaag gacaggaacc ggcttggaat attgaaatat 2280 ttaaggaata
ttttaggttc ctagagtgtt acatgaagaa gggagaaata tccaggaccc 2340
ggatcataga aggctttgta ggcaatgtaa ggagtttgga tttgattcta agtggtatat
2400 tcctatggtt agaaagcaga gggtaggttg aggtgttggg atatgttctt
catgtgtggg 2460 tgccagatag acctcattca ttggatctct ttccctcttc
ccaaacgtaa ggcatcggga 2520 atggttctct aatgtgtaga ggacatatct
aagaaaaags cagacatctt ggatcagctt 2580 atgtcacaga tgtctgtgag
atggctttgg aaaacaaagc tttttattcc agaaatctcc 2640 acctgtaatt
gagggcctgt gggaagatgc tagacttagg tctggccatt cattcaacaa 2700
gaagaacttg gaaatttaga tgtctgccat aactgattta ttgtgatgct tttttaagtt
2760 tcttaattgt gtattggtag agatttccaa agatagttac gtggagaaag
aaaagtatca 2820 ccccctcatt taaaggtgat aaaaatgaat tcttatcttc
tccctccctt ttttttttgg 2880 agacaccagc tcactgtgtt gcctgggctg
acctgggctc agtctatcc 2929 11 3609 DNA Homo sapiens misc_feature 92
n=a, c, g or t 11 tgtgtgtgtg tgtgtggtga aagcacttaa aatataccca
gtaattttca agtgtacaat 60 acattattat agtcaccttg ttgtacaata
antctcttga actgtgtatt tcttagccta 120 tgttgttagt agttaaatcg
acgtaggcca tctagttagc ttaaaatttg tgggctgggc 180 acggtggctc
acgcctgtaa tcctagcaca ctgggaggcc aaggcaggag gatcacttga 240
gtccaggagt ttgagaccag cttgggcaac attgtgagac cctgtctcta caaaattttt
300 tttttaattt gtcatctgtt agatgtgaag ttttttggtc agttataagc
atattatgtg 360 atttagtttt gtttaagctt taaatatgaa aacttctaga
atcataatct tagcttgtca 420 tcttctgaac tctagaattt atctccctag
atgcaaattc ttagcacttt gtagtatttg 480 cgattacatg ttgtacatga
aggaatttct tgtttcttta atctcagaac tttatgtggg 540 atatttattt
gctgtttaaa attttaattt ctcgttgtgg ggcctttttt tttttttttt 600
ttaagacagg tcttgctcca tcacccaagt tggagtgcag tggcaccatc tcagctcact
660 gcaacctctg cccccaggct caagacatct tcccacctca gccttccgag
tagctgggac 720 tgaaggcacc cactaccatg cctggctaat tttttgggtt
ggttggttgg tttgttttga 780 gacggagttt tgctcttatc gcccaggctg
cagtgctatg gtgcgatctc agctcactga 840 aacctttgcc tcccgggttc
aagcgattnt cctgcctcag cctccacaag tagctgagat
900 tacaggctcc tgccaccatg tccagctaat ttttgtattt ttagtagaga
cgggatttta 960 ccatgttgac caggttggtc tcaaattctt gacctcaggt
gatccgcctg cctcagcctc 1020 ccaaagtgct gggattacag gcatgagcca
ccatgcccgg cttcaatttt tttgtagttt 1080 tggtagagat ggggtctcac
cctgttgaca ggctggtctc ttaactcgtg agctcaagca 1140 gtccacccac
ctcagcctcc caaagtgtcg ggatttacag gcctgagcca ctgcccgggc 1200
tccttatggg gcttctttaa taagttcctc cagtgatttt ttttttctta acctttatta
1260 gccctttccc atagcattaa atcatataaa cttcttggtg acttggagca
gtccttcagc 1320 ttttttttct aggactatct ttttgaaatt ttgggatttt
ttttcatttc ccttatcaca 1380 aaatngtctc actattgttt ttgggggaaa
aggcacattt catttgctgg attctttttt 1440 cagagtcagg tttactgccc
acctaaaaaa caacagatgg atgactttca gagagaataa 1500 cccataatat
aaacggattt ctttaaatgt aaatcacttt ttccgtaagt gaccagttat 1560
ccacacctta ttttatattt tagatcctat agcaatggaa ctcagcgatg caaatctgca
1620 aacactaaca gaatatttaa agaaaacact tgatcctgat cctgccatcc
gacgtccagg 1680 taaagaaaat aaacgttttt tggttgatta attccttcag
gacaggtgag agaaacctat 1740 gtattatctg cctccttata aatatatttt
aaaaaattca tacttgggtt ctcaagcgta 1800 gtttataagt gttacagctg
ctataccatg gtgtgttgtt tcttttttga gttaaaatag 1860 cttttatttg
gcatcttcct gggattaaaa agtactagtt ttcggataaa aagtactgac 1920
atgtttagaa tgctttgttc tttgaccttt tcaaaatacc cacttaaaac atgcctgctt
1980 taggccggat gcggtggctc acacctgtaa tcccagcact ttgggaggct
gaggcaggcg 2040 gatcacgacg tcaggagatg gagaccatcc tggctaacac
ggtgaaaccc cgtctctact 2100 aaaagtacaa aaaattagcc aggcgtggtg
gcgggtgcct gtggtcccag ctactcagga 2160 ggctgagtca ggagaatggc
gtgaaccccg gaggcagagc ttgcagtgag ccgagattgc 2220 gccactgcac
tccagcctgg gcgacagagc aagactccgt ctcaaaaaaa aaaaaaagcc 2280
tgctttagag ataaaataat tagggcatag aaaggagagt aattgcctaa aaagtgatta
2340 agtgaatgaa gccaagtaat cagttactgt ctcttaattt taattttcta
ttgggtttga 2400 tttaactaat atattttaag acctagagct ttttttttcc
ttcctgaatc tgaccagatt 2460 ccattaaaaa cgtagaatta aaatattgaa
ctcaggtgtt cacaggactc agctgttaag 2520 ttaaatgagg gagataggcc
aagtggagac tggtaatatc aggagaatac atgccctgat 2580 taagtggcaa
gtactttttg gaactagcta attttgggga ttcagactag ttttgcaaga 2640
tcagatttcc agttttgctt gaaaaataag gatatttata tgaagtgccc tcatttttaa
2700 atgttagcaa gtaacttcaa aagattttgg ggccgggtgc ggtggctcac
gcttgtaatc 2760 ccagcacttt gggaggccga ggtgggcaga tcacgagggc
aggagatcga gaccatcgtg 2820 gctaatatgg tgaaacccct tctttactaa
aaaatacaaa aacttagctg ggtgtgatgg 2880 cacacacctg tagtcccagc
tgctcaagag gctgaggcag gagaattgct tgaacctggg 2940 aggtggaggt
tgcagtgagt tgagattgcg ccactgcact ccagcctggg tgacagagcg 3000
agacgtagtc ccaaaaaaaa aaatattttg aaacacagcc agggttggtg gtcacgcctt
3060 taatcccagt aatttgggag gccaaggcag gaagatccct tgagtccagg
agttcgagac 3120 cagcctgggc aacaaagcaa gaccctgtct ctacaaaaaa
tttaaaaatt agccaagtgt 3180 ggtgatgtgt gcctgtattc ctagcaactg
gggaggctga gacaaaagct tccttataac 3240 aagaggtcgg agctgcagtg
agttatgatc atgtcagtgc actctagcct gggtgacaga 3300 gggaaatctt
