U.S. patent application number 10/765500 was filed with the patent office on 2004-07-15 for antisense modulation of tradd expression.
Invention is credited to Cowsert, Lex M., Monia, Brett P..
Application Number | 20040137501 10/765500 |
Document ID | / |
Family ID | 22503089 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137501 |
Kind Code |
A1 |
Monia, Brett P. ; et
al. |
July 15, 2004 |
Antisense modulation of TRADD expression
Abstract
Antisense compounds, compositions and methods are provided for
modulating the expression of TRADD. The compositions comprise
antisense compounds, particularly antisense oligonucleotides,
targeted to nucleic acids encoding TRADD. Methods of using these
compounds for modulation of TRADD expression and for treatment of
diseases associated with expression of TRADD are provided.
Inventors: |
Monia, Brett P.; (La Costa,
CA) ; Cowsert, Lex M.; (Carlsbad, CA) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
22503089 |
Appl. No.: |
10/765500 |
Filed: |
January 26, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10765500 |
Jan 26, 2004 |
|
|
|
09763748 |
May 29, 2001 |
|
|
|
09763748 |
May 29, 2001 |
|
|
|
PCT/US99/19614 |
Aug 25, 1999 |
|
|
|
09763748 |
May 29, 2001 |
|
|
|
09143212 |
Aug 28, 1998 |
|
|
|
6077672 |
|
|
|
|
Current U.S.
Class: |
435/6.14 ;
514/44A; 536/23.5 |
Current CPC
Class: |
C12N 15/1138 20130101;
C12N 2310/321 20130101; C12N 2310/3341 20130101; C12N 2310/346
20130101; C12N 2310/321 20130101; C12N 2310/341 20130101; A61K
38/00 20130101; Y02P 20/582 20151101; C12N 2310/315 20130101; C12N
2310/3525 20130101 |
Class at
Publication: |
435/006 ;
514/044; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 048/00 |
Claims
What is claimed is:
1. An antisense compound 8 to 30 nucleotides in length targeted to
a nucleic acid molecule encoding human TRADD, wherein said
antisense compound inhibits the expression of human TRADD.
2. The antisense compound of claim 1 which is an antisense
oligonucleotide.
3. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 17,
18, 19, 21, 24, 25, 26, 27, 29, 31, 32, 34, 35, 36, 38, 39, 40, 41,
43, 44, 46, 47, 50, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65,
66, 67, 68, 69, 71, 72, 73, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85
and 86.
4. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises SEQ ID NO: 9, 17, 26, 27, 34, 47, 50, 52,
55, 58, 61, 68, 69, 72, 78, 85 and 86.
5. The antisense compound of claim 0.2 wherein the antisense
oligonucleotide comprises at least one modified internucleoside
linkage.
6. The antisense compound of claim 5 wherein the modified
internucleoside linkage is a phosphorothioate linkage.
7. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified sugar moiety.
8. The antisense compound of claim 7 wherein the modified sugar
moiety is a 2'-O-methoxyethyl sugar moiety.
9. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified nucleobase.
10. The antisense compound of claim 9 wherein the modified
nucleobase is a 5-methylcytosine.
11. The antisense compound of claim 2 which is a chimeric
oligonucleotide.
12. A pharmaceutical composition comprising the antisense compound
of claim 1 and a pharmaceutically acceptable carrier or
diluent.
13. The pharmaceutical composition of claim 12 further comprising a
colloidal dispersion system.
14. The pharmaceutical composition of claim 12 wherein the
antisense compound is an antisense oligonucleotide.
15. A method of inhibiting the expression of TRADD in human cells
or tissues comprising contacting said cells or tissues with the
antisense compound of claim 1 so that expression of TRADD is
inhibited.
16. A method of treating a human having a disease or condition
associated with TRADD comprising administering to said animal a
therapeutically or prophylactically effective amount of the
antisense compound of claim 1 so that expression of TRADD is
inhibited.
17. The method of claim 16 wherein the disease or condition is
septic shock, inflammation, or cancer.
Description
[0001] This application is a continuation of U.S. Ser. No.
09/763,748 filed May 29, 2001, which is the U.S. National Phase of
PCT/US99/19614 filed Aug. 25, 1999, which is a continuation of
09/143,212 filed Aug. 28, 1998, now issued as U.S. Pat. No.
6,077,672, each of which are herein incorporated by reference in
their entireties.
FIELD OF THE INVENTION
[0002] The present invention provides compositions and methods for
modulating the expression of TRADD. In particular, this invention
relates to antisense compounds, particularly oligonucleotides,
specifically hybridizable with nucleic acids encoding human TRADD.
Such oligonucleotides have been shown to modulate the expression of
TRADD.
BACKGROUND OF THE INVENTION
[0003] One of the principal mechanisms by which cellular regulation
is effected is through the transduction of extracellular signals
into intracellular signals that in turn modulate biochemical
pathways. Examples of such extracellular signaling molecules
include growth factors, cytokines, and chemokines. The cell surface
receptors of these molecules and their associated signal
transduction pathways are therefore one of the principal means by
which cellular behavior is regulated. Because cellular phenotypes
are largely influenced by the activity of these pathways, it is
currently believed that a number of disease states and/or disorders
are a result of either aberrant activation or functional mutations
in the molecular components of signal transduction pathways.
Consequently, considerable attention has been devoted to the
characterization of these proteins.
[0004] For example, the polypeptide cytokine tumor necrosis factor
(TNF) is normally produced during infection, injury, or invasion
and serves as a pivotal mediator of the inflammatory response. In
recent years, a number of in vivo animal and human studies have
demonstrated that overexpression of TNF by the host in response to
disease and infection is itself responsible for the pathological
consequences associated with the underlying disease. For example,
septic shock as a result of massive bacterial infection has been
attributed to infection-induced expression of TNF. Thus, systemic
exposure to TNF at levels comparable to those following massive
bacterial infection has been shown to result in a spectrum of
symptoms (shock, tissue injury, capillary leakage, hypoxia,
pulmonary edema, multiple organ failure, and high mortality rate)
that is virtually indistinguishable from septic shock syndrome
(Tracey and Cerami, Ann. Rev. Med., 1994, 45, 491-503). Further
evidence has been provided in animal models of septic shock, in
which it has been demonstrated that systemic exposure to anti-TNF
neutralizing antibodies block bacterial-induced sepsis (Tracey and
Cerami, Ann. Rev. Med., 1994, 45, 491-503). In addition to these
acute effects, chronic exposure to low-dose TNF results in a
syndrome of cachexia marked by anorexia, weight loss, dehydration,
and depletion of whole-body protein and lipid. Chronic production
of TNF has been implicated in a number of diseases including AIDS
and cancer (Tracey and Cerami, Ann. Rev. Med., 1994, 45,
491-503).
[0005] Studies examining the molecular events associated with TNF
exposure have revealed that activation of the transcription factor
NF-kappa-B is a critical component of many of the effects
attributed to TNF (Tewari and Dixit, Curr. Opin. Genet. Dev., 1996,
6, 39-44). More recently, the intracellular protein TRADD has been
identified as a critical link between TNF receptor binding and
downstream activation of NF-kappa-B. Thus, overexpression of native
TRADD was shown to activate NF-kappa-B in the absence of TNF and
dominant negative mutants of TRADD have been shown to block
TNF-induced NF-kappa-B activation (Tewari and Dixit, Curr. Opin.
