U.S. patent application number 15/803484 was filed with the patent office on 2018-09-27 for modulation of transthyretin expression.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Vickie L. Brown-Driver, Ravi Jain.
Application Number | 20180273949 15/803484 |
Document ID | / |
Family ID | 35187558 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180273949 |
Kind Code |
A1 |
Brown-Driver; Vickie L. ; et
al. |
September 27, 2018 |
MODULATION OF TRANSTHYRETIN EXPRESSION
Abstract
Compounds, compositions and methods are provided for modulating
the expression of transthyretin. The compositions comprise
oligonucleotides, targeted to nucleic acid encoding transthyretin.
Methods of using these compounds for modulation of transthyretin
expression and for diagnosis and treatment of diseases and
conditions associated with expression of transthyretin are
provided.
Inventors: |
Brown-Driver; Vickie L.;
(Solana Beach, CA) ; Jain; Ravi; (Fremont,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
35187558 |
Appl. No.: |
15/803484 |
Filed: |
November 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15092476 |
Apr 6, 2016 |
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15803484 |
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14614647 |
Feb 5, 2015 |
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15092476 |
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11097928 |
Apr 1, 2005 |
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14614647 |
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60559863 |
Apr 5, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/315 20130101;
C12N 2310/11 20130101; C12Q 2600/136 20130101; C12N 2310/346
20130101; Y10T 436/143333 20150115; C12N 2310/341 20130101; C12N
2310/321 20130101; C12N 15/1136 20130101; C12N 2310/3341 20130101;
C12Q 2600/158 20130101; C12N 15/113 20130101; C12N 2310/3525
20130101; C12Q 1/6883 20130101; C12N 2310/321 20130101; C12N
2310/3525 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12Q 1/6883 20060101 C12Q001/6883 |
Claims
1-18. (canceled)
19. An antisense compound comprising a modified oligonucleotide 20
to 25 nucleobases in length, wherein the modified oligonucleotide
has at least a 13-nucleobase portion of SEQ ID NO: 75 and is at
least 95% complementary to SEQ ID NO: 4, and wherein the modified
oligonucleotide comprises at least one modified sugar moiety.
20. The antisense compound of claim 19, wherein each "T" of SEQ ID
NO: 75 is a uracil.
21. The antisense compound of claim 19, wherein at least one
modified sugar moiety comprises a 2'-O--CH.sub.3 modification.
22. The antisense compound of claim 20, wherein at least one
modified sugar moiety comprises a 2'-O--CH.sub.3 modification.
23. The antisense compound of claim 19, wherein: the modified
oligonucleotide is 21 nucleobases in length; the modified
oligonucleotide has at least a 13-nucleobase portion of SEQ ID NO:
75 and is at least 95% complementary to SEQ ID NO: 4, wherein each
"T" of SEQ ID NO: 75 is a uracil; and the modified oligonucleotide
comprises at least one 2'-O--CH.sub.3 modified sugar moiety.
24. The antisense compound of claim 19, wherein the antisense
compound is single-stranded.
25. The antisense compound of claim 20, wherein the antisense
compound is single-stranded.
26. The antisense compound of claim 21, wherein the antisense
compound is single-stranded.
27. The antisense compound of claim 22, wherein the antisense
compound is single-stranded.
28. The antisense compound of claim 23, wherein the antisense
compound is single-stranded.
29. A compound comprising a nanoparticle and the antisense compound
of claim 19.
30. A compound comprising a nanoparticle and the antisense compound
of claim 23.
31. A compound comprising a nanoparticle and the antisense compound
of claim 28.
32. A compound comprising a liposome and the antisense compound of
claim 19.
33. A compound comprising a liposome and the antisense compound of
claim 23.
34. A compound comprising a liposome and the antisense compound of
claim 28.
35. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
antisense compound of claim 19, thereby inhibiting expression of
human transthyretin in the cell or tissue.
36. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
antisense compound of claim 23, thereby inhibiting expression of
human transthyretin in the cell or tissue.
37. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
antisense compound of claim 28, thereby inhibiting expression of
human transthyretin in the cell or tissue.
38. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
compound of claim 29, thereby inhibiting expression of human
transthyretin in the cell or tissue.
39. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
compound of claim 30, thereby inhibiting expression of human
transthyretin in the cell or tissue.
40. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
compound of claim 31, thereby inhibiting expression of human
transthyretin in the cell or tissue.
41. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
compound of claim 32, thereby inhibiting expression of human
transthyretin in the cell or tissue.
42. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
compound of claim 33, thereby inhibiting expression of human
transthyretin in the cell or tissue.
43. A method of inhibiting the expression of human transthyretin in
a cell or tissue comprising contacting said cell or tissue with the
compound of claim 34, thereby inhibiting expression of human
transthyretin in the cell or tissue.
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled RTS0531USC7SEQ_ST25.txt, created Nov. 3, 2017, which
is 38 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention provides compositions and methods for
modulating the expression of transthyretin. In particular, this
invention relates to antisense compounds, particularly
oligonucleotide compounds, which, in preferred embodiments,
hybridize with nucleic acid molecules encoding transthyretin. Such
compounds are shown herein to modulate the expression of
transthyretin.
BACKGROUND OF THE INVENTION
[0003] Steroid hormones, thyroid hormones, retinoids, and vitamin D
are small hydrophobic molecules that serve as important signaling
molecules throughout the body. Although all of these molecules are
insoluble in water, they are made soluble for transport in the
bloodstream and other extracellular fluids by binding to specific
carrier proteins, from which they dissociate before entering a
target cell. One such carrier protein is transthyretin.
[0004] Transthyretin (also known as TTR; TTR, prealbumin;
prealbumin, thyroxine; PALB; TBPA; HST2651; amyloidosis 1,
included; dysprealbuminemic euthyroidal hyperthyroxinemia,
included; hyperthytoxinemia, dysprealbuminemic, included;
hyperthytoxinemia, dystransthyretinemic, included; amyloid
polyneuropathy, multiple forms, included; senile systemic
amyloidosis, included) is a homotetrameric transport protein found
in the extracellular fluids of vertebrates (Palha, Clin Chem Lab
Med, 2002, 40, 1292-1300).
[0005] Transthyretin was first identified as the major thyroid
hormone carrier in the cerebrospinal fluid (CSF) and in the serum
(Palha, Clin Chem Lab Med, 2002, 40, 1292-1300; Seibert, J. Biol.
Chem, 1942, 143, 29-38). Transthyretin was cloned from adult human
cDNA libraries and the gene was subsequently mapped to chromosome
region 18q11.2-q12.1 (Mita et al., Biochem Biophys Res Commun,
1984, 124, 558-564; Sparkes et al., Hum Genet, 1987, 75, 151-154;
Whitehead et al., Mol Biol Med, 1984, 2, 411-423).
[0006] The liver and the choroid plexus are the primary sites of
transthyretin synthesis in humans (Palha, Clin Chem Lab Med, 2002,
40, 1292-1300).
[0007] Transthyretin that is synthesized in the liver is secreted
into the blood, whereas transthyretin originating in the choroid
plexus is destined for the CSF. In the choroid plexus,
transthyretin synthesis represents about 20% of total local protein
synthesis and as much as 25% of the total CSF protein (Dickson et
al., J Biol Chem, 1986, 261, 3475-3478). Transthyretin synthesis
has also been identified in the yolk sac of developing rats(Soprano
et al., Proc Natl Acad Sci USA, 1986, 83, 7330-7334); the retina,
ciliar body and optic nerve regions of bovine and rat eyes (Martone
et al., Biochem Biophys Res Commun, 1988, 151, 905-912; Ong et al.,
Biochemistry, 1994, 33, 1835-1842); human and porcine pancreatic
islets (Jacobsson et al., J Histochem Cytochem, 1989, 37, 31-37)
and, in minor amounts, in the stomach, heart, skeletal muscle, and
spleen of rats (Soprano et al., J Biol Chem, 1985, 260,
11793-11798).
[0008] It is currently believed that transthyretin serves as a
hormone reservoir. As demand for thyroid hormone increases,
transthyretin increases the transport and release of hormone to
targets such as brain, kidney, and cardiac tissues, thereby
ensuring a uniform hormone distribution within the cells in each of
these tissues (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300).