gtcttaaaaa aaaaaaaaaa aaggccaggt gcatttgctc acgcctgtaa 3360
tcccagcatt ttgggaagcc aaggcaggcg gatcatgagg tcaagagatc tagaccatcc
3420 tggccaatgt ggtgaaaccc tgtctctact aaaaatacaa aaattagctg
ggcgtggtgg 3480 tgcacggctg tagtcccaga tacttgggag gctgaggcag
gagaattgct tgaatccagg 3540 aagcggaggt tgcagttaac cgaggccact
gcactccagc ctggtgacag agcaagactc 3600 cgtctcaaa 3609 12 12597 DNA
Homo sapiens misc_feature 4964 n=a, c, g or t 12 ctacttggga
ggctgaggtg caagaatcgc ttgaacccag gaggtgaagg ttccagtgag 60
ccaagattgc ccaactgcac tccagcctag gtgacagagt gagactttgt ctcaaaaaaa
120 aaaaaaattg aaatacagtg caagccaaac tacttctggg ccagatatat
cttgtgagca 180 gtcagttatt gatccttgat atgggccata ttaaaatgct
cttgacctgt ttcttcaaaa 240 cttaccaatg tgcatgctca tagaggatgt
tggaagggtt ccatgaggaa gagaatgtgt 300 gttatatatt atgtatgatg
actagaatca tgaacatgtt aatgcatcta tcctcttcta 360 gaaaagtatt
tgtgattttt gagaggtaaa attagtttat tagtgtataa agaagtaatg 420
tgttcctgga taaagggaag ttaatggctc taatctttaa tcttcttaga ctttcaaaga
480 aagctgttgc caacactatt aaaaaatttt tttttttttt tttttttgga
gacggagtct 540 ccctctgtcg cccaggctag agtgcagtgg cgccatcttg
gctcactgca agctccgcct 600 cccgggtcca cgccattctc ctgcctcagc
ctcctgagta gctgggacta caggcgcccg 660 ccaccatgcc tacctaatta
tttgtatttt tagtagagac agggtttcac cgtgttagcc 720 aggatggtct
ccatctccat gacctcgtga tccacccgcc tcagccgtcc caaagtgctg 780
ggattacagg catgagccgt gcccggtgga aaaattatta ttattattta tttatttatt
840 tatttattta tttatttttg agacagagtc tcactcttgc ccaggctgga
gtgcagtggc 900 gccatcttgg ctcactgcaa gctccacctc ccgggttcac
cccattgttg tgcctcagcc 960 tcccgagtag ctgggactaa ggcgcccgcc
accacacccg gctaattttt tgtattttta 1020 gtagagacgg gtgtttcacc
aggttagtca ggatggtctc gatttcctga ccttgtgatc 1080 cactcgtctc
ggcctcccat agtgctggga ttacaggtgt gagccaccgc acccggcaat 1140
tatttttttt ttaataatgt gtgaggcaag agttctgttg ctttatgtat tcaggacaga
1200 gtataaaaat aatctggtat ttctggaagt taggacatag catttctcag
ggatcgagac 1260 tgactgacta gactgactca actagacctt ttgagactcc
ttgagtgcgc agaaacatat 1320 gcatgtgaat attgtgataa gttgaaagaa
atgracacag aaaggracag actcaaactc 1380 acttcctgga tattgattac
aatttaggaa agagacttgg caatttatat gggcttttcc 1440 tcaaatcatg
gatgtagtgt gttgccctgt acaaaatgct ggctactaag tggaggaaac 1500
aaattgttct gtactcatat aaaaccatag tatgtcccct cctgaaaaac atttttcagt
1560 ttaacaattt tcatccttca agaaagacat gataagcaca aagcaaggag
agtttgaaaa 1620 tatcagtgaa actcttggga ctcagtcaga aagagggaag
gtgttaaatg atacatgaaa 1680 ttaatttttc aaaaaatatt ggaatattgg
gtgttgccct atgaagccaa aggtagaaaa 1740 aaattaagag aaatactgta
ctgcccatca catgtgatca ttaacctagg aaatttatcc 1800 cagaatgtaa
tctgggttga aaatctattg gtaaattttt tggataaatt cattgctggt 1860
attaagaagt ttttaggttt ttttttagtt cgacaagaaa gcttagggct ggtaaggtaa
1920 aataaggact gcactaaaac tttcagtagt gaagcctagc tgtgtgatct
tgaacaagtt 1980 acttaacctt agtgccttgg ttttttaaat gctatctact
tttctgggta atagtgacaa 2040 ctaaatgaaa ttttataatt ttacatatat
aatatatatg taaatcgttg gacccatttg 2100 gctaataata ctatattagg
atgaattgac tcatttgctc ttcatcacta gcctataaaa 2160 gaaatattac
tactaatagt actaataata ctcctttctg aaataagacc tgggtatatg 2220
aagtcttgaa gccacttaat ctgcctggga agcagcttct cttgcagatg agatgatcat
2280 ccaattaggc cactgtatag gtttgtgaga atcatgtgag ttaattgatg
gtaaagttat 2340 ttgaaaactg aagcattata taagtttaat gtaacatata
taatccactt aatagataag 2400 aaaagtgagg ccctgtagac ccacaaataa
cctaatcttt tgtgtttttt tgattttttt 2460 ttatatatat atatttgata
tatataaggt ggaaagattt ttatcttagt cttttaacat 2520 gaaaattctt
ttacagctga gaaatttctt gaatctgttg aaggaaatca gaattatcca 2580
ctgttgcttt tgacattact ggagaagtcc caggataatg ttatcaaagt atgtgcttca
2640 gtaacattca aaaactatat taaaaggaac tggagaattg taagtatttt
gtgaatacat 2700 aatttaatac cctgtatgtt tataaggttt atataagcag
tgttcttcaa agataaggca 2760 cgtgcttgta gtcccaacta ctctggaggc
tgaggcggaa ggatcacttg agtccaggag 2820 ttcaaggctg cagtgagcta
tgattgcacc actgtactct agccttttag tgacagaatg 2880 agaccttgtc
tcaaaaaaaa ggattcagat atggatcaaa gataaaatta aacaggaccc 2940
ttttccaaag cttctgttat ttcatttgaa tttatataaa tttgtccagc ttcatacctc
3000 gggttgctag ggagtgagac ttggtcttaa tctccattat gtttggaatt
tatgctttac 3060 tcaccrccta ctgaaaagac attgtgggaa gacagtctga
ataaatgaag tgatttctga 3120 tgttggaaaa ataaagctga gaagaaatca
gatacataaa acatttgcag aatgcaaaat 3180 aatttagatt tcaacttttt
gataaggtat tttgccctat gtcttgtggg atatgtatat 3240 atatactaat
tttgagatta aacctatttt ataacaggtt taatgattag taaattataa 3300
attaagaaac aggcattttt tttccttagc ttaggacagt gattctcaac tgggggcagt
3360 ttggctcacc aggggacatt tggcagtata tttggagaca tcatagctgg
aggattgcta 3420 ctggaatcta gtgggtaaag gccagagatg ctgctaagca
tcctacaatg cacaagatag 3480 ctttccacaa caaagaatta tctgagccaa
aatgtcagat tgagaaagcc tggctcatga 3540 tagagaaaaa gatgaggctt
ttctatagtg atagcagtta tgatgttaca aagtattagc 3600 agttgacaaa
aatcatcagt ttatctaaaa agtcaacttt aaactgctca aaaacatctt 3660
agagtcttac tttttttctc attccataaa agagagagta aatattgttt taagcctgta