Genet. Dev., 1996, 6, 39-44). A second effect of TNF in many cell
types is the induction of apoptosis, or programmed cell death. As
with NF-kappa-B activation, TRADD overexpression has been shown to
mimic TNF induction of apoptosis as well (Darnay and Aggarwal, J.
Leukoc. Biol., 1997, 61, 559-566; Tewari and Dixit, Curr. Opin.
Genet. Dev., 1996, 6, 39-44).
[0006] As a result of these advances in the understanding of TNF
overexpression in certain disease states, there is a great desire
to provide compositions of matter which can inhibit the cellular
effects elicited by TNF. In vitro studies have shown that maximal
cellular responses to TNF are elicited when as little as 10% of TNF
cell membrane receptors are occupied (Tracey and Cerami, Ann. Rev.
Med., 1994, 45, 491-503). These data indicate that downstream
effector proteins such as TRADD are the rate-limiting step of TNF
action and would therefore serve as the most efficient targets for
inhibition of TNF-induced events.
[0007] Currently, there are no known therapeutic agents which
effectively inhibit the synthesis of TRADD. Antisense
oligonucleotides capable of inhibiting TRADD function may therefore
prove to be uniquely useful in a number of therapeutic, diagnostic
and research applications.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to antisense compounds,
particularly oligonucleotides, which are targeted to a nucleic acid
encoding TRADD, and which modulate the expression of TRADD.
Pharmaceutical and other compositions comprising the antisense
compounds of the invention are also provided. Further provided are
methods of modulating the expression of TRADD 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 TRADD 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
[0009] The present invention employs oligomeric antisense
compounds, particularly oligonucleotides, for use in modulating the
function of nucleic acid molecules encoding TRADD, ultimately
modulating the amount of TRADD produced. This is accomplished by
providing antisense compounds which specifically hybridize with one
or more nucleic acids encoding TRADD. As used herein, the terms
"target nucleic acid" and "nucleic acid encoding TRADD" encompass
DNA encoding TRADD, 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 TRADD. 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.
[0010] 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 TRADD. 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 (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
TRADD, regardless of the sequence(s) of such codons.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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, or in the case of in vitro assays, under
conditions in which the assays are performed.
[0016] 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.
[0017] 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 oligonucleotides 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.
[0018] 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.
[0019] 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 30 nucleobases. Particularly preferred are
antisense oligonucleotides comprising from about 8 to about 30
nucleobases (i.e. from about 8 to about 30 linked nucleosides). As
is known in the art, a nucleoside is a base-sugar combination. The
base portion of the nucleoside is normally a heterocyclic base. The
two most common classes of such heterocyclic bases are the purines
and the pyrimidines. Nucleotides are nucleosides that further
include a phosphate group covalently linked to the sugar portion of
the nucleoside. For those nucleosides that include a pentofuranosyl
sugar, the phosphate group can be linked to either the 2', 3' or 5'
hydroxyl moiety of the sugar. In forming oligonucleotides, the
phosphate groups covalently link adjacent nucleosides to one
another to form a linear polymeric compound. In turn the respective
ends of this linear polymeric structure can be further joined to
form a circular structure, however, open linear structures are
generally preferred. Within the oligonucleotide structure, the
phosphate groups are commonly referred to as forming the
internucleoside backbone of the oligonucleotide. The normal linkage
or backbone of RNA and DNA is a 3' to 5' phosphodiester
linkage.
[0020] 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.
[0021] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms are also
included.
[0022] 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; 5,625,050; and
5,697,248, certain of which are commonly owned with this
application, and each of which is herein incorporated by
reference.
[0023] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
[0024] 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; and
5,677,439, certain of which are commonly owned with this
application, and each of which is herein incorporated by
reference.
[0025] 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.
[0026] 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.
[0027] 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)]2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy-group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in U.S. patent application Ser. No. 09/016,520, filed
on Jan. 30, 1998, which is commonly owned with the instant
application and the contents of which are herein incorporated by
reference.
[0028] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugars structures include,
but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920;
and 5,858,221.
[0029] 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, 0.2-aminoadenine, 6-methyl and other alkyl
derivatives of adenine and guanine, 2-propyl and other alkyl
derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and
cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further
nucleobases include those disclosed in U.S. Pat. No. 3,687,808,
those disclosed in The Concise Encyclopedia Of Polymer Science And
Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley &
Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613, and those disclosed by
Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,
pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.
Certain of these nucleobases are particularly useful for increasing
the binding affinity of the oligomeric compounds of the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and O-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and
Lebleu, B., eds., Antisense Research and Applications, CRC Press,
Boca Raton, 1993, pp. 276-278) and are presently preferred base
substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0030] 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; and 5,681,941.
[0031] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] The antisense compounds of the invention are synthesized in
vitro and do not include antisense compositions of biological
origin, or genetic vector constructs designed to direct the in vivo
synthesis of antisense molecules. The compounds of the invention
may also be admixed, encapsulated, conjugated or otherwise
associated with other molecules, molecule structures or mixtures of
compounds, as for example, liposomes, receptor targeted molecules,
oral, rectal, topical or other formulations, for assisting in
uptake, distribution and/or absorption. Representative United
States patents that teach the preparation of such uptake,
distribution and/or absorption assisting formulations include, but
are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
[0037] 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.
[0038] 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 to Imbach
et al.
[0039] 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.
[0040] 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-methylbenzenesufonic 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.
[0041] 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.
[0042] 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 TRADD 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.
[0043] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding TRADD, 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 TRADD 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 TRADD in a sample may also be prepared.
[0044] 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.
[0045] 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.
[0046] Compositions and formulations for oral administration
include powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets or tablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable.
[0047] 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.
[0048] Pharmaceutical compositions and/or formulations comprising
the oligonucleotides of the present invention may also include
penetration enhancers in order to enhance the alimentary delivery
of the oligonucleotides. Penetration enhancers may be classified as
belonging to one of five broad categories, i.e., fatty acids, bile
salts, chelating agents, surfactants and non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8,
91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33). One or more penetration enhancers from one
or more of these broad categories may be included. Various fatty
acids and their derivatives which act as penetration enhancers
include, for example, oleic acid, lauric acid, capric acid,
myristic acid, palmitic acid, stearic acid, linoleic acid,
linolenic acid, dicaprate, tricaprate, recinleate, monoolein
(a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid,
arichidonic acid, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono-
and di-glycerides and physiologically acceptable salts thereof
(i.e., oleate, laurate, caprate, myristate, palmitate, stearate,
linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, 8:2, 91-192; Muranishi, Critical Reviews in
Therapeutic Drug Carrier Systems, 1990, 7:1, 1-33; El-Hariri et
al., J. Pharm. Pharmacol., 1992, 44, 651-654). Examples of some
presently preferred fatty acids are sodium caprate and sodium
laurate, used singly or in combination at concentrations of 0.5 to
5%.
[0049] The physiological roles of bile include the facilitation of
dispersion and absorption of lipids and fat-soluble vitamins
(Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological
Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill,
New York, N.Y., 1996, pages 934-935). Various natural bile salts,
and their synthetic derivatives, act as penetration enhancers.
Thus, the term "bile salt" includes any of the naturally occurring
components of bile as well as any of their synthetic derivatives. A
presently preferred bile salt is chenodeoxycholic acid (CDCA)
(Sigma Chemical Company, St. Louis, Mo.), generally used at
concentrations of 0.5 to 2%.