Transthyretin transports the thyroid hormones triiodothyronine
(T.sub.3) and thyroxine (T.sub.4) as well as the
retinol/retinol-binding protein complex. A mouse strain deficient
in transthyretin is viable and fertile, yet exhibits significantly
depressed levels of serum retinol, retinol-binding protein, and
thyroid hormone, confirming transthyretin's role in maintaining
normal levels of these metabolites in circulating plasma (Episkopou
et al., Proc Natl Acad Sci USA, 1993, 90, 2375-2379). In addition
to serving as a transport protein, transthyretin has been reported
to have a variety of other functions, including: inhibiting
interleukin-1 production in monocytes and endothelial cells (Borish
et al., Inflammation, 1992, 16, 471-484); involvement in the
metabolism of the environmental pollutant polyhalogenated biphenyl
(Brouwer and van den Berg, Toxicol Appl Pharmacol, 1986, 85,
301-312); and binding pterins (Ernstrom et al., FEBS Lett, 1995,
360, 177-182). Furthermore, in recent years a link between
transthyretin and lipoprotein biology has become increasingly
apparent. A fraction of plasma transthyretin circulates in high
density lipoproteins (HDL) through binding to apolipoprotein A-1
(Sousa et al., J Biol Chem, 2000, 275, 38176-38181), and
transthyretin has been shown to proteolytically process
apolipoprotein A-1 (Liz et al., J Biol Chem, 2004). Furthermore,
transthyretin reabsorption by the kidneys is mediated by the
lipoprotein receptor megalin (Sousa et al., J Biol Chem, 2000, 275,
38176-38181). This reabsorption serves as a means for preventing
hormone loss in urine. Finally, the major site of degradation for
both transthyretin and lipoproteins is the liver. There is
considerable evidence that hepatic uptake of both transthyretin and
lipoproteins is mediated by an as yet unidentified lipoprotein
receptor, suggesting a shared degradation pathway (Sousa and
Saraiva, J Biol Chem, 2001, 276, 14420-14425).
[0009] Transthyretin is associated with both local and systemic
amyloidosis, a disorder characterized by extracellular systemic
deposition of mutated or wild-type transthyretin as amyloid fibrils
(Cornwell et al., Biochem Biophys Res Commun, 1988, 154, 648-653;
Saraiva et al., J Clin Invest, 1984, 74, 104-119; Yazaki et al.,
Muscle Nerve, 2003, 28, 438-442), leading to organ dysfunction and
death. Senile systemic amyloidosis is a sporadic disorder resulting
from the extracellular deposition of wild-type transthyretin
fibrils in cardiac and other tissues. Over 80 mutations in
transthyretin are associated with familial amyloidotic
polyneuropathy and cardiomyopathy. In most of these cases,
inheritance is autosomal dominant (Reixach et al., Proc Natl Acad
Sci USA, 2004, 101, 2817-2822). Jiang et al (Jiang et al., Proc
Natl Acad Sci USA, 2001, 98, 14943-14948) demonstrated that the
variant with a valine to isoleucine mutation at amino acid 122
(Val122Ile), which is among the most common amyloidogenic mutations
worldwide, increases the velocity of rate-limiting tetramer
dissociation, thereby resulting in accelerated amyloidogenesis.
This finding suggests the possibility that treatments for
transthyretin-related amyloidoses may include small molecules that
stabilize the tetrameric form (Adamski-Werner et al., J Med Chem,
2004, 47, 355-374; Altland and Winter, Neurogenetics, 1999, 2,
183-188). Small molecule stabilizers were also shown to be of use
in preventing the formation of amyloid fibrils of the wildtype
transthyretin (Reixach et al., Proc Natl Acad Sci USA, 2004, 101,
2817-2822). Other common transthyretin mutations associated with
amyloidosis include Val30Met and Glu61Lys. In vitro studies have
shown success using ribozymes to specifically target and inhibit
the expression of the Glu61Lys and Val30Met variants (Propsting et
al., Biochem Biophys Res Commun, 1999, 260, 313-317; Tanaka et al.,
J Neurol Sci, 2001, 183, 79-84). Single-stranded oligonucleotides
have been used both in vitro and in vivo to correct single-base
mutation (Val30Met) in transthyretin to the wild-type form through
targeted recombination (Nakamura et al., Gene Ther, 2004). The
success of this therapy was limited, however, with gene conversion
rates of 11% in vitro and 9% in vivo. These levels are not
sufficient for suppression of the variant transthyretin in clinical
terms (Nakamura et al., Gene Ther, 2004).
[0010] Thus liver transplantation is currently the only available
therapy for familial amyloidotic polyneuropathy. However, this
therapy is associated with several problems, and does not address
conditions which are caused by transthyretin variant production in
tissues other than liver (Yazaki et al., Muscle Nerve, 2003, 28,
438-442). Consequently, there remains an unmet need for agents
capable of effectively modulating transthyretin expression
(Nakamura et al., Gene Ther, 2004; Tanaka et al., J Neurol Sci,
2001, 183, 79-84).
[0011] The PCT publication WO200259621 and the US pre-grant
publication 20020160394 claim pharmaceutical compositions for
treating obesity, comprising an antisense oligonucleotide that
hybridizes to a polynucleotide encoding transthyretin and reduces
expression of the polynucleotide. Also claimed is the use of said
oligonucleotide in the manufacture of a medicament for treating
obesity (Wu, 2002).
[0012] The U.S. Pat. No. 5,744,368 discloses a primer of 22
nucleotides in length targeted to Exon 4 of transthyretin
(Goldgaber et al., 1998).
[0013] Antisense technology is an effective means of reducing the
expression of specific gene products and therefore is uniquely
useful in a number of therapeutic, diagnostic, and research
applications for the modulation of transthyretin expression. The
present invention provides compositions and methods for modulating
transthyretin expression.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to antisense compounds,
especially nucleic acid and nucleic acid-like oligomers, which are
targeted to a nucleic acid encoding transthyretin, and which
modulate the expression of transthyretin. Pharmaceutical and other
compositions comprising the compounds of the invention are also
provided. Further provided are methods of screening for modulators
of transthyretin and methods of modulating the expression of
transthyretin in cells, tissues or animals comprising contacting
said cells, tissues or animals with one or more of the compounds or
compositions of the invention. Methods of treating an animal,
particularly a human, suspected of having or being prone to a
disease or condition associated with expression of transthyretin
are also set forth herein. Such methods comprise administering a
therapeutically or prophylactically effective amount of one or more
of the compounds or compositions of the invention to the person in
need of treatment.
DETAILED DESCRIPTION OF THE INVENTION
A. Overview of the Invention
[0015] The present invention employs antisense compounds,
preferably oligonucleotides and similar species for use in
modulating the function or effect of nucleic acid molecules
encoding transthyretin. This is accomplished by providing
oligonucleotides which specifically hybridize with one or more
nucleic acid molecules encoding transthyretin. As used herein, the
terms "target nucleic acid" and "nucleic acid molecule encoding
transthyretin" have been used for convenience to encompass DNA
encoding transthyretin, RNA (including pre-mRNA and mRNA or
portions thereof) transcribed from such DNA, and also cDNA derived
from such RNA. The hybridization of a compound of this invention
with its target nucleic acid is generally referred to as
"antisense". Consequently, the preferred mechanism believed to be
included in the practice of some preferred embodiments of the
invention is referred to herein as "antisense inhibition." Such
antisense inhibition is typically based upon hydrogen bonding-based
hybridization of oligonucleotide strands or segments such that at
least one strand or segment is cleaved, degraded, or otherwise
rendered inoperable. In this regard, it is presently preferred to
target specific nucleic acid molecules and their functions for such
antisense inhibition.
[0016] The functions of DNA to be interfered with can include
replication and transcription. Replication and transcription, for
example, can be from an endogenous cellular template, a vector, a
plasmid construct or otherwise. The functions of RNA to be
interfered with can include functions such as translocation of the
RNA to a site of protein translation, translocation of the RNA to
sites within the cell which are distant from the site of RNA
synthesis, translation of protein from the RNA, splicing of the RNA
to yield one or more RNA species, and catalytic activity or complex
formation involving the RNA which may be engaged in or facilitated
by the RNA. One preferred result of such interference with target
nucleic acid function is modulation of the expression of
transthyretin. In the context of the present invention,
"modulation" and "modulation of expression" mean either an increase
(stimulation) or a decrease (inhibition) in the amount or levels of
a nucleic acid molecule encoding the gene, e.g., DNA or RNA.
Inhibition is often the preferred form of modulation of expression
and mRNA is often a preferred target nucleic acid.
[0017] In the context of this invention, "hybridization" means the
pairing of complementary strands of oligomeric compounds. In the
present invention, the preferred mechanism of pairing involves
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases (nucleobases) of the strands of oligomeric
compounds. For example, adenine and thymine are complementary
nucleobases which pair through the formation of hydrogen bonds.
Hybridization can occur under varying circumstances.