3720 tgatttctca agtatcacca tgagtggtaa agaaatctta gaggccgggc
ttggtggccc 3780 atgcctgtaa tttcagcact ttgggaggct gaggtgggtg
gattgcttga gctcagcaat 3840 tcaagaccag cttgggcaat aaagtgagat
cccatctttc caaaaaatac aaaaattagc 3900 gaggcatatg gtggtgcacg
gcctgtagtc ccagccatta gggaggctga gatgagggga 3960 tcacttgtgc
ctgggaggca gaggttgcaa tgaactgaga tggtgccact gcacttcagc 4020
ttgggcaaca gtgccagaca ctgtctttta aatctcagaa tgtttaaaag tccatctcta
4080 ctggtagaca tgccagccct ctccctctat tttgatttgt aaaactagtg
agacttaaca 4140 gaatctcttt gggtgatata atctaggatg cattctaact
agtaatggtc aagaaatttt 4200 cacagagggc tgggtatggt ggctcacacc
tgtaatccca gcactttagg aggctcagat 4260 gggaggattg cttgaggcca
ggagttggag accagcctgg acaacaaagt gagacccctc 4320 ttctctacaa
aaaaatttaa aattagccag gtgtggtcgt gtgtggtgtg catctgtggt 4380
ccttgggagg ctgcagtggg agggattgtc ttgagtctgg gaggtgaagg ctgcagtgag
4440 ccaaggtttt gtgctaccct ccagcctggg tgacagatgg aaacccagtc
tcaaaaaaaa 4500 aaaaagaata aaaaaaattg gaaagatttt tgttctaaag
tgagagttaa attgaagctt 4560 ttgaggatgt gtatggatta agtagcatat
atctgtaaag attttaatta wgkgtcctga 4620 ttagtttaaa tatgaaatgg
aaaaaaaawt tttttttttt tttttttttt tttttttttt 4680 tttttttttt
tttgagaggg agtcttgctc tgttgcccag gctggaatgc aatggtgcga 4740
tcttggctca ttgcagcctc tgcctctcag gtccaaatga ttgtcctgcc tcagcctcct
4800 gagtagttag tgttacaggt gcccaccacc atgttcagct aattttgtat
ttttagtaga 4860 aatggtttca ccatgttggc caggctggtc tcaaactcct
gaccttgagt gagccatcgc 4920 acctggccta aaatggaaat tttttttttt
tttttttttt gtangagaca gagtcactct 4980 wgttgcccag gctggagtgc
agcggcacca tctcggctcc ctgcaacctc cacctcctgg 5040 gttcaagcaa
ttctcctgct tcagcctccc gagtaactgg gattataggt gcctgccacc 5100
atgtccggct aatttttgta tgtttagtag agacatggtt tcaccatgtt ggtcaggctg
5160 gtctcaaact cctgacctca ggtgatccac ctgccttggc ctctcaaagt
gctgggatta 5220 taggcgtgag ccactgcgcc cagtctaaaa tggaaattct
taattggcat caaggagtaa 5280 gggaagttca ctaatttgta tgtaaaatat
ttgcatttgg gaggcaatgt acgtttttct 5340 gggaaaagga ttcacgctgt
catcagatta ttgaaggaat ctgtgaccca ttcatgtaga 5400 gggaaactga
agaaagacca aaccttcctc tatttgtttt cttagaaaat tagtaaataa 5460
aaaaacagtt atgttttacc taaaatcatg tggacatgaa tgtggatttt gtgttacttc
5520 ctcagttact gctttgcttt taggttgaag atgaaccaaa caaaatttgt
gaagccgatc 5580 gagtggccat taaagccaac atagtgcact tgatgcttag
cagcccagag caaattcaga 5640 agcaggtaat gtcgctccac tttttagatg
ggctcctctg taaagctctg atctaattaa 5700 tcttttcctc tcctagttaa
gtgatgcaat tagcattatt ggcagagaag attttccaca 5760 gaaatggcct
gacttgctga cagaaatggt gaatcgcttt cagagtggag atttccatgt 5820
tattaatgga gtcctccgta cagcacattc attatttaaa aggtattgat gcatagattc
5880 atgtttttaa aatacttcct aaagttttat ttgcttgtgt aaacagttgt
gtttttgtga 5940 ctgttgtcat tcctttgaat ctgatcatct tggaatgaga
gcagaagttt ctgtatkgcg 6000 tttgtttctc ctcaactagg atgtttattt
catattgctc tattacctgt gaaatactta 6060 gtcttgataa actgcttgat
ccctttaaaa agaaaactgg ctgggcatga tggctcacac 6120 ctataatcct
agcactttag gagggcaaag tgggcggatc acctgaggtc aggagtttga 6180
gaccagcctg gccaacatgg taaaaccccg tctctactaa aaatacaaaa aaaactagcc
6240 aggcatggtg gtatgtgcct gtcatcccag ctactcagga ggctgagaca
ggagaatcac 6300 ttgagcctgg gaggtggagg ctgcagtgag ccgagatcaa
accactgcac tccagcctgg 6360 gcgacagagc gagactccgt ctcaaaaaaa
aaaaaaaaaa aaaaaaaaaa agaattccct 6420 ttgaatttcg gaatttcgga
aacccatcta ttcatagatg tccaatcagc atctctcctt 6480 gaagacctaa
agtgagtaaa taaaaaaaac ttgtaaattt atctgtttta ccttaatttt 6540
ttagataccg tcatgaattt aagtcaaacg agttatggac tgaaattaag cttgttctgg
6600 atgcctttgc tttgcctttg actaatcttt ttaaggtatg gaatgcatct
tggtgatatt 6660 tttaaattaa tattttaaat tgcttgaatg tttgtataca
tgttaagaga atgctttgaa 6720 agcttatttg ataaataatg tttagagcat
ttcttttgaa aaatttcaaa cctacacaga 6780 aatagagatg ctatagtgaa
ctcccatcta ttattacaca aatttaatgg ttagcaacgt 6840 tttattttct
ctgtcccctt tcttaggaat gttataaaag caaatcccaa gcatcatatc 6900
atttcaccca agtattttaa agtatgtgtc tctagaggaa gaaatacata tttttatttg
6960 actatgtttg gagtttaatt aattatatat ttatacatta ttttacagct
ttacaggata 7020 ctttcctgaa tgtgttcttg gtgtttgtga aaatcctata
agatttaaag aaatatatat 7080 atactttttt tcttttttct tttttaaaga
cagggtctca ctctcattgc ccaggctgga 7140 gtgcagtggg gcatgattat
agctcactgc agcctcaact acccaagctc aggtgattct 7200 cccacctcag
cattccaagt agctgggact acaggtgcac actaccatgc ctggctgctt 7260
tttctctttt tttttccctc tctctttttt tttttttttt tttttttttt gagatggagt
7320 ttcgctcttg ctcactgcaa cctccgcctc ccgggttcaa gcaattctcc
gagtacactg 7380 ggattacagc ctgtgccact gggcctggcc agtaaaacct
gttaatcgca tttagaataa 7440 taaagttgga aacaacctaa ataccaagtg
gcagatgaat tgtttaataa gagcatataa 7500 agatagtttt gtgtaatata
tttttatata tgcacagatt gggaagacgt agttcagaat 7560 attaacactg
gactttttag tgccattatg aacagttcaa aaatacatac tttaacctat 7620
tttccacaat ctnttaagtg actgtattat ttaaaagcca aggctgggca tggtggctca
7680 cgcctgtaat cccagcactt tgagaggccg aggcgggcgg atcacgaggt