[0050] Complex formulations comprising one or more penetration
enhancers may be used. For example, bile salts may be used in
combination with fatty acides to make complex formulations.
Preferred combinations include CDCA combined with sodium caprate or
sodium laurate (generally 0.5 to 5%).
[0051] Chelating agents include, but are not limited to, disodium
ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,
sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl
derivatives of collagen, laureth-9 and N-amino acyl derivatives of
beta-diketones (enamines)(Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, 8:2, 92-192; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1,
1-33; Buur et al., J. Control Rel., 1990, 14, 43-51). Chelating
agents have the added advantage of also serving as DNase
inhibitors.
[0052] Surfactants include, for example, sodium lauryl sulfate,
polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, 8:2, 92-191); and perfluorochemical emulsions, such as FC-43
(Takahashi et al., J. Pharm. Phamacol., 1988, 40, 252-257).
[0053] Non-surfactants include, for example, unsaturated cyclic
ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
8:2, 92-191); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0054] As used herein, "carrier compound" refers to a nucleic acid,
or analog thereof, which is inert (i.e., does not possess
biological activity per se) but is recognized as a nucleic acid by
in vivo processes that reduce the bioavailability of a nucleic acid
having biological activity by, for example, degrading the
biologically active nucleic acid or promoting its removal from
circulation. The coadministration of a nucleic acid and a carrier
compound, typically with an excess of the latter substance, can
result in a substantial reduction of the amount of nucleic acid
recovered in the liver, kidney or other extracirculatory
reservoirs, presumably due to competition between the carrier
compound and the nucleic acid for a common receptor. For example,
the recovery of a partially phosphorothioated oligonucleotide in
hepatic tissue is reduced when it is coadministered with
polyinosinic acid, dextran sulfate, polycytidic acid or
4-acetamido-4'-isothiocyano-stilbene-2,2'-di- sulfonic acid (Miyao
et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
[0055] In contrast to a carrier compound, a "pharmaceutically
acceptable carrier" (excipient) is a pharmaceutically acceptable
solvent, suspending agent or any other pharmacologically inert
vehicle for delivering one or more nucleic acids to an animal. The
pharmaceutically acceptable carrier may be liquid or solid and is
selected with the planned manner of administration in mind so as to
provide for the desired bulk, consistency, etc., when combined with
a nucleic acid and the other components of a given pharmaceutical
composition. Typical pharmaceutically acceptable carriers include,
but are not limited to, binding agents (e.g., pregelatinized maize
starch, 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.); disintegrates (e.g., starch, sodium starch glycolate, etc.);
or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained
release oral delivery systems and/or enteric coatings for orally
administered dosage forms are described in U.S. Pat. Nos.
4,704,295; 4,556,552; 4,309,406; and 4,309,404.
[0056] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional
compatible pharmaceutically-active materials such as, e.g.,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the composition of present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the invention.
[0057] Regardless of the method by which the antisense compounds of
the invention are introduced into a patient, colloidal dispersion
systems may be used as delivery vehicles to enhance the in vivo
stability of the compounds and/or to target the compounds to a
particular organ, tissue or cell type. Colloidal dispersion systems
include, but are not limited to, macromolecule complexes,
nanocapsules, microspheres, beads and lipid-based systems including
oil-in-water emulsions, micelles, mixed micelles, liposomes and
lipid:oligonucleotide complexes of uncharacterized structure. A
preferred colloidal dispersion system is a plurality of liposomes.
Liposomes are microscopic spheres having an aqueous core surrounded
by one or more outer layer(s) made up of lipids arranged in a
bilayer configuration (see, generally, Chonn et al., Current Op.
Biotech., 1995, 6, 698-708).
[0058] Certain embodiments of the invention provide for liposomes
and other 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, anticancer
drugs such as daunorubicin, dactinomycin, doxorubicin, bleomycin,
mitomycin, nitrogen mustard, chlorambucil, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),
5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),
colchicine, vincristine, vinblastine, etoposide, teniposide,
cisplatin and diethylstilbestrol (DES). See, generally, The Merck
Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds.,
1987, Rahway, N.J., pages 1206-1228). Antiinflammatory 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.
[0059] 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. Examples of antisense oligonucleotides include, but are not
limited to, those directed to the following targets as disclosed in
the indicated U.S. Patents, or pending U.S. applications, which are
commonly owned with the instant application and are hereby
incorporated by reference, or the indicated published PCT
applications: raf (WO 96/39415, WO 95/32987 and U.S. Pat. No.
5,563,255, issued Oct. 8, 1996, and 5,656,612, issued Aug. 12,
1997), the p120 nucleolar antigen (WO 93/17125 and U.S. Pat. No.
5,656,743, issued Aug. 12, 1997), protein kinase C (WO 95/02069, WO
95/03833 and WO 93/19203), multidrug resistance-associated protein
(WO 95/10938 and U.S. Pat. No. 5,510,239, issued Mar. 23, 1996),
subunits of transcription factor AP-1 (pending application U.S.
Ser. No. 08/837,201, filed Apr. 14, 1997), Jun kinases (pending
application U.S. Ser. No. 08/910,629, filed Aug. 13, 1997), MDR-1
(multidrug resistance glycoprotein; pending application U.S. Ser.
No. 08/731,199, filed Sep. 30, 1997), HIV (U.S. Pat. No. 5,166,195,
issued Nov. 24, 1992 and 5,591,600, issued Jan. 7, 1997),
herpesvirus (U.S. Pat. No. 5,248,670, issued Sep. 28, 1993 and U.S.
Pat. No. 5,514,577, issued May 7, 1996), cytomegalovirus (U.S. Pat.
No. 5,442,049, issued Aug. 15, 1995 and 5,591,720, issued Jan. 7,
1997), papillomavirus (U.S. Pat. No. 5,457,189, issued Oct. 10,
1995), intercellular adhesion molecule-1 (ICAM-1) (U.S. Pat. No.
5,514,788, issued May 7, 1996), 5-lipoxygenase (U.S. Pat. No.
5,530,114, issued Jun. 25, 1996) and influenzavirus (U.S. Pat. No.
5,580,767, issued Dec. 3, 1996). Two or more combined compounds may
be used together or sequentially.
[0060] 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.
[0061] 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
[0062] Nucleoside Phosphoramidites for Oligonucleotide Synthesis
Deoxy and 2'-alkoxy amidites
[0063] 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.
[0064] 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.).
[0065] 2'-Fluoro amidites
[0066] 2'-Fluorodeoxyadenosine amidites
[0067] 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.
[0068] 2'-Fluorodeoxyguanosine
[0069] The synthesis of 2'-deoxy-2'-fluoroguanosine was
accomplished using tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosylguani- ne as starting material, and
conversion to the intermediate
diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS
group was followed by protection of the hydroxyl group with THP to
give diisobutyryl di-THP protected arabinofuranosylguanine.
Selective O-deacylation and triflation was followed by treatment of
the crude product with fluoride, then deprotection of the THP
groups. Standard methodologies were used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidi- tes.
[0070] 2'-Fluorouridine
[0071] 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.
[0072] 2'-Fluorodeoxycytidine
[0073] 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.
[0074] 2'-O-(2-Methoxyethyl) Modified Amidites
[0075] 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.