[0018] An antisense compound is specifically hybridizable when
binding of the compound to the target nucleic acid interferes with
the normal function of the target nucleic acid to cause a loss of
activity, and there is a sufficient degree of complementarity to
avoid non-specific binding of the antisense compound to non-target
nucleic acid sequences under conditions in which specific binding
is desired, i.e., under physiological conditions in the case of in
vivo assays or therapeutic treatment, and under conditions in which
assays are performed in the case of in vitro assays.
[0019] In the present invention the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which a compound of the invention will hybridize to its target
sequence, but to a minimal number of other sequences. Stringent
conditions are sequence-dependent and will be different in
different circumstances and in the context of this invention,
"stringent conditions" under which oligomeric compounds hybridize
to a target sequence are determined by the nature and composition
of the oligomeric compounds and the assays in which they are being
investigated.
[0020] "Complementary," as used herein, refers to the capacity for
precise pairing between two nucleobases of an oligomeric compound.
For example, if a nucleobase at a certain position of an
oligonucleotide (an oligomeric compound), is capable of hydrogen
bonding with a nucleobase at a certain position of a target nucleic
acid, said target nucleic acid being a DNA, RNA, or oligonucleotide
molecule, then the position of hydrogen bonding between the
oligonucleotide and the target nucleic acid is considered to be a
complementary position. The oligonucleotide and the further DNA,
RNA, or oligonucleotide molecule are complementary to each other
when a sufficient number of complementary positions in each
molecule are occupied by nucleobases which can hydrogen bond with
each other. Thus, "specifically hybridizable" and "complementary"
are terms which are used to indicate a sufficient degree of precise
pairing or complementarity over a sufficient number of nucleobases
such that stable and specific binding occurs between the
oligonucleotide and a target nucleic acid.
[0021] 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. Moreover, an
oligonucleotide may hybridize over one or more segments such that
intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure or hairpin structure).
It is preferred that the antisense compounds of the present
invention comprise at least 70%, or at least 75%, or at least 80%,
or at least 85% sequence complementarity to a target region within
the target nucleic acid, more preferably that they comprise at
least 90% sequence complementarity and even more preferably
comprise at least 95% or at least 99% sequence complementarity to
the target region within the target nucleic acid sequence to which
they are targeted. For example, an antisense compound in which 18
of 20 nucleobases of the antisense compound are complementary to a
target region, and would therefore specifically hybridize, would
represent 90 percent complementarity. In this example, the
remaining noncomplementary nucleobases may be clustered or
interspersed with complementary nucleobases and need not be
contiguous to each other or to complementary nucleobases. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs
(basic local alignment search tools) and PowerBLAST programs known
in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410;
Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0022] Percent homology, sequence identity or complementarity, can
be determined by, for example, the Gap program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, Madison Wis.), using default settings,
which uses the algorithm of Smith and Waterman (Adv. Appl. Math.,
1981, 2, 482-489). In some preferred embodiments, homology,
sequence identity or complementarity, is between about 80% and
about 90%. In some preferred embodiments, homology, sequence
identity or complementarity, is about 90%, about 92%, about 94%,
about 95%, about 96%, about 97%, about 98%, about 99% or about
100%.
B. Compounds of the Invention
[0023] According to the present invention, antisense compounds
include antisense oligomeric compounds, antisense oligonucleotides,
ribozymes, external guide sequence (EGS) oligonucleotides,
alternate splicers, primers, probes, and other oligomeric compounds
which hybridize to at least a portion of the target nucleic acid.
As such, these compounds may be introduced in the form of
single-stranded, double-stranded, circular or hairpin oligomeric
compounds and may contain structural elements such as internal or
terminal bulges or loops. Once introduced to a system, the
compounds of the invention may elicit the action of one or more
enzymes or structural proteins to effect modification of the target
nucleic acid.
[0024] One non-limiting example of such an enzyme is RNAse H, a
cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. It is known in the art that single-stranded antisense
compounds which are "DNA-like" elicit RNAse H. Activation of RNase
H, therefore, results in cleavage of the RNA target, thereby
greatly enhancing the efficiency of oligonucleotide-mediated
inhibition of gene expression. Similar roles have been postulated
for other ribonucleases such as those in the RNase III and
ribonuclease L family of enzymes.
[0025] While the preferred form of antisense compound is a
single-stranded antisense oligonucleotide, in many species the
introduction of double-stranded structures, such as double-stranded
RNA (dsRNA) molecules, has been shown to induce potent and specific
antisense-mediated reduction of the function of a gene or its
associated gene products. This phenomenon occurs in both plants and
animals and is believed to have an evolutionary connection to viral
defense and transposon silencing.
[0026] The first evidence that dsRNA could lead to gene silencing
in animals came in 1995 from work in the nematode, Caenorhabditis
elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et
al. have shown that the primary interference effects of dsRNA are
posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,
1998, 95, 15502-15507). The posttranscriptional antisense mechanism
defined in Caenorhabditis elegans resulting from exposure to
double-stranded RNA (dsRNA) has since been designated RNA
interference (RNAi). This term has been generalized to mean
antisense-mediated gene silencing involving the introduction of
dsRNA leading to the sequence-specific reduction of endogenous
targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811).
Recently, it has been shown that it is, in fact, the
single-stranded RNA oligomers of antisense polarity of the dsRNAs
which are the potent inducers of RNAi (Tijsterman et al., Science,
2002, 295, 694-697).
[0027] The antisense compounds of the present invention also
include modified compounds in which a different base is present at
one or more of the nucleotide positions in the compound. For
example, if the first nucleotide is an adenosine, modified
compounds may be produced which contain thymidine, guanosine or
cytidine at this position. This may be done at any of the positions
of the antisense compound. These compounds are then tested using
the methods described herein to determine their ability to inhibit
expression of transthyretin mRNA.
[0028] In the context of this invention, the term "oligomeric
compound" refers to a polymer or oligomer comprising a plurality of
monomeric units. 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, chimeras,
analogs and homologs 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 a target
nucleic acid and increased stability in the presence of
nucleases.
[0029] While oligonucleotides are a preferred form of the antisense
compounds of this invention, the present invention comprehends
other families of antisense compounds as well, including but not
limited to oligonucleotide analogs and mimetics such as those
described herein.
[0030] The antisense compounds in accordance with this invention
preferably comprise from about 8 to about 80 nucleobases (i.e. from
about 8 to about 80 linked nucleosides). One of ordinary skill in
the art will appreciate that the invention embodies compounds of 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, or 80 nucleobases in length.
[0031] In one preferred embodiment, the antisense compounds of the
invention are 13 to 50 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies compounds of 13
to 50 nucleobases in length, inclusive as detailed above.
[0032] In another preferred embodiment, the antisense compounds of
the invention are 15 to 30 nucleobases in length. One having
ordinary skill in the art will appreciate that this embodies
compounds of 15 to 30 nucleobases in length, inclusive as detailed
above.
[0033] Particularly preferred compounds are oligonucleotides from
about 13 to about 50 nucleobases, even more preferably those
comprising from about 15 to about 30 nucleobases.
[0034] Antisense compounds 8-80 nucleobases in length comprising a
stretch of at least eight (8) consecutive nucleobases selected from
within the illustrative antisense compounds are considered to be
suitable antisense compounds as well.
[0035] Exemplary preferred antisense compounds include
oligonucleotide sequences that comprise at least the 8 consecutive
nucleobases from the 5'-terminus of one of the illustrative
preferred antisense compounds (the remaining nucleobases being a
consecutive stretch of the same oligonucleotide beginning
immediately upstream of the 5'-terminus of the antisense compound
which is specifically hybridizable to the target nucleic acid and
continuing until the oligonucleotide contains about 8 to about 80
nucleobases). Similarly preferred antisense compounds are
represented by oligonucleotide sequences that comprise at least the
8 consecutive nucleobases from the 3'-terminus of one of the
illustrative preferred antisense compounds (the remaining
nucleobases being a consecutive stretch of the same oligonucleotide
beginning immediately downstream of the 3'-terminus of the
antisense compound which is specifically hybridizable to the target
nucleic acid and continuing until the oligonucleotide contains
about 8 to about 80 nucleobases). It is also understood that
preferred antisense compounds may be represented by oligonucleotide
sequences that comprise at least 8 consecutive nucleobases from an
internal portion of the sequence of an illustrative preferred
antisense compound, and may extend in either or both directions
until the oligonucleotide contains about 8 to about 80
nucleobases.
[0036] One having skill in the art armed with the preferred
antisense compounds illustrated herein will be able, without undue
experimentation, to identify further preferred antisense
compounds.