caggagatcg 7740 agaccaccat ggctaacgtg gtgtcaggag atcgagacca
ccatggctaa cgtggtgaaa 7800 ccccgtctct actaaaaata caaaaaacaa
aaacaaaatt agccgggcgt ggtggcaggc 7860 acctatagtc ccagctactt
gggaggctga ggcaggagaa tggtgtgaac ccaggaggtg 7920 gagcttgcag
cgagccgaga ttgtgccact gcgctccagc ctggacgaca gagcaagact 7980
ccgtctcaaa aaaataaata agtaaaatat gaatagtctc agcttagtgc aaggctgttt
8040 cactaagatc agcatgctgg gtttatgatg cactaaaacc atgttgctaa
attcctttcc 8100 aaggccacta ttgaactctg cagtacccat gcaaatgatg
cctctgccct gaggattctg 8160 ttttcttccc tgatcctgat ctcaaaattg
ttctatagtt taaactttca ggtaagttca 8220 tttgatttct tgcttttggt
tcttactctt tgattttaaa tagggttttc tttctgtttg 8280 ataaagactt
ctttgccagc attgattttt ctgaaagaaa aggttttttc tgactctatt 8340
tatctatagt gttctgttct acagtagttc tttcagatgc taatgagatg tttcatggaa
8400 aaaagcagta ttctcttata tctagtaggt tagcaaaacc ccacatccta
actcactctt 8460 ggagagtcac cctgtacact gttttactgg tggcattaag
aagctagatt tctgaggcct 8520 tcctctttcg ttcatttata tgggtattaa
catttgggga atcatgtttt ggttaggggt 8580 tgctgtccag cttatacaaa
atgactttta ttggaagcca tatgagggag acaaaagact 8640 tgaagtacca
gactttgaaa ggggtaggaa ctagtagtat ttcagaggaa acatacccac 8700
agggctatag cagggaatgc ctagttaccc tgttgctact gagatagtgg cctccaatca
8760 ttctttctgt tcttaatcat taattcaaga ttcaggacca agaaaggggt
gttaattggt 8820 acaaactgtt gctatactgc gatgcaagga agggcctagc
ccattcattg tttgtagcca 8880 gtacctggac ttatcttcac aacatgaata
aactcatagt ggtagggaaa gcattcccca 8940 aaaggaaatt gggttgtttt
agctagaata ggagatgaac agcccaaaag gacatgtccc 9000 acatcagtgt
tcagctggat agcagtttga gaagttctta cttaaatgag tactactatt 9060
tttgcttaca ccttttgaat tctgagaagt caggtttaat gaagctcttg agaacttgtc
9120 tggcagggag accaaggaaa gaggcataat ctaccctcag gggacttgta
ccctcattaa 9180 gaaggcagta ctatgccaca agaaatcagt aactaaaaga
caattagttt atctgccttg 9240 gtggtcatct tacaataaac gtagttcaga
aaagaaaggg gatgtattag tatgcactga 9300 gcttgagctt tgtaaggaaa
gtcttcatat agtcttagct gaaccttgaa ggaagattta 9360 gattaggatg
agaactagag gtggcatcat ttatagcata caaacagagg cagaagtcag 9420
aatgctttat gcagctgcca gatgtagctg gagcagagaa ttcttgtcag aataggtaat
9480 gctcaggctg tgaagcctgt gtataaaatg ttttgtccac gtggtaaggg
aacttataaa 9540 agtggtgttc ttaatatttt aatcaaaagt ttcaagtcag
tgtttattct gtaggatctc 9600 cctgaatttt ttgaagataa tatggaaact
tggatgaata attttcatac tctcttaaca 9660 ttggataata agcttttaca
aactgatgta agtatttaaa atgtcgcctg agtggtcttt 9720 ttctttcatt
aagagttatt tggctacagt ctggaaacct atttactctt gcattgttaa 9780
aagataaaat ttcaatactg taataatatt tgattctatt tgatgttaca tgtttggtgt
9840 gtgtagtata cagaattact cttatcatgg gcttggctta agccatagtg
tacatataag 9900 gctgtttcat tacattattg aaagtaggtc tttaaagtag
tgacattatg gatatttcat 9960 gaaataaaat tatttttcat tgaagatact
aagaaaactt aaaactctat aatggtagcc 10020 cctttttaaa aatcatctgg
ctaggcatgg tggttcacac ctgtaatccc agcactttgg 10080 gcggccaagg
tggggaggat cccttgagcc caggagttca agaccagcct tggcaatgta 10140
aggagacccc catcactaca aaaaataaaa ataaaattag ccagatgtgt tggctcatgc
10200 taatggtcac agctactcag aaggcttagt tgtgaggatc ccttgacctt
gagcccagga 10260 gggcaaggct gcaatgagct gtgaccatgc cactgcactc
cagcctggga gacagaggga 10320 gacctcgtct cagaaaaaga aaaaattatc
cacagctcga gaaagagaga aaatggtaaa 10380 agattgtaat ttgtaaatat
ttctgatatg ggttttatat tgtgtacttt taaacatata 10440 acacttattc
tgtgctactc atttgttaat atatttatga ccttctttcc gaagagacaa 10500
aagacaaaat attaaaaata tttcaatacc aaataaacta aaaattagaa ttggtgattc
10560 aaatcaggtt ttaagagtgg ggtacagaaa gaaattgtta ctatcgttgt
acaacagatt 10620 ttgtctcaga gtttgactat attaatagga actttaaaag
atgtagctct cataaaataa 10680 ttatttcagt ccaatgggag ggaaaaacac
tccttcagaa aacaaaactt ttcttcacac 10740 aaaagtcaca aggaaatttt
ttggatagaa tcttacaagg acctttttgc ccagttaacc 10800 atgtgctacc
ttgtgctact atttatgcct taagtaatca gtgtgaggga gttttggttt 10860
tggctttggt ttttgttctt actctgatgt cttcccttgt ctcactctgt caccaaggct
10920 ggagtgcagt ggcatgatca tgacttactg cagcttcgac ctccctgggc
tcaagtgatc 10980 ctcccacctc agcctcccaa gtagcaggga ctatgggtat
gcaccaccat gcccagctga 11040 ttttttttat tttgggggct tgctgtgttg
cccaggctag actcaaaact cctgggcttg 11100 gcccgtcgca gtggcccacg
cctataatcc cagcactttg ggaggccgag gccatcctgg 11160 ctaacacggt
gaaaccccgt ctctactaaa aaaaaaaaat acaaaaatta gccgggcgtg 11220
atggcgggtg cctgtagtcc cagctacttg ggaggcctga ggcaggagaa tgacgtgaac
11280 ccaggaggcg gagcttgcag tgagctgaga tcgcgccact gcactccagc
ctgggcaaca 11340 gagggagact gcgtctcaaa aaaaaaaaaa aaaaaccact
cctggcctca ggccatcctc 11400 cagcctcagc ctcccagagg gttgggatta
ccggcgtgag ccactacact cagccagaat 11460 atactgtttg aatctaatac
ggaaatattt tgtaattcag caacttattt gaaagacttg 11520 gtgaaaagat
tggtggtggc atagctgtag tttacaataa ctgatttgat ttggatttaa 11580
ttgataaaga gtctagttca ggaatttcag ttactcctct tacttatgtt agcattagag
11640 ttgtatgttg gagtttttgt tttcttttat gtgaggatga agaggaagcc
ggcttattgg 11700 agctcttaaa atcccagatt tgtgataatg ccgcactcta