[0076]
2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]
[0077] 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.).
[0078] 2'-O-Methoxyethyl-5-methyluridine
[0079] 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.
[0080] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0081] 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%).
[0082]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0083] 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 tic by first quenching the tic
sample with the addition of MeOH. Upon completion of the reaction,
as judged by tic, 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.
[0084]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triaz-
oleuridine
[0085] 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 .degree. C. and stirred for 0.5 h using an overhead
stirrer. POCl.sub.3 was added dropwise, over a 30 minute period, to
the stirred solution maintained at 0-10.degree. C., and the
resulting mixture stirred for an additional 2 hours. The first
solution was added dropwise, over a 45 minute period, to the later
solution. The resulting reaction mixture was stored overnight in a
cold room. Salts were filtered from the reaction mixture and the
solution was evaporated. The residue was dissolved in EtOAc (1 L)
and the insoluble solids were removed by filtration. The filtrate
was washed with 1.times.300 mL of NaHCO.sub.3 and 2.times.300 mL of
saturated NaCl, dried over sodium sulfate and evaporated. The
residue was triturated with EtOAc to give the title compound.
[0086] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0087] A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5--
methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and
NH.sub.4OH (30 mL) was stirred at room temperature for 2 hours. The
dioxane solution was evaporated and the residue azeotroped with
MeOH (2.times.200 mL). The residue was dissolved in MeOH (300 mL)
and transferred to a 2 liter stainless steel pressure vessel. MeOH
(400 mL) saturated with NH.sub.3 gas was added and the vessel
heated to 100.degree. C. for 2 hours (tlc showed complete
conversion). The vessel contents were evaporated to dryness and the
residue was dissolved in EtOAc (500 mL) and washed once with
saturated NaCl (200 mL). The organics were dried over sodium
sulfate and the solvent was evaporated to give 85 g (95%) of the
title compound.
[0088]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0089] 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.
[0090]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine--
3'-amidite
[0091]
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.
[0092] 2'-(Aminooxyethyl) Nucleoside Amidites and
2'-(dimethylaminooxyethy- l) Nucleoside Amidites
[0093] Aminooxyethyl and dimethylaminooxyethyl amidites are
prepared as per the methods of U.S. patent application Ser. No.
10/037,143, filed Feb. 14, 1998, and Ser. No. 09/016,520, filed
Jan. 30, 1998, each of which is commonly owned with the instant
application and is herein incorporated by reference.
Example 2
[0094] Oligonucleotide Synthesis
[0095] 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.
[0096] 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 hr), 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.
[0097] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0098] 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.
[0099] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein
incorporated by reference.
[0100] 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.
[0101] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0102] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0103] 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
[0104] Oligonucleoside Synthesis
[0105] 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
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289, all of which are herein
incorporated by reference.
[0106] 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.
[0107] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 4
[0108] PNA Synthesis
[0109] 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
[0110] Synthesis of Chimeric Oligonucleotides
[0111] 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".
[0112] [2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0113] Chimeric oligonucleotides having 2'-Q-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 380B, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
increasing the wait step after the delivery of tetrazole and base
to 600 s repeated four times for RNA and twice for 2'-O-methyl. The
fully protected oligonucleotide is cleaved from the support and the
phosphate group is deprotected in 3:1 Ammonia/Ethanol at room
temperature overnight then lyophilized to dryness. Treatment in
methanolic ammonia for 24 hrs at room temperature is then done to
deprotect all bases and sample was again lyophilized to dryness.
The pellet is resuspended in 1M TBAF in THF for 24 hrs at room
temperature to deprotect the 2' positions. The reaction is then
quenched with 1M TEAA and the sample is then reduced to {fraction
(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 spectrometer.
[0114] [2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)]
Chimeric Phosphorothioate Oligonucleotides
[0115] [2'-O-(2-methoxyethyl)]-[2'-deoxy]-[-2'-O-(methoxyethyl)]
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
[0116] [2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy
Phosphorothioate]-[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0117] [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.
[0118] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 6
[0119] Oligonucleotide Isolation
[0120] 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
[0121] Oligonucleotide Synthesis--96 Well Plate Format
[0122] 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.
[0123] 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
[0124] Oligonucleotide Analysis--96 Well Plate Format
[0125] 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/AC.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
[0126] Cell Culture and Oligonucleotide Treatment
[0127] 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 four cell types are provided for
illustrative purposes, but other cell types can be routinely
used.
[0128] T-24 Cells:
[0129] The 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.
[0130] 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.
[0131] A549 Cells:
[0132] 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
trysinization and dilution when they reached 90% confluence.
[0133] NHDF Cells:
[0134] 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.
[0135] HEK Cells:
[0136] 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.
[0137] Treatment With Antisense Compounds:
[0138] 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 uL of OPTI-MEM.TM.-1
containing 3.75 .mu.g/mL LIPOFECTIN.TM. (Gibco BRL) and the desired
oligonucleotide at a final concentration of 150 nM. After 4 hours
of treatment, the medium was replaced with fresh medium. Cells were
harvested 16 hours after oligonucleotide treatment.
Example 10
[0139] Analysis of Oligonucleotide Inhibition of TRADD
Expression
[0140] Antisense modulation of TRADD expression can be assayed in a
variety of ways known in the art. For example, TRADD 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. Other methods of
PCR are also known in the art.
[0141] TRADD protein levels 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 TRADD 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.
[0142] 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
[0143] Poly(A)+ mRNA Isolation
[0144] 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. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0145] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
[0146] Total RNA Isolation
[0147] Total mRNA 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 pippeting 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.
Example 13
[0148] Real-Time Quantitative PCR Analysis of TRADD mRNA Levels
[0149] Quantitation of TRADD mRNA levels was determined by
real-time quantitative PCR using the ABI PRISM.TM. 7700 Sequence
Detection System (PE-Applied Biosystems, Foster City, Calif.)
according to manufacturer's instructions. This is a closed-tube,
non-gel-based, fluorescence detection system which allows
high-throughput quantitation of polymerase chain reaction (PCR)
products in real-time. As opposed to standard PCR, in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., JOE or FAM, obtained from either Operon
Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster
City, Calif.) is attached to the 5' end of the probe and a quencher
dye (e.g., TAMRA, obtained from either Operon Technologies Inc.,
Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is
attached to the 3' end of the probe. When the probe and dyes are
intact, reporter dye emission is quenched by the proximity of the
3' quencher dye. During amplification, annealing of the probe to
the target sequence creates a substrate that can be cleaved by the
5'-exonuclease activity of Taq polymerase. During the extension
phase of the PCR amplification cycle, cleavage of the probe by Taq
polymerase releases the reporter dye from the remainder of the
probe (and hence from the quencher moiety) and a sequence-specific
fluorescent signal is generated. With each cycle, additional
reporter dye molecules are cleaved from their respective probes,
and the fluorescence intensity is monitored at regular (six-second)
intervals by laser optics built into the ABI PRISM.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.
[0150] PCR reagents were obtained from PE-Applied Biosystems,
Foster City, Calif. RT-PCR reactions were carried out by adding 25
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
poly(A) mRNA solution. The RT reaction was carried out by
incubation for 30 minutes at 48.degree. C. Following a 10 minute
incubation at 95.degree. C. to activate the AMPLITAQ GOLD.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). TRADD probes and primers were
designed to hybridize to the human TRADD sequence, using published
sequence information (GenBank accession number L41690, incorporated
herein as SEQ ID NO:1).