C. Targets of the Invention
[0037] "Targeting" an antisense compound to a particular nucleic
acid molecule, in the context of this invention, can be a multistep
process. The process usually begins with the identification of a
target nucleic acid whose function is to be modulated. This target
nucleic acid 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
nucleic acid encodes transthyretin.
[0038] The targeting process usually also includes determination of
at least one target region, segment, or site within the target
nucleic acid for the antisense interaction to occur such that the
desired effect, e.g., modulation of expression, will result. Within
the context of the present invention, the term "region" is defined
as a portion of the target nucleic acid having at least one
identifiable structure, function, or characteristic. Within regions
of target nucleic acids are segments. "Segments" are defined as
smaller or sub-portions of regions within a target nucleic acid.
"Sites," as used in the present invention, are defined as positions
within a target nucleic acid.
[0039] Since, as is known in the art, the translation initiation
codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in
the corresponding DNA molecule), the translation initiation codon
is also referred to as the "AUG codon," the "start codon" or the
"AUG start codon". A minority of genes have a translation
initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG,
and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo.
Thus, the terms "translation initiation codon" and "start codon"
can encompass many codon sequences, even though the initiator amino
acid in each instance is typically methionine (in eukaryotes) or
formylmethionine (in prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA transcribed from a gene encoding
transthyretin, regardless of the sequence(s) of such codons. It is
also known in the art that a translation termination codon (or
"stop codon") of a gene may have one of three sequences, i.e.,
5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are
5'-TAA, 5'-TAG and 5'-TGA, respectively).
[0040] 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. Consequently, the "start codon region" (or
"translation initiation codon region") and the "stop codon region"
(or "translation termination codon region") are all regions which
may be targeted effectively with the antisense compounds of the
present invention.
[0041] 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. Within the context of the
present invention, a preferred region is the intragenic region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of a gene.
[0042] 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 site 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
site. It is also preferred to target the 5' cap region.
[0043] 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.
Targeting splice sites, i.e., intron-exon junctions or exon-intron
junctions, may also be particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also preferred target sites. mRNA transcripts
produced via the process of splicing of two (or more) mRNAs from
different gene sources are known as "fusion transcripts". It is
also known that introns can be effectively targeted using antisense
compounds targeted to, for example, DNA or pre-mRNA.
[0044] It is also known in the art that alternative RNA transcripts
can be produced from the same genomic region of DNA. These
alternative transcripts are generally known as "variants". More
specifically, "pre-mRNA variants" are transcripts produced from the
same genomic DNA that differ from other transcripts produced from
the same genomic DNA in either their start or stop position and
contain both intronic and exonic sequence.
[0045] Upon excision of one or more exon or intron regions, or
portions thereof during splicing, pre-mRNA variants produce smaller
"mRNA variants". Consequently, mRNA variants are processed pre-mRNA
variants and each unique pre-mRNA variant must always produce a
unique mRNA variant as a result of splicing. These mRNA variants
are also known as "alternative splice variants". If no splicing of
the pre-mRNA variant occurs then the pre-mRNA variant is identical
to the mRNA variant.
[0046] It is also known in the art that variants can be produced
through the use of alternative signals to start or stop
transcription and that pre-mRNAs and mRNAs can possess more that
one start codon or stop codon. Variants that originate from a
pre-mRNA or mRNA that use alternative start codons are known as
"alternative start variants" of that pre-mRNA or mRNA. Those
transcripts that use an alternative stop codon are known as
"alternative stop variants" of that pre-mRNA or mRNA. One specific
type of alternative stop variant is the "polyA variant" in which
the multiple transcripts produced result from the alternative
selection of one of the "polyA stop signals" by the transcription
machinery, thereby producing transcripts that terminate at unique
polyA sites. Within the context of the invention, the types of
variants described herein are also preferred target nucleic
acids.
[0047] The locations on the target nucleic acid to which the
preferred antisense compounds hybridize are hereinbelow referred to
as "preferred target segments." As used herein the term "preferred
target segment" is defined as at least an 8-nucleobase portion of a
target region to which an active antisense compound is targeted.
While not wishing to be bound by theory, it is presently believed
that these target segments represent portions of the target nucleic
acid which are accessible for hybridization.
[0048] While the specific sequences of certain preferred target
segments are set forth herein, one of skill in the art will
recognize that these serve to illustrate and describe particular
embodiments within the scope of the present invention. Additional
preferred target segments may be identified by one having ordinary
skill.
[0049] Target segments 8-80 nucleobases in length comprising a
stretch of at least eight (8) consecutive nucleobases selected from
within the illustrative preferred target segments are considered to
be suitable for targeting as well.
[0050] Target segments can include DNA or RNA sequences that
comprise at least the 8 consecutive nucleobases from the
5'-terminus of one of the illustrative preferred target segments
(the remaining nucleobases being a consecutive stretch of the same
DNA or RNA beginning immediately upstream of the 5'-terminus of the
target segment and continuing until the DNA or RNA contains about 8
to about 80 nucleobases). Similarly preferred target segments are
represented by DNA or RNA sequences that comprise at least the 8
consecutive nucleobases from the 3'-terminus of one of the
illustrative preferred target segments (the remaining nucleobases
being a consecutive stretch of the same DNA or RNA beginning
immediately downstream of the 3'-terminus of the target segment and
continuing until the DNA or RNA contains about 8 to about 80
nucleobases). It is also understood that preferred antisense target
segments may be represented by DNA or RNA sequences that comprise
at least 8 consecutive nucleobases from an internal portion of the
sequence of an illustrative preferred target segment, and may
extend in either or both directions until the oligonucleotide
contains about 8 to about 80 nucleobases. One having skill in the
art armed with the preferred target segments illustrated herein
will be able, without undue experimentation, to identify further
preferred target segments.
[0051] Once one or more target regions, segments or sites have been
identified, antisense compounds are chosen which are sufficiently
complementary to the target, i.e., hybridize sufficiently well and
with sufficient specificity, to give the desired effect.
[0052] The oligomeric antisense compounds may also be targeted to
regions of the target nucleobase sequence (e.g., such as those
disclosed in Example 13) comprising nucleobases 1-80, 81-160,
161-240, 241-320, 321-400, 401-480, 481-560, 561-640, 641-650, or
any combination thereof.
[0053] Oligomeric compounds targeted to nucleobases 3880-3899 of
SEQ ID NO: 11, or to nucleobases 6-25, 59-78, 91-119, 126-152,
170-189, 197-216, 217-236, 232-251, 250-269, 264-297, 323-361,
425-469, 460-532, 532-619 of SEQ ID NO: 4 are also suitable
embodiments.
D. Screening and Target Validation
[0054] In a further embodiment, the "preferred target segments"
identified herein may be employed in a screen for additional
compounds that modulate the expression of transthyretin.
"Modulators" are those compounds that decrease or increase the
expression of a nucleic acid molecule encoding transthyretin and
which comprise at least an 8-nucleobase portion which is
complementary to a preferred target segment. The screening method
comprises the steps of contacting a preferred target segment of a
nucleic acid molecule encoding transthyretin with one or more
candidate modulators, and selecting for one or more candidate
modulators which decrease or increase the expression of a nucleic
acid molecule encoding transthyretin. Once it is shown that the
candidate modulator or modulators are capable of modulating (e.g.
either decreasing or increasing) the expression of a nucleic acid
molecule encoding transthyretin, the modulator may then be employed
in further investigative studies of the function of transthyretin,
or for use as a research, diagnostic, or therapeutic agent in
accordance with the present invention.
[0055] The preferred target segments of the present invention may
be also be combined with their respective complementary antisense
compounds of the present invention to form stabilized
double-stranded (duplexed) oligonucleotides.
[0056] Such double stranded oligonucleotide moieties have been
shown in the art to modulate target expression and regulate
translation as well as RNA processsing via an antisense mechanism.
Moreover, the double-stranded moieties may be subject to chemical
modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and
Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263,
103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et
al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et
al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,
2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200).
For example, such double-stranded moieties have been shown to
inhibit the target by the classical hybridization of antisense
strand of the duplex to the target, thereby triggering enzymatic
degradation of the target (Tijsterman et al., Science, 2002, 295,
694-697).
[0057] The antisense compounds of the present invention can also be
applied in the areas of drug discovery and target validation. The
present invention comprehends the use of the compounds and
preferred target segments identified herein in drug discovery
efforts to elucidate relationships that exist between transthyretin
and a disease state, phenotype, or condition. These methods include
detecting or modulating transthyretin comprising contacting a
sample, tissue, cell, or organism with the compounds of the present
invention, measuring the nucleic acid or protein level of
transthyretin and/or a related phenotypic or chemical endpoint at
some time after treatment, and optionally comparing the measured
value to a non-treated sample or sample treated with a further
compound of the invention. These methods can also be performed in
parallel or in combination with other experiments to determine the
function of unknown genes for the process of target validation or
to determine the validity of a particular gene product as a target
for treatment or prevention of a particular disease, condition, or
phenotype.