tgcacaaaag tacgatgaag 11760 aattccagcg atacctgcct cgttttgtta
cagccatctg gaatttacta gttacaacgg 11820 gtcaagaggt taaatatgat
ttggtaagat gatggtggag acaaataatt aaaagacatt 11880 ctctccctat
ctcctccaag aaataagctg ttgagttttg cttttaaaaa tattcttgtt 11940
tttgtgtttt gttccagttg gtaagtaatg caattcaatt tctggcttca gtttgtgaga
12000 gacctcatta taagaatcta tttgaggacc agaacacgct gacaagtatc
tgtgaaaagg 12060 ttattgtgcc taacatggaa tttagaggta attatggcaa
aagtatatta gtataaatct 12120 actaagtctg tgtttgtttt ttgtaacata
ttcagtctaa ttcatttatt actggataaa 12180 acttgtatgt catctatttc
ttattttcta aaccaagaca gaaaatgtag tatctgtaat 12240
gagttttgtt gtgccctgtg agggttttct catggaaata ttaaataaaa cttcaaaaat
12300 tcctttacac taaaaaaata aactatctgg aatttctatt tagagaaaga
atttgatttt 12360 gtttcattta ttatttcaat gaatttttag ctggctttat
ttatttatat atttatttga 12420 gacaggggtc tcgctctgtc acccaggctg
gagtgcagtg atgcaatctt ggctcactgc 12480 aaccttagcc tcccaggctc
aagtgatcct cctacctcag tcttctgagt agctgggacc 12540 acaggcacac
atcaccatgc ctggctaatt tcttgtattt ttggtagaga cagggtt 12597 13 5037
DNA Homo sapiens misc_feature 801 n=a, c, g or t 13 gagagagaga
gggagtctcg ctctgtctcc caggctggag tgcagtggca tgattttggc 60
tcactgcaac ctccacttcc caggttcaag cgattctcaa gcctcagcct cccgagtagc
120 tgggactaca gatgtgcacc acatacctgg ctaattttgc tatttttaat
ggagatgggt 180 tttgccatgt tagccaggtt ggtctcagac tctagcctca
agtgatccgc cagcctcggc 240 ctcccaaagt gctggggtta caggtgtgag
ccaccaagca tggcctctgc tactgatttc 300 taagagaaag agaccccaaa
cagttctctc tcagactcac ctgctccatt catgtcagct 360 cactgtttct
tcctcttagt caacctcttt aattattttt taaaccttga gccttctaga 420
aatactcagt atttcccagc taaattttgt tgccaacccg tcctaatttt aaaaggaagg
480 gaaatttaga ttatttttaa atgtagagag ctatctcggg gttgtctaag
catcttaaaa 540 tttagttgat gaattagtgc gcttttttcc ccccagtcgc
tctcttattc tgaagtttaa 600 ctaaactcta tttgtttttg ctgtttttgt
attttacagc tatcatgaga agtttttctc 660 tcctacaaga agccataatc
ccctacatcc ctactctcat cactcagctt acacagaagc 720 tattagctgt
tagtaaggta atagagccaa ttttgaaagt ggggttcctt ttttatttgc 780
tccaaactgt ttgggcctca naactctttg gcattacgtg aagctttaga gaaggtctgt
840 gaattcttcc tgaccagcca ctaatggact tttctgaaag tgtgggcatg
ctgttagatt 900 ggagacgtaa tagcagtcac ctttaagtgt gtgctagaag
cttgaataca ttttcattct 960 cattacaaaa gtaacatgtt tattgtagaa
aaagaaagtg cagatgatcc aaaattacct 1020 taaatggagt taagtaggct
ttgcactaaa tggatataaa agaggctgtc tggacttcta 1080 tgaaatgatg
attaaaatcc tttgtgttgt cttttcttcc ttactcttat ttctcaaaag 1140
gaaagttcac tctgatcaaa attttacaga tacttagatt catgtttatt aacactaaat
1200 tttagaaaat aaatccgttt aaggctgaca gaattccttt ttagatgcta
ttcttgatct 1260 tatgagaatt agatcaattc ataggaagtg ttggagggat
tttggagaac agagtgatta 1320 ttattcaacc gagttagggc taaagggtct
ttggactacc ttagggcctt gagggatgga 1380 gcggggaggg aacccaagtc
tccgcctccc atatgtatta ccatctcatg ttctgattga 1440 gttagatttg
tatgctagta gccttccaag aacttgttta aagaaaaatg gggttttgtt 1500
gcttaaaaag aacataaaaa actactcatt tgggggatta tcagtaagta gacatagaca
1560 ttgaggccca cagaagttaa ggctagtggc attactagga cttagtattt
cttctctgcg 1620 agggcacgtg ggcttaagac aagttacagc tctgggatct
gaggtttgta taagcaactc 1680 tgtaactctg aagtattttt ttcatggtgg
actttgggcc atttaaaaaa aaattcaaat 1740 gaacaagcac tagggtataa
ttggtgtttt tttcttaatt ttgattttta aaaaataaga 1800 tggcctctat
tttaaatgtg ttttataaag gttaggtgtg gattattagc ataattaggt 1860
tttttttttt taatttcaga acccaagcaa acctcacttt aatcactaca tgtttgaagc
1920 aatatgttta tccataagaa taacttgcaa agctaaccct gctgctgttg
taaattttga 1980 ggaggctttg tttttggtgt ttactgaaat cttacaaaat
gatgtgcaag gtaagttaac 2040 ggaaattatt ttctttgtaa tggaataaaa
ttaacatggc tataaaatgc agcccaccta 2100 agcgatgtgg gccttttgtg
tcagtcattc cttctgaagc tcacagctct gtttttactt 2160 ttatcaacag
aatttattcc atacgtcttt caagtgatgt ctttgcttct ggaaacacac 2220
aaaaatgaca tcccgtcttc ctatatggcc ttatttcctc atctccttca gccagtgctt
2280 tgggaaagaa caggaaatat tcctgctcta gtgaggcttc ttcaagcatt
cttagaacgc 2340 ggttcaaaca caatagcaag tgctgcagct gacaaaattg
tgcgtcaggt tttgatataa 2400 ctgtaatttt ataaagtagc ttggagaaac
tggggatggc agatgtttga aattttttta 2460 tttaaaataa atttaaggct
gggggcagtg gctcaagccc accactttgg gagcctctag 2520 tgggaggatc
ccttgagccg aggagtttaa gaccagccta ggcaacgtgg tgagatctat 2580
ctctacaaag gaaaaaaaag ctttttaatt agttggacac agtggagcat gcctgtggtc
2640 ccatctactt gggaggctaa ggtgagaggg tcgcttgagc ccaggagttt
aaggctgcag 2700 tgagccatga tctcaccatt gcactccatc ctgggcaaca
gagctagacc ctgtctctta 2760 aaaaaaataa taataataat aacggaaata
attagtagaa aatgttatgg tatgttttat 2820 cagaattctg tatcctttac
ttatttatat ccatgatgga aaattttaaa aacagagcca 2880 aacaggttga
ttttaaaaac ttaactttgg tcgggcgtgg tggctcacac ctgtaatctc 2940
agcactttgg gaggccgagg tgggcagatc acctgaggtc aggagttcaa gaccagcctg
3000 gccaacatgg cgaaaccccg tctctactaa aaataaaaat tacgtgggtg
tggtggcacg 3060 tgcctgtaat cccagctact cagaaggctg aggcaggaga
atcgcttgaa cctgggaagc 3120 agaggttgca gtgagccaag atcgccccat