[0151] For TRADD the PCR primers were: forward primer:
ACGAGGAGCGCTGTTTGAGT (SEQ ID No. 2) reverse primer:
TCCAGCTCAGCCAGTTCTTCAT (SEQ ID No. 3) and the PCR probe was:
FAM-CCAGCAGCCCGACCGGCTC-TAMRA (SEQ ID No. 4) 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.
[0152] For GAPDH the PCR primers were: forward primer:
GAAGGTGAAGGTCGGAGTC (SEQ ID No. 5) reverse primer:
GAAGATGGTGATGGGATTTC (SEQ ID No. 6) and the PCR probe was: 5'
JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID No. 7) 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
[0153] Northern Blot Analysis of TRADD mRNA Levels
[0154] 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.).
[0155] Membranes were probed using QUICKHYB.TM. hybridization
solution (Stratagene, La Jolla, Calif.) using manufacturer's
recommendations for stringent conditions with a TRADD specific
probe prepared by PCR using the forward primer ACGAGGAGCGCTGTTTGAGT
(SEQ ID No. 2) and the reverse primer TCCAGCTCAGCCAGTTCTTCAT (SEQ
ID No. 3). To normalize for variations in loading and transfer
efficiency membranes were stripped and probed for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.). 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
[0156] Antisense Inhibition of TRADD Expression--phosphorothioate
oligodeoxynucleotides
[0157] In accordance with the present invention, a series of
oligonucleotides were designed to target different regions of the
human TRADD RNA, using published sequences (GenBank accession
number L41690, incorporated herein as SEQ ID NO: 1). The
oligonucleotides are shown in Table 1. Target sites are indicated
by nucleotide numbers, as given in the sequence source reference
(Genbank accession no. L41690), to which the oligonucleotide binds.
All compounds in Table 1 are oligodeoxynucleotides with
phosphorothioate backbones (internucleoside linkages) throughout.
The compounds were analyzed for effect on TRADD mRNA levels by
quantitative real-time PCR as described in other examples herein.
Data are averages from three experiments. If present, "N.D."
indicates "no data".
1TABLE 1 Inhibition of TRADD mRNA levels by phosphorothioate
oligodeoxynucleotides % TARGET Inhi- SEQ ID ISIS# REGION SITE
SEQUENCE bition NO. 19799 Coding 1 ggttcccacgcccgccag 0 8 19800
Coding 31 ctcacctcctggccgcct 24 9 19801 Coding 40
agctgccatctcacctcc 0 10 19802 Coding 61 ctcttcgtgcccattttg 11 11
19803 Coding 67 cacccactcttcgtgccc 0 12 19804 Coding 73
gctgcccacccactcttc 0 13 19805 Coding 80 ggtatgcgctgcccaccc 0 14
19806 Coding 90 tccacaaacaggtatgcg 0 15 19807 Coding 96
gaggactccacaaacagg 7 16 19808 Coding 103 gtccagcgaggactccac 64 17
19809 Coding 110 ccaccttgtccagcgagg 41 18 19810 Coding 123
gcatccgacaggaccacc 7 19 19811 Coding 129 gcgtaggcatccgacagg 3 20
19812 Coding 133 gtgcgcgtaggcatccga 0 21 19813 Coding 193
cccgccgctctctgccaa 0 22 19814 Coding 224 ggatcttcagcatctgca 0 23
19815 Coding 230 tgcggtggatcttcagca 0 24 19816 Coding 240
tgcgggtcgctgcggtgg 31 25 19817 Coding 269 gcccgcagaatcgcagct 25 26
19818 Coding 293 ggaggaagcggccacagg 32 27 19819 Coding 362
agtgctgggcgagcgcgg 0 28 19820 Coding 372 agcggcaccgagtgctgg 0 29
19821 Coding 378 agttgcagcggcaccgag 0 30 19822 Coding 384
agctccagttgcagcggc 18 31 19823 Coding 420 gccagcaaagcgtccagc 0 32
19824 Coding 448 gatgcaactcaaacagcg 0 33 19825 Coding 456
tgggctaggatgcaactc 33 34 19826 Coding 461 gctgctgggctaggatgc 16 35
19827 Coding 468 cggtcgggctgctgggct 0 36 19828 Coding 492
tcagccagttcttcatcc 0 37 19829 Coding 510 cgcagcgcatcctccagc 1 38
19830 Coding 516 agatttcgcagcgcatcc 0 39 19831 Coding 526
gccgcacttcagatttcg 0 40 19832 Coding 532 ccccgagccgcacttcag 23 41
19833 Coding 563 ccgaagcgacctccccgt 0 42 19834 Coding 596
ccgacagagagggcaccg 0 43 19835 Coding 617 gcggcggcggcggcttca 0 44
19836 Coding 624 ggtggcggcggcggcggc 1 45 19837 Coding 629
gggcaggtggcggcggcg 30 46 19838 Coding 636 aaagtctgggcaggtggc 44 47
19839 Coding 644 ggaacagaaaagtctggg 7 48 19840 Coding 650
gaccctggaacagaaaag 10 49 19841 Coding 662 tcactacaggctgaccct 26 50
19842 Coding 666 cgattcactacaggctga 14 51 19843 Coding 685
gtccttcaggctcagcgg 25 52 19844 Coding 692 tctgttggtccttcaggc 0 53
19845 Coding 720 catttgagacccacagag 17 54 19846 Coding 727
cttgcgccatttgagacc 30 55 19847 Coding 741 agtgagcgccccaccttg 2 56
19848 Coding 763 cagcgcccggcagcctcg 0 57 19849 Coding 783
gagtccagcgccgggtcc 52 58 19850 Coding 794 cgtaggccagcgagtcca 0 59
19851 Coding 803 gctcgtactcgtaggcca 0 60 19852 Coding 810
ccctcgcgctcgtactcg 58 61 19853 Coding 822 tgctcgtacagtccctcg 0 62
19854 Coding 851 gcacgaagcgccgcagca 0 63 19855 Coding 930
tcctctgccaggctggtg 0 64 19856 Coding 939 cccagcaagtcctctgcc 25 65
19857 Coding 964 caggccgccattgggatc 0 66 19858 Stop 986
tggctgcacccctggtct 0 67 19859 3' UTR 1004 tccaggttctccaaaagc 54 68
19860 3' UTR 1016 accctaaggccatccagg 36 69 19861 3' UTR 1032
aatagccgcagaaggaac 17 70 19862 3' UTR 1063 tcagggtcccgtggatgg 0 71
19863 3' UTR 1079 taggccaagtggagtttc 42 72 19864 3' UTR 1093
gcaggtccagcagatagg 10 73 19865 3' UTR 1117 gggaaggcaatcaactct 0 74
19866 3' UTR 1161 gaggcagaatccccaatg 0 75 19867 3' UTR 1176
tctatcaaagtacctgag 0 76 19868 3' UTR 1188 cccaccccacactctatc 53 77
19869 3' UTR 1219 aaggtgaggctgatctcc 28 78 19870 3' UTR 1228
ggatgggagaaggtgagg 45 79 19871 3' UTR 1262 aaactgtaagggctggct 9 80
19872 3' UTR 1286 aaagatcaaggtgcttca 0 81 19873 3' UTR 1303
atgaagtccaggacacca 0 82 19874 3' UTR 1339 ctgttttacttcactgca 0 83
19875 3' UTR 1351 caagattgattcctgttt 6 84 19876 3' UTR 1378
ccacgctgagtgtgagct 32 85 19877 3' UTR 1409 actttattatcattgctt 34 86
19878 3' UTR 1418 ccgtgttatactttatta 0 87
[0158] As shown in Table 1, SEQ ID NOs 9, 17, 18, 25, 26, 27, 34,
41, 46, 47, 50, 52, 55, 58, 61, 65, 68, 69, 72, 77, 78, 79, 85 and
86 demonstrated at least 20% inhibition of TRADD expression in this
assay and are therefore preferred.