E. Kits, Research Reagents, Diagnostics, and Therapeutics
[0058] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. Furthermore, 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 or to distinguish between functions of various
members of a biological pathway.
[0059] For use in kits and diagnostics, the compounds of the
present invention, either alone or in combination with other
compounds or therapeutics, can be used as tools in differential
and/or combinatorial analyses to elucidate expression patterns of a
portion or the entire complement of genes expressed within cells
and tissues.
[0060] As one nonlimiting example, expression patterns within cells
or tissues treated with one or more antisense compounds are
compared to control cells or tissues not treated with antisense
compounds and the patterns produced are analyzed for differential
levels of gene expression as they pertain, for example, to disease
association, signaling pathway, cellular localization, expression
level, size, structure or function of the genes examined. These
analyses can be performed on stimulated or unstimulated cells and
in the presence or absence of other compounds which affect
expression patterns.
[0061] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression)(Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. U S. A., 2000, 97, 1976-81), protein
arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (To, Comb. Chem. High
Throughput Screen, 2000, 3, 235-41).
[0062] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding transthyretin. For example, oligonucleotides
that are shown to hybridize with such efficiency and under such
conditions as disclosed herein as to be effective transthyretin
inhibitors will also be effective primers or probes under
conditions favoring gene amplification or detection, respectively.
These primers and probes are useful in methods requiring the
specific detection of nucleic acid molecules encoding transthyretin
and in the amplification of said nucleic acid molecules for
detection or for use in further studies of transthyretin.
Hybridization of the antisense oligonucleotides, particularly the
primers and probes, of the invention with a nucleic acid encoding
transthyretin 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 transthyretin in a sample may also be prepared.
[0063] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense compounds have been employed as therapeutic moieties in
the treatment of disease states in animals, including humans.
Antisense oligonucleotide drugs, including ribozymes, have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that
antisense compounds can be useful therapeutic modalities that can
be configured to be useful in treatment regimes for the treatment
of cells, tissues and animals, especially humans.
[0064] For therapeutics, an animal, preferably a human, suspected
of having a disease or disorder which can be treated by modulating
the expression of transthyretin is treated by administering
antisense compounds in accordance with this invention. For example,
in one non-limiting embodiment, the methods comprise the step of
administering to the animal in need of treatment, a therapeutically
effective amount of a transthyretin inhibitor. The transthyretin
inhibitors of the present invention effectively inhibit the
activity of the transthyretin protein or inhibit the expression of
the transthyretin protein. In one embodiment, the activity or
expression of transthyretin in an animal is inhibited by about 10%.
Preferably, the activity or expression of transthyretin in an
animal is inhibited by about 30%. More preferably, the activity or
expression of transthyretin in an animal is inhibited by 50% or
more. Thus, the oligomeric antisense compounds modulate expression
of transthyretin mRNA by at least 10%, by at least 20%, by at least
25%, by at least 30%, by at least 40%, by at least 50%, by at least
60%, by at least 70%, by at least 75%, by at least 80%, by at least
85%, by at least 90%, by at least 95%, by at least 98%, by at least
99%, or by 100%.
[0065] For example, the reduction of the expression of
transthyretin may be measured in serum, adipose tissue, liver or
any other body fluid, tissue or organ of the animal. Preferably,
the cells contained within said fluids, tissues or organs being
analyzed contain a nucleic acid molecule encoding transthyretin
protein and/or the transthyretin protein itself.
[0066] The antisense compounds of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of a
compound to a suitable pharmaceutically acceptable diluent or
carrier. Use of the compounds and methods of the invention may also
be useful prophylactically.
F. Modifications
[0067] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base sometimes referred to as a "nucleobase" or simply
a "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 compound can be
further joined to form a circular compound, however, linear
compounds are generally preferred. In addition, linear compounds
may have internal nucleobase complementarity and may therefore fold
in a manner as to produce a fully or partially double-stranded
compound. Within oligonucleotides, 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.
Modified Internucleoside Linkages (Backbones)
[0068] 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.
[0069] Preferred modified oligonucleotide backbones containing a
phosphorus atom therein include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkyl-phosphotriaminoalkylphosphotriesters, methyl and other
alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphos-phonates, thionoalkylphosphotriesters,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Preferred oligonucleotides having inverted
polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0070] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S.: 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899;
5,721,218; 5,672,697 and 5,625,050, certain of which are commonly
owned with this application, and each of which is herein
incorporated by reference.
[0071] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
[0072] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S.: 5,034,506; 5,166,315; 5,185,444; 5,214,134;
5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;
5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;
5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;
5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269
and 5,677,439, certain of which are commonly owned with this
application, and each of which is herein incorporated by
reference.
Modified Sugar and Internucleoside Linkages-Mimetics
[0073] In other preferred antisense compounds, e.g.,
oligonucleotide mimetics, both the sugar and the internucleoside
linkage (i.e. the backbone), of the nucleotide units are replaced
with novel groups. The nucleobase units are maintained for
hybridization with an appropriate target nucleic acid. One such
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.: 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.
[0074] 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.
Modified Sugars
[0075] Modified antisense compounds may also contain one or more
substituted sugar moieties. Preferred are antisense compounds,
preferably antisense oligonucleotides, comprising 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-Co-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.sub.3].sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, poly-alkylamino, substituted silyl, an RNA
cleaving group, a reporter group, an intercalator, a group for
improving the pharmacokinetic properties of an oligonucleotide, or
a group for improving the pharmacodynamic properties of an
oligonucleotide, and other substituents having similar properties.
A preferred modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2, also described in
examples hereinbelow.
[0076] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Antisense compounds may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S.: 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and
5,700,920, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0077] A further preferred modification of the sugar includes
Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is
linked to the 3' or 4' carbon atom of the sugar ring, thereby
forming a bicyclic sugar moiety. The linkage is preferably a
methylene (--CH.sub.2--).sub.n group bridging the 2' oxygen atom
and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation
thereof are described in WO 98/39352 and WO 99/14226.
Natural and Modified Nucleobases
[0078] Antisense compounds may also include nucleobase (often
referred to in the art as heterocyclic base or simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
uracil (U). Modified nucleobases include other synthetic and
natural nucleobases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine
and other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine
and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases
include tricyclic pyrimidines such as phenoxazine
cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the 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. and
are presently preferred base substitutions, even more particularly
when combined with 2'-O-methoxyethyl sugar modifications.
[0079] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653;
5,763,588; 6,005,096; and 5,681,941, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference, and U.S. Pat. No. 5,750,692, which is
commonly owned with the instant application and also herein
incorporated by reference.
Conjugates
[0080] Another modification of the antisense compounds of the
invention involves chemically linking to the antisense compound one
or more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. These
moieties or conjugates can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugate groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve uptake,
enhance resistance to degradation, and/or strengthen
sequence-specific hybridization with the target nucleic acid.
Groups that enhance the pharmacokinetic properties, in the context
of this invention, include groups that improve uptake,
distribution, metabolism or excretion of the compounds of the
present invention. Representative conjugate groups are disclosed in
International Patent Application PCT/US92/09196, filed Oct. 23,
1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which
are incorporated herein by reference. Conjugate moieties include
but are not limited to lipid moieties such as a cholesterol moiety,
cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a
thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl
residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a
polyamine or a polyethylene glycol chain, or adamantane acetic
acid, a palmityl moiety, or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety. Antisense compounds of
the invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) which is incorporated herein by reference in its
entirety.
[0081] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S.: 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.
Chimeric Compounds
[0082] 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.
[0083] 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. Chimeric antisense oligonucleotides are thus a form of
antisense 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, increased stability 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-mediated inhibition of gene
expression. The cleavage of RNA:RNA hybrids can, in like fashion,
be accomplished through the actions of endoribonucleases, such as
RNAseL which cleaves both cellular and viral RNA. Cleavage of the
RNA target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known
in the art.
[0084] 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.: 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference in its entirety.
G. Formulations
[0085] 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.: 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.
[0086] 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.
[0087] 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. For oligonucleotides,
preferred examples of pharmaceutically acceptable salts and their
uses are further described in U.S. Pat. No. 6,287,860, which is
incorporated herein in its entirety. Sodium salts have been shown
to be suitable forms of oligonucleotide drugs.
[0088] 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. 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.
[0089] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0090] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0091] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, foams and
liposome-containing formulations. The pharmaceutical compositions
and formulations of the present invention may comprise one or more
penetration enhancers, carriers, excipients or other active or
inactive ingredients.