tgcactctag cctgggcaac aagagtgaaa 3180 ctccgtctca aagaaaaaaa
aaattacttt aatggctacg cgggaggaat tgttccccat 3240 atgtttcaga
aataattaaa gagaaaagga tagattatta cagtatgaac tctgttttaa 3300
gacatatatc atgtttaact tttcgaataa ttattcctgt taaccatttt ctgttggatc
3360 tcatttctta acagcctggg ttactaggtg tctttcagaa gctgattgca
tccaaagcaa 3420 atgaccacca aggtttttat cttctaaaca gtataataga
gcacatgcct ccgtgagtat 3480 gactagaact ttgtgcattt atttagaaat
tttgttaggt gctcagaaaa gtcctaaatt 3540 tatattgttg atttttttta
attctttagt gaatcagttg accaatatag gaaacaaatc 3600 ttcattctgc
tattccagag acttcagaat tccaaaacaa ccaagtttat caagagtaag 3660
taaaatcatc tggatgttct acagaagtaa tgaaagaagg tagctaaaac ccttagactt
3720 tatggttgca gatacatttt ttatccgttt taaactttat gcaaaaaata
cttggccacg 3780 tgtggaggtt catgcctgta atcgcagcac tttgggtggc
caagggaggc cgatcgcttg 3840 agctcaggag ttcaagacca gcctgggcaa
catggcgaaa ccctgtccct acgaaaagta 3900 caacaaaatt agccaggcat
ggtggcatgt gcctgtagtt gcagctgctt gggaggctga 3960 agtaggagca
ttacctaagt cccagaggtt gaggctgcag tgagccaaga ttacacccac 4020
tgcactccat cctaggtgat agagtgagac cctgtctcaa aaaaaaaaaa gaaaatattg
4080 taagggcctg gtgcagtggc tcttatttgt aatcccagca ctttgggagg
tgaaagtggg 4140 agtattcctt gagttcagga gttaacgacc agcctgggca
acatggtaag accctgtctc 4200 tacaaaacag ccaagcatgg tggtgtgtgc
ctgtagcccc agctactcag gaggctgagg 4260 tgggaggatc acttgaccct
gggaagttaa ggctgcgtga gctgtgatca cgctcctgca 4320 cccagcctag
gtgatagagt gaaaccttct cacaagaaaa aagaaaatat tttaagatgt 4380
ttgtgttctc caaacattaa gttagtgtta gtcatattgt gttccagagg ttggtttatg
4440 gttggatacc aaaacatttt ttcagtagag agaatattat aaggaaatat
aaatcaaaga 4500 gagacagcat gtaaaggtct atgacagaaa gacaagtttt
ctatttacta gaaggcaggt 4560 attgatgcca cagcctttgg atgttaatga
ttaagcatag gtttattttg cagacttatc 4620 agttaccagt ttagtgcgtg
agttctctat gctgatattc tgcatcttct ttcaacaggt 4680 tttttagtct
ttattaattt gtattgcata aaatatgggg cactagcact acaagaaata 4740
tttgatggta tacaaccaaa gtaagtttgt ttttattatt ttttaaaggg aagaaaaatg
4800 ttttgacttt ttttttagta caaatcagta tctctgtctt atagatgatg
atgtggttct 4860 tggtatggag atagtgtcta tggttttcaa aatattctta
ggtattggga gtaaagaaaa 4920 agtaatcctg gcccagccat ggtggctcac
gcccgtaatc ccacactctg agaggctgag 4980 gagggcggat ttagctcagg
agtttgagac cagcctgggc aatataagtg agactcc 5037 14 1919 DNA Homo
sapiens misc_feature 1015 n=a, c, g or t 14 gtgaggcgga gcggcggcag
gagcgggtag tgccagctac ggtccgcggc tggggttccc 60 tcctccgttt
ctgtatcccc acgagatcct atagca atg gaa ctc agc gat gca 114 Met Glu
Leu Ser Asp Ala 1 5 aat ctg caa aca cta aca gaa tat tta aag aaa aca
ctt gat cct gat 162 Asn Leu Gln Thr Leu Thr Glu Tyr Leu Lys Lys Thr
Leu Asp Pro Asp 10 15 20 cct gcc atc cga cgt cca gct gag aaa ttt
ctt gaa tct gtt gaa gga 210 Pro Ala Ile Arg Arg Pro Ala Glu Lys Phe
Leu Glu Ser Val Glu Gly 25 30 35 aat cag aat tat cca ctg ttg ctt
ttg aca tta ctg gag aag tcc cag 258 Asn Gln Asn Tyr Pro Leu Leu Leu
Leu Thr Leu Leu Glu Lys Ser Gln 40 45 50 gat aat gtt atc aaa gta
tgt gct tca gta aca ttc aaa aac tat att 306 Asp Asn Val Ile Lys Val
Cys Ala Ser Val Thr Phe Lys Asn Tyr Ile 55 60 65 70 aaa agg aac tgg
aga att gtt gaa gat gaa cca aac aaa att tgt gaa 354 Lys Arg Asn Trp
Arg Ile Val Glu Asp Glu Pro Asn Lys Ile Cys Glu 75 80 85 gcc gat
cga gtg gcc att aaa gcc aac ata gtg cac ttg atg ctt agc 402 Ala Asp
Arg Val Ala Ile Lys Ala Asn Ile Val His Leu Met Leu Ser 90 95 100
agc cca gag caa att cag aag cag tta agt gat gca att agc att att 450
Ser Pro Glu Gln Ile Gln Lys Gln Leu Ser Asp Ala Ile Ser Ile Ile 105
110 115 ggc aga gaa gat ttt cca cag aaa tgg cct gac ttg ctg aca gaa
atg 498 Gly Arg Glu Asp Phe Pro Gln Lys Trp Pro Asp Leu Leu Thr Glu
Met 120 125 130 gtg aat cgc ttt cag agt gga gat ttc cat gtt att aat
gga gtc ctc 546 Val Asn Arg Phe Gln Ser Gly Asp Phe His Val Ile Asn
Gly Val Leu 135 140 145 150 cgt aca gca cat tca tta ttt aaa aga tac
cgt cat gaa ttt aag tca 594 Arg Thr Ala His Ser Leu Phe Lys Arg Tyr
Arg His Glu Phe Lys Ser 155 160 165 aac gag tta tgg act gaa att aag
ctt gtt ctg gat gcc ttt gct ttg 642 Asn Glu Leu Trp Thr Glu Ile Lys
Leu Val Leu Asp Ala Phe Ala Leu 170 175 180 cct ttg act aat ctt ttt
aag gta tgg aat gca tct tgg tga tatttttaaa 694 Pro Leu Thr Asn Leu
Phe Lys Val Trp Asn Ala Ser Trp 185 190 195 ttaatatttt aaattgcttg
aatgtttgta tacatgttaa gagaatgctt tgaaagctta 754 tttgatagat
aatgtttaga gcatttcttt tgaaaaattt caaacctaca cagaaataga 814
gatgctatag tgaactccca tctattatta cacaaattta atggttagca acgttttatt
874 ttctctgttc cctttcttag gaatgttata aaagcaaatc ccaagcatca
tatcatttca 934 cccaagtatt ttaaagtatg tgtctctaaa ggaagaaata
catattttta tttgactatg 994 tttggagttt aattaattat ntatttatac
attattttac agtttacagg atactttcct 1054 gaatgtgttc ttggtgtttg
tgaaaatcct ataagattaa agaaatatat atatactttt 1114 tttctttttt
cttttttaaa gacagggtct cactctcatt gcccaggctg gagtgcagtg 1174
agctattaca gtgccactgc aatccagcct gggcaacaga gcgaggtccc gtttcttaaa