Example 16
[0159] Antisense Inhibition of TRADD Expression--phosphorothioate
2'-MOE gapmer oligonucleotides
[0160] In accordance with the present invention, a second series of
oligonucleotides targeted to human TRADD were synthesized. The
oligonucleotide sequences are shown in Table 2. Target sites are
indicated by nucleotide numbers, as given in the sequence source
reference (Genbank accession no. L41690), to which the
oligonucleotide binds.
[0161] All compounds in Table 2 are chimeric oligonucleotides
("gapmers") 18 nucleotides in length, composed of a central "gap"
region consisting of ten 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by four-nucleotide "wings". The
wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
throughout the oligonucleotide. Cytidine residues in the 2'-MOE
wings are 5-methylcytidines.
[0162] Data were obtained by real-time quantitative PCR as
described in other examples herein and are averaged from three
experiments. If present, "N.D." indicates "no data".
2TABLE 2 Inhibition of TRADD mRNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap % TARGET Inhi- SEQ ID ISIS# REGION SITE SEQUENCE bition NO.
20039 Coding 1 ggttcccacgcccgccag 0 8 20040 Coding 31
ctcacctcctggccgcct 39 9 20041 Coding 40 agctgccatctcacctcc 51 10
20042 Coding 61 ctcttcgtgcccattttg 53 11 20043 Coding 67
cacccactcttcgtgccc 50 12 20044 Coding 73 gctgcccacccactcttc 74 13
20045 Coding 80 ggtatgcgctgcccaccc 88 14 20046 Coding 90
tccacaaacaggtatgcg 67 15 20047 Coding 96 gaggactccacaaacagg N.D. 16
20048 Coding 103 gtccagcgaggactccac 70 17 20049 Coding 110
ccaccttgtccagcgagg N.D. 18 20050 Coding 123 gcatccgacaggaccacc 70
19 20051 Coding 129 gcgtaggcatccgacagg N.D. 20 20052 Coding 133
gtgcgcgtaggcatccga 79 21 20053 Coding 193 cccgccgctctctgccaa 13 22
20054 Coding 224 ggatcttcagcatctgca N.D. 23 20055 Coding 230
tgcggtggatcttcagca 50 24 20056 Coding 240 tgcgggtcgctgcggtgg 15 25
20057 Coding 269 gcccgcagaatcgcagct 43 26 20058 Coding 293
ggaggaagcggccacagg 66 27 20059 Coding 362 agtgctgggcgagcgcgg 23 28
20060 Coding 372 agcggcaccgagtgctgg 66 29 20061 Coding 378
agttgcagcggcaccgag 24 30 20062 Coding 384 agctccagttgcagcggc 63 31
20063 Coding 420 gccagcaaagcgtccagc 75 32 20064 Coding 448
gatgcaactcaaacagcg 13 33 20065 Coding 456 tgggctaggatgcaactc 44 34
20066 Coding 461 gctgctgggctaggatgc 47 35 20067 Coding 468
cggtcgggctgctgggct 40 36 20068 Coding 492 tcagccagttcttcatcc 0 37
20069 Coding 510 cgcagcgcatcctccagc 78 38 20070 Coding 516
agatttcgcagcgcatcc 48 39 20071 Coding 526 gccgcacttcagatttcg 38 40
20072 Coding 532 ccccgagccgcacttcag 10 41 20073 Coding 563
ccgaagcgacctccccgt 1 42 20074 Coding 596 ccgacagagagggcaccg 46 43
20075 Coding 617 gcggcggcggcggcttca 84 44 20076 Coding 624
ggtggcggcggcggcggc 24 45 20077 Coding 629 gggcaggtggcggcggcg 11 46
20078 Coding 636 aaagtctgggcaggtggc 47 47 20079 Coding 644
ggaacagaaaagtctggg 16 48 20080 Coding 650 gaccctggaacagaaaag N.D.
49 20081 Coding 662 tcactacaggctgaccct 46 50 20082 Coding 666
cgattcactacaggctga 17 51 20083 Coding 685 gtccttcaggctcagcgg 69 52
20084 Coding 692 tctgttggtccttcaggc 40 53 20085 Coding 720
catttgagacccacagag 1 54 20086 Coding 727 cttgcgccatttgagacc 40 55
20087 Coding 741 agtgagcgccccaccttg 70 56 20088 Coding 763
cagcgcccggcagcctcg 38 57 20089 Coding 783 gagtccagcgccgggtcc 56 58
20090 Coding 794 cgtaggccagcgagtcca 43 59 20091 Coding 803
gctcgtactcgtaggcca 82 60 20092 Coding 810 ccctcgcgctcgtactcg 76 61
20093 Coding 822 tgctcgtacagtccctcg 62 62 20094 Coding 851
gcacgaagcgccgcagca 0 63 20095 Coding 930 tcctctgccaggctggtg 69 64
20096 Coding 939 cccagcaagtcctctgcc 29 65 20097 Coding 964
caggccgccattgggatc 57 66 20098 Stop 986 tggctgcacccctggtct 64 67
20099 3' UTR 1004 tccaggttctccaaaagc 50 68 20100 3' UTR 1016
accctaaggccatccagg 49 69 20101 3' UTR 1032 aatagccgcagaaggaac 0 70
20102 3' UTR 1063 tcagggtcccgtggatgg 75 71 20103 3' UTR 1079
taggccaagtggagtttc 60 72 20104 3' UTR 1093 gcaggtccagcagatagg 79 73
20105 3' UTR 1117 gggaaggcaatcaactct 0 74 20106 3' UTR 1161
gaggcagaatccccaatg 60 75 20107 3' UTR 1176 tctatcaaagtacctgag 16 76
20108 3' UTR 1188 cccaccccacactctatc 0 77 20109 3' UTR 1219
aaggtgaggctgatctcc 77 78 20110 3' UTR 1228 ggatgggagaaggtgagg 14 79
20111 3' UTR 1262 aaactgtaagggctggct 67 80 20112 3' UTR 1286
aaagatcaaggtgcttca 50 81 20113 3' UTR 1303 atgaagtccaggacacca 53 82
20114 3' UTR 1339 ctgttttacttcactgca 74 83 20115 3' UTR 1351
caagattgattcctgttt 68 84 20116 3' UTR 1378 ccacgctgagtgtgagct 81 85
20117 3' UTR 1409 actttattatcattgctt 72 86 20118 3' UTR 1418
ccgtgttatactttatta 0 87
[0163] As shown in Table 2, SEQ ID NOs 9, 10, 11, 12, 13, 14, 15,
17, 19, 21, 24, 26, 27, 29, 31, 32, 34, 35, 36, 38, 39, 40, 43, 44,
47, 50, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 64, 66, 67, 68, 69,
71, 72, 73, 75, 78, 80, 81, 82, 83, 84, 85 and 86 demonstrated at
least 30% inhibition of TRADD expression in this experiment and are
therefore preferred.