[0092] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. Emulsions may contain additional components in
addition to the dispersed phases, and the active drug which may be
present as a solution in either the aqueous phase, oily phase or
itself as a separate phase. Microemulsions are included as an
embodiment of the present invention. Emulsions and their uses are
well known in the art and are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety.
[0093] Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome"
means a vesicle composed of amphiphilic lipids arranged in a
spherical bilayer or bilayers. Liposomes are unilamellar or
multilamellar vesicles which have a membrane formed from a
lipophilic material and an aqueous interior that contains the
composition to be delivered. Cationic liposomes are positively
charged liposomes which are believed to interact with negatively
charged DNA molecules to form a stable complex. Liposomes that are
pH-sensitive or negatively-charged are believed to entrap DNA
rather than complex with it. Both cationic and noncationic
liposomes have been used to deliver DNA to cells.
[0094] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Liposomes and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety.
[0095] The pharmaceutical formulations and compositions of the
present invention may also include surfactants. Surfactants and
their uses are further described in U.S. Pat. No. 6,287,860, which
is incorporated herein in its entirety.
[0096] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides. Penetration enhancers and
their uses are further described in U.S. Pat. No. 6,287,860, which
is incorporated herein in its entirety.
[0097] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0098] Preferred formulations for topical administration include
those in which the oligonucleotides of the invention are in
admixture with a topical delivery agent such as lipids, liposomes,
fatty acids, fatty acid esters, steroids, chelating agents and
surfactants. Preferred lipids and liposomes include neutral (e.g.
dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl
choline DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl
ethanolamine DOTMA).
[0099] For topical or other administration, oligonucleotides of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Preferred fatty acids and esters,
pharmaceutically acceptable salts thereof, and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety. Topical formulations are described in
detail in U.S. patent application Ser. No. 09/315,298 filed on May
20, 1999, which is incorporated herein by reference in its
entirety.
[0100] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Preferred surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Preferred bile
acids/salts and fatty acids and their uses are further described in
U.S. Pat. No. 6,287,860, which is incorporated herein in its
entirety. Also preferred are combinations of penetration enhancers,
for example, fatty acids/salts in combination with bile
acids/salts. A particularly preferred combination is the sodium
salt of lauric acid, capric acid and UDCA. Further penetration
enhancers include polyoxyethylene-9-lauryl ether,
polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention
may be delivered orally, in granular form including sprayed dried
particles, or complexed to form micro or nanoparticles.
Oligonucleotide complexing agents and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein
in its entirety. Oral formulations for oligonucleotides and their
preparation are described in detail in U.S. application Ser. No.
09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20,
1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is
incorporated herein by reference in their entirety.
[0101] 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.
[0102] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include but are not limited to cancer chemotherapeutic drugs such
as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,
mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine,
procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea,
deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil
(5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX),
colchicine, taxol, vincristine, vinblastine, etoposide (VP-16),
trimetrexate, irinotecan, topotecan, gemcitabine, teniposide,
cisplatin and diethylstilbestrol (DES). When used with the
compounds of the invention, such chemotherapeutic agents may be
used individually (e.g., 5-FU and oligonucleotide), sequentially
(e.g., 5-FU and oligonucleotide for a period of time followed by
MTX and oligonucleotide), or in combination with one or more other
such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide,
or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory
drugs, including but not limited to nonsteroidal anti-inflammatory
drugs and corticosteroids, and antiviral drugs, including but not
limited to ribivirin, vidarabine, acyclovir and ganciclovir, may
also be combined in compositions of the invention. Combinations of
antisense compounds and other non-antisense drugs are also within
the scope of this invention. Two or more combined compounds may be
used together or sequentially.
[0103] 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. Alternatively, compositions of the invention may contain
two or more antisense compounds targeted to different regions of
the same nucleic acid target. Numerous examples of antisense
compounds are known in the art. Two or more combined compounds may
be used together or sequentially.
H. Dosing
[0104] The formulation of therapeutic compositions and their
subsequent administration (dosing) 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.
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 at desired intervals. 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.
[0105] 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. Each of the
references, GenBank accession numbers, and the like recited in the
present application is incorporated herein by reference in its
entirety.
EXAMPLES
Example 1--Synthesis of Nucleoside Phosphoramidites
[0106] Synthesis of nucleoside phorsphoramidates, including
amidites and their intermediates were prepared as described in U.S.
Pat. No. 6,426,220 and published PCT WO 02/36743, both of which are
incorporated herein by reference.
Example 2--Oligonucleotide and Oligonucleoside Synthesis
[0107] 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.
[0108] Oligonucleotides: Unsubstituted and substituted
phosphodiester (P.dbd.O) oligonucleotides are synthesized on an
automated DNA synthesizer (Applied Biosystems model 394) using
standard phosphoramidite chemistry with oxidation by iodine.
[0109] Phosphorothioates (P.dbd.S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions:
thiation was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time was increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligonucleotides were recovered by precipitating with >3 volumes
of ethanol from a 1 M NH.sub.4OAc solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270, herein incorporated by reference.
[0110] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863. 3'-Deoxy-3'-methylene phosphonate
oligonucleotides are prepared as described in U.S. Pat. No.
5,610,289 or 5,625,050. Phosphoramidite oligonucleotides are
prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No.
5,366,878. 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). 3'-Deoxy-3'-amino phosphoramidate oligonucleotides
are prepared as described in U.S. Pat. No. 5,476,925.
Phosphotriester oligonucleotides are prepared as described in U.S.
Pat. No. 5,023,243. Borano phosphate oligonucleotides are prepared
as described in U.S. Pat. Nos. 5,130,302 and 5,177,198.
Oligonucleosides: Methylenemethylimino linked oligonucleosides,
also identified as MMI linked oligonucleosides,
methylenedimethylhydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289. Formacetal and thioformacetal
linked oligonucleosides are prepared as described in U.S. Pat. Nos.
5,264,562 and 5,264,564. Ethylene oxide linked oligonucleosides are
prepared as described in U.S. Pat. No. 5,223,618. All patents and
applications are incorporated herein by reference.
Example 3--RNA Synthesis
[0111] In general, RNA synthesis chemistry is based on the
selective incorporation of various protecting groups at strategic
intermediary reactions. Methods of RNA synthesis are well known in
the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado,
1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120,
11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem.
Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H.
Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta
Chem. Scand, 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom
Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res.,
1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2315-2331).
[0112] RNA antisense compounds (RNA oligonucleotides) of the
present invention can be synthesized by the methods herein or
purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once
synthesized, complementary RNA antisense compounds can then be
annealed by methods known in the art to form double stranded
(duplexed) antisense compounds. For example, duplexes can be formed
by combining 30 .mu.l of each of the complementary strands of RNA
oligonucleotides (50 uM RNA oligonucleotide solution) and 15 .mu.l
of 5.times. annealing buffer (100 mM potassium acetate, 30 mM
HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1
minute at 90.degree. C., then 1 hour at 37.degree. C. The resulting
duplexed antisense compounds can be used in kits, assays, screens,
or other methods to investigate the role of a target nucleic acid,
or for diagnostic or therapeutic purposes.
Example 4--Synthesis of Chimeric Compounds
[0113] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0114] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, 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
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spetrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry.
[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.
[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy
Phosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0116] [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,
oxidation 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.
[0117] 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 5--Design and Screening of Duplexed Antisense Compounds
Targeting Transthyretin
[0118] In accordance with the present invention, a series of
nucleic acid duplexes comprising the antisense compounds of the
present invention and their complements can be designed to target
transthyretin. The nucleobase sequence of the antisense strand of
the duplex comprises at least an 8-nucleobase portion of an
oligonucleotide in Table 1. The ends of the strands may be modified
by the addition of one or more natural or modified nucleobases to
form an overhang. The sense strand of the dsRNA is then designed
and synthesized as the complement of the antisense strand and may
also contain modifications or additions to either terminus. For
example, in one embodiment, both strands of the dsRNA duplex would
be complementary over the central nucleobases, each having
overhangs at one or both termini.
[0119] For example, a duplex comprising an antisense strand having
the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 134) and having a
two-nucleobase overhang of deoxythymidine(dT) would have the
following structure:
TABLE-US-00001 cgagaggcggacgggaccgTT Antisense Strand (SEQ ID NO:
135) ||||||||||||||||||| TTgctctccgcctgccctggc Complement (SEQ ID
NO: 136)
[0120] In another embodiment, a duplex comprising an antisense
strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:
134) may be prepared with blunt ends (no single stranded overhang)
as shown:
TABLE-US-00002 cgagaggcggacgggaccg Antisense Strand (SEQ ID NO:
134) ||||||||||||||||||| gctctccgcctgccctggc Complement (SEQ ID NO:
137)
[0121] The RNA duplex can be unimolecular or bimolecular; i.e, the
two strands can be part of a single molecule or may be separate
molecules.