1234 aaacatatat gtgtgtggcg tgtgtatata tatgtatata tattttttca
ttgtattatt 1294 gcggagactt tcaaacatat atagaaagag cataatgaag
cctgcatgtg cccagcttca 1354 ataattacca atatcttgcc agttttgttt
cgtttctcct ttgattctct gtattgagca 1414 agtcttagac atcatacgtt
tcccgcgtaa gtaccttatt ctacatcatt aaccagtaag 1474 gactttttaa
ttaaccacaa taccactatc acacctaata atagtaattc cttatggatc 1534
ttttctttag acctattttt gaaggcataa aagcagttga gtttctggag aatttttgga
1594 tggtgattaa tgacttgact ggctgctctt cccagagctg tggcagctct
ccccccgtag 1654 aagatggggt ttgtattggc gcaccaagat ctccaacagc
cagtgtgtgt ttcccatttc 1714 ctgtaggttc catcaatggt gagcaccagc
ctgaatgcag aagcgctcca gtatctccaa 1774 gggtaccttc aggcagccag
tgtgacactg ctttaaactg catttttctn aatgggctaa 1834 acccagatgg
tttcctagga aatcacaggc ttctgagcac agctgcatta aaacaaagga 1894
agttttcctt ttgaacttgt cacga 1919 15 1418 DNA Homo sapiens
misc_feature 1205 n=a, c, g or t 15 tcagaagctg attgcatcca
aagcaaatga ccaccaaggt ttttatcttc taaacagtat 60 aatagagcac atg cct
cct gaa tca gtt gac caa tat agg aaa caa atc 109 Met Pro Pro Glu Ser
Val Asp Gln Tyr Arg Lys Gln Ile 1 5 10 ttc att ctg cta ttc cag aga
ctt cag aat tcc aaa aca acc aag ttt 157 Phe Ile Leu Leu Phe Gln Arg
Leu Gln Asn Ser Lys Thr Thr Lys Phe 15 20 25 atc aag agt ttt tta
gtc ttt att aat ttg tat tgc ata aaa tat ggg 205 Ile Lys Ser Phe Leu
Val Phe Ile Asn Leu Tyr Cys Ile Lys Tyr Gly 30 35 40 45 gca cta gca
cta caa gaa ata ttt gat ggt ata caa cca aaa atg ttt 253 Ala Leu Ala
Leu Gln Glu Ile Phe Asp Gly Ile Gln Pro Lys Met Phe 50 55 60 gga
atg gtt ttg gaa aaa att att att cct gaa att cag aag gta tct 301 Gly
Met Val Leu Glu Lys Ile Ile Ile Pro Glu Ile Gln Lys Val Ser 65 70
75 gga aat gta gag aaa aag atc tgt gcg gtt ggc ata acc aaa tta cta
349 Gly Asn Val Glu Lys Lys Ile Cys Ala Val Gly Ile Thr Lys Leu Leu
80 85 90 aca gaa tgt ccc cca atg atg gac act gag tat acc aaa ctg
tgg act 397 Thr Glu Cys Pro Pro Met Met Asp Thr Glu Tyr Thr Lys Leu
Trp Thr 95 100 105 cca tta tta cag tct ttg att ggt ctt ttt gag tta
ccc gaa gat gat 445 Pro Leu Leu Gln Ser Leu Ile Gly Leu Phe Glu Leu
Pro Glu Asp Asp 110 115 120 125 acc att cct gat gag gaa cat ttt att
gac ata gaa gat aca cca gga 493 Thr Ile Pro Asp Glu Glu His Phe Ile
Asp Ile Glu Asp Thr Pro Gly 130 135 140 tat cag act gcc ttc tca cag
ttg gca ttt gct ggg aaa aaa gag cat 541 Tyr Gln Thr Ala Phe Ser Gln
Leu Ala Phe Ala Gly Lys Lys Glu His 145 150 155 gat cct gta ggt caa
atg gtg aat aac ccc aaa att cac ctg gca cag 589 Asp Pro Val Gly Gln
Met Val Asn Asn Pro Lys Ile His Leu Ala Gln 160 165 170 tca ctt cac
aag ttg tct acc gcc tgt cca gga agg acc tat ttt tga 637 Ser Leu His
Lys Leu Ser Thr Ala Cys Pro Gly Arg Thr Tyr Phe 175 180 185
aggcataaaa gcagttgagt ttctggagaa tttttggatg gtgattaatg acttgactgg
697 ctgctcttcc cagagctgtg gcagctctcc cgtagaagat ggggtttgta
ttggcgcacc 757 aagatctcca acagccagtg tgtgtttccc atctcttgta
ggttccatca atggtgagca 817 ccagcctgaa tgcagaagcg ctccagtatc
tccaagggta ccttcaggca gccagtgtga 877 cactgcttta aactgcattt
ttctaatggg ctaaacccag atggtttcct aggaaatcac 937 aggcttctga
gcacagctgc attaaaacaa aggaagttct ccttttgaac ttgtcacgaa 997
ttccatcttg taaaggatat taaatgttgc tttaacctga accttgagca aattagttgg
1057 tttgtgtgat catacagtta tgtgggtggc ttctagtttg caacttcaag
ggacaagtat 1117 taatagttca gtgtatggcg ttggtttgtg ttgagcgttt
gcacggtttg gataatctta 1177 aattttgacg gacactgtgg agactttnct
gttactaaat ccttttgttt tgaagctgtt 1237 gctatttgta tttctcttgt
cctttatatt ttttgtctgt ttatttacgc ttttattgga 1297 aatgtgaata
agtaaagaat tacttgtgtt acttgccaag cagtgcacat ttcatagttt 1357
caaatctgta atcagcaata aaaatcctaa aatatgtacc taaaaaaaaa aaaaaaaaaa
1417 a 1418 16 20 DNA Artificial Sequence Antisense Oligonucleotide
16 ggaagaataa tacttgtgca 20 17 20 DNA Artificial Sequence Antisense
Oligonucleotide 17 tcccagcact ttgggaggct 20 18 20 DNA Artificial
Sequence Antisense Oligonucleotide 18 cgaggtatga agctggacaa 20 19
20 DNA Artificial Sequence Antisense Oligonucleotide 19 atgtctccaa
atatactgcc 20 20 20 DNA Artificial Sequence Antisense
Oligonucleotide 20 gcgacattac ctgcttctga 20 21 20 DNA Artificial
Sequence Antisense Oligonucleotide 21 tgaaataaac atcctagttg 20 22
20 DNA Artificial Sequence Antisense Oligonucleotide 22 cagggagatc
ctacagaata 20 23 20 DNA Artificial Sequence Antisense
Oligonucleotide 23 ggaataaatt ctgttgataa 20 24 20 DNA Artificial
Sequence Antisense Oligonucleotide 24 aaccccggca aaatggcgcg 20 25
20 DNA Artificial Sequence Antisense Oligonucleotide 25 aaccccagcc
gcggaccgta 20 26 20 DNA Artificial Sequence Antisense
Oligonucleotide 26 ctgagttcca ttgctatagg 20 27 20 DNA Artificial
Sequence Antisense Oligonucleotide 27 agaaatttct cagctggacg 20 28
20 DNA Artificial Sequence Antisense Oligonucleotide 28 attctgattt
ccttcaacag 20 29 20 DNA Artificial Sequence Antisense
Oligonucleotide 29 atgtcaaaag caacagtgga 20 30 20 DNA Artificial