Example 17
[0164] Western Blot Analysis of TRADD Protein Levels
[0165] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 hr 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 TRADD 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
87 1 1435 DNA Homo sapiens CDS (1)..(987) 1 ctg gcg ggc gtg gga acc
cag gcc ccg ccg agg cgg cca gga ggt gag 48 Leu Ala Gly Val Gly Thr
Gln Ala Pro Pro Arg Arg Pro Gly Gly Glu 1 5 10 15 atg gca gct ggg
caa aat ggg cac gaa gag tgg gtg ggc agc gca tac 96 Met Ala Ala Gly
Gln Asn Gly His Glu Glu Trp Val Gly Ser Ala Tyr 20 25 30 ctg ttt
gtg gag tcc tcg ctg gac aag gtg gtc ctg tcg gat gcc tac 144 Leu Phe
Val Glu Ser Ser Leu Asp Lys Val Val Leu Ser Asp Ala Tyr 35 40 45
gcg cac ccc cag cag aag gtg gca gtg tac agg gct ctg cag gct gcc 192
Ala His Pro Gln Gln Lys Val Ala Val Tyr Arg Ala Leu Gln Ala Ala 50
55 60 ttg gca gag agc ggc ggg agc ccg gac gtg ctg cag atg ctg aag
atc 240 Leu Ala Glu Ser Gly Gly Ser Pro Asp Val Leu Gln Met Leu Lys
Ile 65 70 75 80 cac cgc agc gac ccg cag ctg atc gtg cag ctg cga ttc
tgc ggg cgg 288 His Arg Ser Asp Pro Gln Leu Ile Val Gln Leu Arg Phe
Cys Gly Arg 85 90 95 cag ccc tgt ggc cgc ttc ctc cgc gcc tac cgc
gag ggg gcg ctg cgc 336 Gln Pro Cys Gly Arg Phe Leu Arg Ala Tyr Arg
Glu Gly Ala Leu Arg 100 105 110 gcc gcg ctg cag agg agc ctg gcg gcc
gcg ctc gcc cag cac tcg gtg 384 Ala Ala Leu Gln Arg Ser Leu Ala Ala
Ala Leu Ala Gln His Ser Val 115 120 125 ccg ctg caa ctg gag ctg cgc
gcc ggc gcc gag cgg ctg gac gct ttg 432 Pro Leu Gln Leu Glu Leu Arg
Ala Gly Ala Glu Arg Leu Asp Ala Leu 130 135 140 ctg gcg gac gag gag
cgc tgt ttg agt tgc atc cta gcc cag cag ccc 480 Leu Ala Asp Glu Glu
Arg Cys Leu Ser Cys Ile Leu Ala Gln Gln Pro 145 150 155 160 gac cgg
ctc cgg gat gaa gaa ctg gct gag ctg gag gat gcg ctg cga 528 Asp Arg
Leu Arg Asp Glu Glu Leu Ala Glu Leu Glu Asp Ala Leu Arg 165 170 175
aat ctg aag tgc ggc tcg ggg gcc cgg ggt ggc gac ggg gag gtc gct 576
Asn Leu Lys Cys Gly Ser Gly Ala Arg Gly Gly Asp Gly Glu Val Ala 180
185 190 tcg gcc ccc ttg cag ccc ccg gtg ccc tct ctg tcg gag gtg aag
ccg 624 Ser Ala Pro Leu Gln Pro Pro Val Pro Ser Leu Ser Glu Val Lys
Pro 195 200 205 ccg ccg ccg ccg cca cct gcc cag act ttt ctg ttc cag
ggt cag cct 672 Pro Pro Pro Pro Pro Pro Ala Gln Thr Phe Leu Phe Gln
Gly Gln Pro 210 215 220 gta gtg aat cgg ccg ctg agc ctg aag gac caa
cag acg ttc gcg cgc 720 Val Val Asn Arg Pro Leu Ser Leu Lys Asp Gln
Gln Thr Phe Ala Arg 225 230 235 240 tct gtg ggt ctc aaa tgg cgc aag
gtg ggg cgc tca ctg cag cga ggc 768 Ser Val Gly Leu Lys Trp Arg Lys
Val Gly Arg Ser Leu Gln Arg Gly 245 250 255 tgc cgg gcg ctg cgg gac
ccg gcg ctg gac tcg ctg gcc tac gag tac 816 Cys Arg Ala Leu Arg Asp
Pro Ala Leu Asp Ser Leu Ala Tyr Glu Tyr 260 265 270 gag cgc gag gga
ctg tac gag cag gcc ttc cag ctg ctg cgg cgc ttc 864 Glu Arg Glu Gly
Leu Tyr Glu Gln Ala Phe Gln Leu Leu Arg Arg Phe 275 280 285 gtg cag
gcc gag ggc cgc cgc gcc acg ctg cag cgc ctg gtg gag gca 912 Val Gln
Ala Glu Gly Arg Arg Ala Thr Leu Gln Arg Leu Val Glu Ala 290 295 300
ctc gag gag aac gag ctc acc agc ctg gca gag gac ttg ctg ggc ctg 960
Leu Glu Glu Asn Glu Leu Thr Ser Leu Ala Glu Asp Leu Leu Gly Leu 305
310 315 320 acc gat ccc aat ggc ggc ctg gcc tag accaggggtg
cagccagctt 1007 Thr Asp Pro Asn Gly Gly Leu Ala 325 ttggagaacc
tggatggcct tagggttcct tctgcggcta ttgctgaacc cctgtccatc 1067
cacgggaccc tgaaactcca cttggcctat ctgctggacc tgctggggca gagttgattg
1127 ccttccccag gagccagacc actgggggtg catcattggg gattctgcct
caggtacttt 1187 gatagagtgt ggggtggggg ggacttgctt tggagatcag
cctcaccttc tcccatccca 1247 gaagcggggc ttacagccag cccttacagt
ttcactcatg aagcaccttg atctttggtg 1307 tcctggactt catcctgggt
gctgcagata ctgcagtgaa gtaaaacagg aatcaatctt 1367 gcctgccccc
agctcacact cagcgtggga ccccgaatgt taagcaatga taataaagta 1427
taacacgg 1435 2 20 DNA Artificial Sequence Synthetic 2 acgaggagcg
ctgtttgagt 20 3 22 DNA Artificial Sequence Synthetic 3 tccagctcag
ccagttcttc at 22 4 19 DNA Artificial Sequence Synthetic 4
ccagcagccc gaccggctc 19 5 19 DNA Artificial Sequence Synthetic 5
gaaggtgaag gtcggagtc 19 6 20 DNA Artificial Sequence Synthetic 6
gaagatggtg atgggatttc 20 7 20 DNA Artificial Sequence Synthetic 7
caagcttccc gttctcagcc 20 8 18 DNA Artificial Sequence Synthetic 8
ggttcccacg cccgccag 18 9 18 DNA Artificial Sequence Synthetic 9
ctcacctcct ggccgcct 18 10 18 DNA Artificial Sequence Synthetic 10
agctgccatc tcacctcc 18 11 18 DNA Artificial Sequence Synthetic 11
ctcttcgtgc ccattttg 18 12 18 DNA Artificial Sequence Synthetic 12
cacccactct tcgtgccc 18 13 18 DNA Artificial Sequence Synthetic 13
gctgcccacc cactcttc 18 14 18 DNA Artificial Sequence Synthetic 14
ggtatgcgct gcccaccc 18 15 18 DNA Artificial Sequence Synthetic 15
tccacaaaca ggtatgcg 18 16 18 DNA Artificial Sequence Synthetic 16