[0122] RNA strands of the duplex can be synthesized by methods
disclosed herein or purchased from Dharmacon Research Inc.,
(Lafayette, Colo.). Once synthesized, the complementary strands are
annealed. The single strands are aliquoted and diluted to a
concentration of 50 uM. Once diluted, 30 uL of each strand is
combined with 15 uL of a 5.times. solution of annealing buffer. The
final concentration of said buffer is 100 mM potassium acetate, 30
mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume
is 75 uL. This solution is incubated for 1 minute at 90.degree. C.
and then centrifuged for 15 seconds. The tube is allowed to sit for
1 hour at 37.degree. C. at which time the dsRNA duplexes are used
in experimentation. The final concentration of the dsRNA duplex is
20 uM. This solution can be stored frozen (-20.degree. C.) and
freeze-thawed up to 5 times. Once prepared, the duplexed antisense
compounds are evaluated for their ability to modulate transthyretin
expression.
[0123] When cells reach 80% confluency, they are treated with
duplexed antisense compounds of the invention. For cells grown in
96-well plates, wells are washed once with 200 .mu.L OPTI-MEM-1
reduced-serum medium (Gibco BRL) and then treated with 130 .mu.L of
OPTI-MEM-1 containing 12 .mu.g/mL LIPOFECTIN (Gibco BRL) and the
desired duplex antisense compound at a final concentration of 200
nM. After 5 hours of treatment, the medium is replaced with fresh
medium. Cells are harvested 16 hours after treatment, at which time
RNA is isolated and target reduction measured by RT-PCR.
Example 6--Oligonucleotide Isolation
[0124] After cleavage from the controlled pore glass solid support
and deblocking in concentrated ammonium hydroxide at 55.degree. C.
for 12-16 hours, the oligonucleotides or oligonucleosides are
recovered by precipitation out of 1 M NH.sub.4OAc with >3
volumes of ethanol. Synthesized oligonucleotides were analyzed by
electrospray mass spectroscopy (molecular weight determination) and
by capillary gel electrophoresis and judged to be at least 70% full
length material. The relative amounts of phosphorothioate and
phosphodiester linkages obtained in the synthesis was determined by
the ratio of correct molecular weight relative to the -16 amu
product (+/-32+/-48). For some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7--Oligonucleotide Synthesis--96 Well Plate Format
[0125] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 96-well format using
methods known to those skilled in the art.
Example 8--Oligonucleotide Analysis--96-Well Plate Format
[0126] The concentration of oligonucleotide in each well was
assessed by dilution of samples and UV absorption spectroscopy
using methods known to those skilled in the art.
Example 9--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. This can be readily determined by methods routine in
the art, for example Northern blot analysis, ribonuclease
protection assays, or RT-PCR. The following cell types are provided
for illustrative purposes, but other cell types can be routinely
used, provided that the target is expressed in the cell type
chosen. All cell types were cultured under standard conditions,
using methods known to those skilled in the art.
[0128] T-24 Cells:
[0129] The human transitional cell bladder carcinoma cell line T-24
was obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). 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.
[0130] A549 Cells:
[0131] The human lung carcinoma cell line A549 was obtained from
the American Type Culture Collection (ATCC) (Manassas, Va.).
[0132] NHDF Cells:
[0133] Human neonatal dermal fibroblast (NHDF) were obtained from
the Clonetics Corporation (Walkersville, Md.).
[0134] HEK Cells:
[0135] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville, Md.).
[0136] HepG2 Cells:
[0137] The human hepatoblastoma cell line HepG2 was obtained from
the American Type Culture Collection (Manassas, Va.). Cells were
seeded into 96-well plates (Falcon-Primaria #3872) at a density of
7000 cells/well for use in RT-PCR analysis. For Northern blotting
or other analyses, cells may be seeded onto 100 mm or other
standard tissue culture plates and treated similarly, using
appropriate volumes of medium and oligonucleotide.
[0138] Treatment with Antisense Compounds:
[0139] When cells reached 65-75% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 100 .mu.L, OPTI-MEM.TM.-1 reduced-serum medium
(Invitrogen Corporation, Carlsbad, Calif.) and then treated with
130 .mu.L, of OPTI-MEM.TM.-1 containing 3.75 .mu.g/mL
LIPOFECTIN.TM. (Invitrogen Corporation, Carlsbad, Calif.) and the
desired concentration of oligonucleotide. Cells are treated and
data are obtained in triplicate. After 4-7 hours of treatment at
37.degree. C., the medium was replaced with fresh medium. Cells
were harvested 16-24 hours after oligonucleotide treatment.
[0140] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is selected from either ISIS 13920
(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human
H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is
targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are
2'-O-(2-methoxyethyl) gapmers (2'-O-(2-methoxyethyl) nucleotides
shown in bold) with a phosphorothioate backbone. For mouse or rat
cells the positive control oligonucleotide is ISIS 15770,
ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2'-O-(2-methoxyethyl) gapmers
(2'-O-(2-methoxyethyl) nucleotides shown in bold) with a
phosphorothioate backbone which is targeted to both mouse and rat
c-raf. The concentration of positive control oligonucleotide that
results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for
ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the
screening concentration for new oligonucleotides in subsequent
experiments for that cell line. If 80% inhibition is not achieved,
the lowest concentration of positive control oligonucleotide that
results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then
utilized as the oligonucleotide screening concentration in
subsequent experiments for that cell line. If 60% inhibition is not
achieved, that particular cell line is deemed as unsuitable for
oligonucleotide transfection experiments. The concentrations of
antisense oligonucleotides used herein are from 50 nM to 300
nM.
Example 10--Analysis of Oligonucleotide Inhibition of Transthyretin
Expression
[0141] Antisense modulation of transthyretin expression can be
assayed in a variety of ways known in the art. For example,
transthyretin 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. The preferred method of RNA analysis of the present
invention is the use of total cellular RNA as described in other
examples herein. Methods of RNA isolation are well known in the
art. Northern blot analysis is also routine in the art. Real-time
quantitative (PCR) can be conveniently accomplished using the
commercially available ABI PRISM.TM. 7600, 7700, or 7900 Sequence
Detection System, available from PE-Applied Biosystems, Foster
City, Calif. and used according to manufacturer's instructions.
[0142] Protein levels of transthyretin can be quantitated in a
variety of ways well known in the art, such as immunoprecipitation,
Western blot analysis (immunoblotting), enzyme-linked immunosorbent
assay (ELISA) or fluorescence-activated cell sorting (FACS).
Antibodies directed to transthyretin 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 monoclonal or polyclonal antibody generation methods
well known in the art.
Example 11--Design of Phenotypic Assays for the Use of
Transthyretin Inhibitors
[0143] Phenotypic assays-Once transthyretin inhibitors have been
identified by the methods disclosed herein, the compounds are
further investigated in one or more phenotypic assays, each having
measurable endpoints predictive of efficacy in the treatment of a
particular disease state or condition. Phenotypic assays, kits and
reagents for their use are well known to those skilled in the art
and are herein used to investigate the role and/or association of
transthyretin in health and disease. Phenotypic assay can be
purchased from any one of several commercial vendors.
Example 12--RNA Isolation
[0144] Poly(A)+ mRNA Isolation.
[0145] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.
Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA
isolation are routine in the art. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C., was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0146] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions
[0147] Total RNA was isolated using an RNEASY 96.TM. kit and
buffers purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures.
[0148] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 13--Real-Time Quantitative PCR Analysis of Transthyretin
mRNA Levels
[0149] Quantitation of transthyretin mRNA levels was accomplished
by real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700,
or 7900 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., FAM or JOE, obtained from either PE-Applied
Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda,
Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is
attached to the 5' end of the probe and a quencher dye (e.g.,
TAMRA, obtained from either PE-Applied Biosystems, Foster City,
Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 3' end of
the probe. When the probe and dyes are intact, reporter dye
emission is quenched by the proximity of the 3' quencher dye.
During amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular intervals by laser
optics built into the ABI PRISM.TM. 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] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0151] PCR reagents were obtained from Invitrogen Corporation,
(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20
.mu.L PCR cocktail (2.5.times. PCR buffer minus MgCl.sub.2, 6.6 mM
MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM
each of forward primer and reverse primer, 125 nM of probe, 4 Units
RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse
transcriptase, and 2.5.times. ROX dye) to 96-well plates containing
30 .mu.L total RNA solution (20-200 ng). 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
PLATINUM.RTM. Taq, 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).