Sequence Antisense Oligonucleotide 30 tcctgggact tctccagtaa 20 31
20 DNA Artificial Sequence Antisense Oligonucleotide 31 aatggccact
cgatcggctt 20 32 20 DNA Artificial Sequence Antisense
Oligonucleotide 32 tctgggctgc taagcatcaa 20 33 20 DNA Artificial
Sequence Antisense Oligonucleotide 33 catcacttaa ctgcttctga 20 34
20 DNA Artificial Sequence Antisense Oligonucleotide 34 ctgtggaaaa
tcttctctgc 20 35 20 DNA Artificial Sequence Antisense
Oligonucleotide 35 gtcaggccat ttctgtggaa 20 36 20 DNA Artificial
Sequence Antisense Oligonucleotide 36 tgtcagcaag tcaggccatt 20 37
20 DNA Artificial Sequence Antisense Oligonucleotide 37 aatgtgctgt
acggaggact 20 38 20 DNA Artificial Sequence Antisense
Oligonucleotide 38 atgacggtat cttttaaata 20 39 20 DNA Artificial
Sequence Antisense Oligonucleotide 39 atagtggcct taaaaagatt 20 40
20 DNA Artificial Sequence Antisense Oligonucleotide 40 cagagttcaa
tagtggcctt 20 41 20 DNA Artificial Sequence Antisense
Oligonucleotide 41 aagtttccat attaccttcc 20 42 20 DNA Artificial
Sequence Antisense Oligonucleotide 42 tccaagtttc catattacct 20 43
20 DNA Artificial Sequence Antisense Oligonucleotide 43 gaaattattc
atccaagttt 20 44 20 DNA Artificial Sequence Antisense
Oligonucleotide 44 gctccaataa gccggcttcc 20 45 20 DNA Artificial
Sequence Antisense Oligonucleotide 45 cacaaatctg ggattttaag 20 46
20 DNA Artificial Sequence Antisense Oligonucleotide 46 gccagaaatt
gaattgcatt 20 47 20 DNA Artificial Sequence Antisense
Oligonucleotide 47 gttctggtcc tcaaatagat 20 48 20 DNA Artificial
Sequence Antisense Oligonucleotide 48 cagctctaaa ttccatgtta 20 49
20 DNA Artificial Sequence Antisense Oligonucleotide 49 attcctgtca
caggtccctc 20 50 20 DNA Artificial Sequence Antisense
Oligonucleotide 50 tattcctgca gcatggaatt 20 51 20 DNA Artificial
Sequence Antisense Oligonucleotide 51 actaggtaga tggctgcatc 20 52
20 DNA Artificial Sequence Antisense Oligonucleotide 52 ataccgtcag
ctttaaggac 20 53 20 DNA Artificial Sequence Antisense
Oligonucleotide 53 tgaagatgat taatcaagag 20 54 20 DNA Artificial
Sequence Antisense Oligonucleotide 54 gagctttgaa aaggtttgtt 20 55
20 DNA Artificial Sequence Antisense Oligonucleotide 55 ctcatgatag
ctttcataat 20 56 20 DNA Artificial Sequence Antisense
Oligonucleotide 56 tcttactaac agctaatagc 20 57 20 DNA Artificial
Sequence Antisense Oligonucleotide 57 gcaagttatt cttatggata 20 58
20 DNA Artificial Sequence Antisense Oligonucleotide 58 aacagcagca
gggttagctt 20 59 20 DNA Artificial Sequence Antisense
Oligonucleotide 59 tccaaacatt tttggttgta 20 60 20 DNA Artificial
Sequence Antisense Oligonucleotide 60 agtaatttgg ttatgccaac 20 61
20 DNA Artificial Sequence Antisense Oligonucleotide 61 acattctgtt
agtaatttgg 20 62 20 DNA Artificial Sequence Antisense
Oligonucleotide 62 tggtgtatct tctatgtcaa 20 63 20 DNA Artificial
Sequence Antisense Oligonucleotide 63 ttgtgaagtg actgtgccag 20 64
20 DNA Artificial Sequence Antisense Oligonucleotide 64 tagacaactt
gtgaagtgac 20 65 20 DNA Artificial Sequence Antisense
Oligonucleotide 65 attgatggaa cccttcctgg 20 66 20 DNA Artificial
Sequence Antisense Oligonucleotide 66 actggagcgc ttctgcattc 20 67
20 DNA Artificial Sequence Antisense Oligonucleotide 67 atactggagc
gcttctgcat 20 68 20 DNA Artificial Sequence Antisense
Oligonucleotide 68 aatgcagttt aaagcagtgt 20 69 20 DNA Artificial
Sequence Antisense Oligonucleotide 69 taatgcagct gtgctcagaa 20 70
20 DNA Artificial Sequence Antisense Oligonucleotide 70 agcaacattt
aatatccttt 20 71 20 DNA Artificial Sequence Antisense
Oligonucleotide 71 aaggttcagg ttaaagcaac 20 72 20 DNA Artificial
Sequence Antisense Oligonucleotide 72 acacaaacca actaatttgc 20 73
20 DNA Artificial Sequence Antisense Oligonucleotide 73 gaagccaccc
acataactgt 20 74 20 DNA Artificial Sequence Antisense
Oligonucleotide 74 tagaagccac ccacataact 20 75 20 DNA Artificial
Sequence Antisense Oligonucleotide 75 gtgcaaacgc tcaacacaaa 20 76
20 DNA Artificial Sequence Antisense Oligonucleotide 76 cgtcaaaatt
taagattatc 20 77 20 DNA Artificial Sequence Antisense
Oligonucleotide 77 tgcactgctt ggcaagtaac 20 78 20 DNA Artificial
Sequence Antisense Oligonucleotide 78 agatttgaaa ctatgaaatg 20 79
20 DNA Artificial Sequence Antisense Oligonucleotide 79 ctgattacag
atttgaaact 20 80 20 DNA Artificial Sequence Antisense
Oligonucleotide 80 taggattttt attgctgatt 20 81 20 DNA Artificial
Sequence Antisense Oligonucleotide 81 gcattccata ccttaaaaag 20 82
20 DNA Artificial Sequence Antisense Oligonucleotide 82 aagtccttac
tggttaatga 20 83 20 DNA Artificial Sequence Antisense
Oligonucleotide 83 ggagatcttg gtgcgccaat 20 84 20 DNA Artificial
Sequence Antisense Oligonucleotide 84 ccattgatgg aacctacagg 20 85
20 DNA Artificial Sequence Antisense Oligonucleotide 85 atgccttcaa
aaataggtcc 20 86 20 DNA Artificial Sequence Antisense
Oligonucleotide 86 gatggaacct acaagagatg 20
* * * * *