gaggactcca caaacagg 18 17 18 DNA Artificial Sequence Synthetic 17
gtccagcgag gactccac 18 18 18 DNA Artificial Sequence Synthetic 18
ccaccttgtc cagcgagg 18 19 18 DNA Artificial Sequence Synthetic 19
gcatccgaca ggaccacc 18 20 18 DNA Artificial Sequence Synthetic 20
gcgtaggcat ccgacagg 18 21 18 DNA Artificial Sequence Synthetic 21
gtgcgcgtag gcatccga 18 22 18 DNA Artificial Sequence Synthetic 22
cccgccgctc tctgccaa 18 23 18 DNA Artificial Sequence Synthetic 23
ggatcttcag catctgca 18 24 18 DNA Artificial Sequence Synthetic 24
tgcggtggat cttcagca 18 25 18 DNA Artificial Sequence Synthetic 25
tgcgggtcgc tgcggtgg 18 26 18 DNA Artificial Sequence Synthetic 26
gcccgcagaa tcgcagct 18 27 18 DNA Artificial Sequence Synthetic 27
ggaggaagcg gccacagg 18 28 18 DNA Artificial Sequence Synthetic 28
agtgctgggc gagcgcgg 18 29 18 DNA Artificial Sequence Synthetic 29
agcggcaccg agtgctgg 18 30 18 DNA Artificial Sequence Synthetic 30
agttgcagcg gcaccgag 18 31 18 DNA Artificial Sequence Synthetic 31
agctccagtt gcagcggc 18 32 18 DNA Artificial Sequence Synthetic 32
gccagcaaag cgtccagc 18 33 18 DNA Artificial Sequence Synthetic 33
gatgcaactc aaacagcg 18 34 18 DNA Artificial Sequence Synthetic 34
tgggctagga tgcaactc 18 35 18 DNA Artificial Sequence Synthetic 35
gctgctgggc taggatgc 18 36 18 DNA Artificial Sequence Synthetic 36
cggtcgggct gctgggct 18 37 18 DNA Artificial Sequence Synthetic 37
tcagccagtt cttcatcc 18 38 18 DNA Artificial Sequence Synthetic 38
cgcagcgcat cctccagc 18 39 18 DNA Artificial Sequence Synthetic 39
agatttcgca gcgcatcc 18 40 18 DNA Artificial Sequence Synthetic 40
gccgcacttc agatttcg 18 41 18 DNA Artificial Sequence Synthetic 41
ccccgagccg cacttcag 18 42 18 DNA Artificial Sequence Synthetic 42
ccgaagcgac ctccccgt 18 43 18 DNA Artificial Sequence Synthetic 43
ccgacagaga gggcaccg 18 44 18 DNA Artificial Sequence Synthetic 44
gcggcggcgg cggcttca 18 45 18 DNA Artificial Sequence Synthetic 45
ggtggcggcg gcggcggc 18 46 18 DNA Artificial Sequence Synthetic 46
gggcaggtgg cggcggcg 18 47 18 DNA Artificial Sequence Syntheetic 47
aaagtctggg caggtggc 18 48 18 DNA Artificial Sequence Synthetic 48
ggaacagaaa agtctggg 18 49 18 DNA Artificial Sequence Synthetic 49
gaccctggaa cagaaaag 18 50 18 DNA Artificial Sequence Synthetic 50
tcactacagg ctgaccct 18 51 18 DNA Artificial Sequence Synthetic 51
cgattcacta caggctga 18 52 18 DNA Artificial Sequence Synthetic 52
gtccttcagg ctcagcgg 18 53 18 DNA Artificial Sequence Synthetic 53
tctgttggtc cttcaggc 18 54 18 DNA Artificial Sequence Synthetic 54
catttgagac ccacagag 18 55 18 DNA Artificial Sequence Synthetic 55
cttgcgccat ttgagacc 18 56 18 DNA Artificial Sequence Synthetic 56
agtgagcgcc ccaccttg 18 57 18 DNA Artificial Sequence Synthetic 57
cagcgcccgg cagcctcg 18 58 18 DNA Artificial Sequence Synthetic 58
gagtccagcg ccgggtcc 18 59 18 DNA Artificial Sequence Synthetic 59
cgtaggccag cgagtcca 18 60 18 DNA Artificial Sequence Synthetic 60
gctcgtactc gtaggcca 18 61 18 DNA Artificial Sequence Synthetic 61
ccctcgcgct cgtactcg 18 62 18 DNA Artificial Sequence Synthetic 62
tgctcgtaca gtccctcg 18 63 18 DNA Artificial Sequence Synthetic 63
gcacgaagcg ccgcagca 18 64 18 DNA Artificial Sequence Synthetic 64
tcctctgcca ggctggtg 18 65 18 DNA Artificial Sequence Synthetic 65
cccagcaagt cctctgcc 18 66 18 DNA Artificial Sequence Synthetic 66
caggccgcca ttgggatc 18 67 18 DNA Artificial Sequence Synthetic 67
tggctgcacc cctggtct 18 68 18 DNA Artificial Sequence Synthetic 68
tccaggttct ccaaaagc 18 69 18 DNA Artificial Sequence Synthetic 69
accctaaggc catccagg 18 70 18 DNA Artificial Sequence Synthetic 70
aatagccgca gaaggaac 18 71 18 DNA Artificial Sequence Synthetic 71
tcagggtccc gtggatgg 18 72 18 DNA Artificial Sequence Synthetic 72
taggccaagt ggagtttc 18 73 18 DNA Artificial Sequence Synthetic 73
gcaggtccag cagatagg 18 74 18 DNA Artificial Sequence Synthetic 74
gggaaggcaa tcaactct 18 75 18 DNA Artificial Sequence Synthetic 75
gaggcagaat ccccaatg 18 76 18 DNA Artificial Sequence Synthetic 76
tctatcaaag tacctgag 18 77 18 DNA Artificial Sequence synthetic 77
cccaccccac actctatc 18 78 18 DNA Artificial Sequence Synthetic 78
aaggtgaggc tgatctcc 18 79 18 DNA Artificial Sequence Synthetic 79
ggatgggaga aggtgagg 18 80 18 DNA Artificial Sequence Synthetic 80
aaactgtaag ggctggct 18 81 18 DNA Artificial Sequence Synthetic 81
aaagatcaag gtgcttca 18 82 18 DNA Artificial Sequence Synthetic 82
atgaagtcca ggacacca 18 83 18 DNA Artificial Sequence Synthetic 83
ctgttttact tcactgca 18 84 18 DNA Artificial Sequence Synthetic 84
caagattgat tcctgttt 18 85 18 DNA Artificial Sequence Synthetic 85
ccacgctgag tgtgagct 18 86 18 DNA Artificial Sequence Synthetic 86
actttattat cattgctt 18 87 18 DNA Artificial Sequence Synthetic 87
ccgtgttata ctttatta 18
* * * * *