[0152] Gene target quantities obtained by real time RT-PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RiboGreen.TM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).
[0153] In this assay, 170 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 485 nm and emission at 530
nm.
[0154] Probes and primers to human transthyretin were designed to
hybridize to a human transthyretin sequence, using published
sequence information (GenBank accession number BCO20791.1,
incorporated herein as SEQ ID NO: 4). For human transthyretin the
PCR primers were:
forward primer: CCCTGCTGAGCCCCTACTC (SEQ ID NO: 5) reverse primer:
TCCCTCATTCCTTGGGATTG (SEQ ID NO: 6) and the PCR probe was:
FAM-ATTCCACCACGGCTGTCGTCA-TAMRA (SEQ ID NO: 7) where FAM is the
fluorescent dye and TAMRA is the quencher dye. For human GAPDH the
PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8)
reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR
probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 10)
where JOE is the fluorescent reporter dye and TAMRA is the quencher
dye.
Example 14--Northern Blot Analysis of Transthyretin mRNA Levels
[0155] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM.
(TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared
following manufacturer's recommended protocols. Twenty micrograms
of total RNA was fractionated by electrophoresis through 1.2%
agarose gels containing 1.1% formaldehyde using a MOPS buffer
system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the
gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.TM.
UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then
probed using QUICKHYB.TM. hybridization solution (Stratagene, La
Jolla, Calif.) using manufacturer's recommendations for stringent
conditions.
[0156] To detect human transthyretin, a human transthyretin
specific probe was prepared by PCR using the forward primer
CCCTGCTGAGCCCCTACTC (SEQ ID NO: 5) and the reverse primer
TCCCTCATTCCTTGGGATTG (SEQ ID NO: 6). To normalize for variations in
loading and transfer efficiency membranes were stripped and probed
for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA
(Clontech, Palo Alto, Calif.).
[0157] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software V3.3 (Molecular
Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels
in untreated controls.
Example 15--Antisense Inhibition of Human Transthyretin Expression
by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings
and a Deoxy Gap
[0158] In accordance with the present invention, a series of
antisense compounds was designed to target different regions of the
human transthyretin RNA, using published sequences (GenBank
accession number BC020791.1, incorporated herein as SEQ ID NO: 4,
and nucleotides 2009236 to 2017289 of the sequence with GenBank
accession number NT_010966.10, incorporated herein as SEQ ID NO:
11). The compounds are shown in Table 1. "Target site" indicates
the first (5'-most) nucleotide number on the particular target
sequence to which the compound binds. All compounds in Table 1 are
chimeric oligonucleotides ("gapmers") 20 nucleotides in length,
composed of a central "gap" region consisting often
2'-deoxynucleotides, which is flanked on both sides (5' and 3'
directions) by five-nucleotide "wings". The wings are composed of
2'-O-(2-methoxyethyl) nucleotides, also known as 2'-MOE
nucleotides. The internucleoside (backbone) linkages are
phosphorothioate (P.dbd.S) throughout the oligonucleotide. All
cytidine residues are 5-methylcytidines. The compounds were
analyzed for their effect on human transthyretin mRNA levels by
quantitative real-time PCR as described in other examples herein.
Data are averages from two experiments in which HepG2 cells were
treated with 50 nM of the antisense oligonucleotides of the present
invention. The positive control ISIS 18078 (SEQ ID NO: 2) was used
for this assay. If present, "N.D." indicates "no data".
TABLE-US-00003 TABLE 1 Inhibition of human transthyretin mRNA
levels by chimeric phosphorothioate oligonucleotides having 2'-MOE
wings and a deoxy gap TARGET SEQ SEQ TARGET % ID ISIS # REGION ID
NO SITE SEQUENCE INHIB NO 304237 Exon 1: 11 596
aaacactcaccgtagggcca 6 12 Intron 1 junction 304238 Intron 1: 11
1520 caccggtgccctgggtgtag 0 13 Exon 2 junction 304239 Intron 2 11
1718 tgagcctctctctaccaagt 0 14 304240 Exon 3: 11 3880
gtatactcacctctgcatgc 33 15 Intron 3 junction 304241 Intron 3 11
4039 ttctcagagtgttgtgaatt 0 16 304242 Intron 3 11 6252
actctgcataaatacatttt 0 17 304243 Intron 3 11 6967
tcttgttttgcaaattcacg 0 18 304244 Intron 3 11 7192
tgaataccacctatgagaga 0 19 304245 5'UTR 4 6 ctgccaagaatgagtggact 33
20 304246 Start Codon 4 18 tgagaagccatcctgccaag 6 21 304247 Start
Codon 4 25 cagacgatgagaagccatcc 2 22 304248 Coding 4 30
aggagcagacgatgagaagc 10 23 304249 Coding 4 59 acacaaataccagtccagca
33 24 304250 Coding 4 60 gacacaaataccagtccagc 0 25 304251 Coding 4
66 gcctcagacacaaataccag 14 26 304252 Coding 4 75
gtagggccagcctcagacac 3 27 304253 Coding 4 86 caccggtgcccgtagggcca
16 28 304254 Coding 4 91 ggattcaccggtgcccgtag 32 29 304255 Coding 4
100 aggacacttggattcaccgg 47 30 304256 Coding 4 105
atcagaggacacttggattc 0 31 304257 Coding 4 110 tgaccatcagaggacacttg
21 32 304258 Coding 4 114 actttgaccatcagaggaca 16 33 304259 Coding
4 126 acagcatctagaactttgac 33 34 304260 Coding 4 133
gcctcggacagcatctagaa 34 35 304261 Coding 4 146 tgatggcaggactgcctcgg
16 36 304262 Coding 4 170 ttctgaacacatgcacggcc 41 37 304263 Coding
4 185 tgtcatcagcagcctttctg 8 38 304264 Coding 4 197
atggctcccaggtgtcatca 34 39 304265 Coding 4 203 aggcaaatggctcccaggtg
15 40 304266 Coding 4 210 ttcccagaggcaaatggctc 0 41 304267 Coding 4
217 actggttttcccagaggcaa 56 42 304268 Coding 4 222
gactcactggttttcccaga 0 43 304269 Coding 4 232 cagctctccagactcactgg
44 44 304270 Coding 4 239 gcccatgcagctctccagac 14 45 304271 Coding
4 244 tgtgagcccatgcagctctc 3 46 304272 Coding 4 250
ctcagttgtgagcccatgca 36 47 304273 Coding 4 257 attcctcctcagttgtgagc
10 48 304274 Coding 4 264 tctacaaattcctcctcagt 34 49 304275 Coding
4 278 ctttgtatatcccttctaca 43 50 304276 Coding 4 298
agatttggtgtctatttcca 1 51 304277 Coding 4 314 caagtgccttccagtaagat
14 52 304278 Coding 4 323 gggagatgccaagtgccttc 53 53 304279 Coding
4 342 tctgcatgctcatggaatgg 42 54 304280 Coding 4 353
tgaataccacctctgcatgc 7 55 304281 Coding 4 360 ttggctgtgaataccacctc
5 56 304282 Coding 4 369 ccggagtcgttggctgtgaa 16 57 304283 Coding 4
401 tcagcagggcggcaatggtg 1 58 304284 Coding 4 425
ccgtggtggaataggagtag 63 59 304285 Coding 4 427 agccgtggtggaataggagt
53 60 304286 Coding 4 431 cgacagccgtggtggaatag 56 61 304287 Coding
4 438 ttggtgacgacagccgtggt 92 62 304288 Coding 4 440
gattggtgacgacagccgtg 70 63 304289 Coding 4 442 gggattggtgacgacagccg
73 64 304290 Coding 4 443 tgggattggtgacgacagcc 83 65 304291 Coding
4 449 attccttgggattggtgacg 45 66 304292 Stop Codon 4 450
cattccttgggattggtgac 27 67 304293 Stop Codon 4 451
tcattccttgggattggtga 20 68 304294 Stop Codon 4 460
agaagtccctcattccttgg 37 69 304295 3'UTR 4 472 gtccactggaggagaagtcc
47 70 304296 3'UTR 4 481 gtccttcaggtccactggag 86 71 304297 3'UTR 4
489 catccctcgtccttcaggtc 76 72 304298 3'UTR 4 501
tacatgaaatcccatccctc 52 73 304299 3'UTR 4 507 cttggttacatgaaatccca
78 74 304300 3'UTR 4 513 aatactcttggttacatgaa 52 75 304301 3'UTR 4
526 ttagtaaaaatggaatactc 20 76 304302 3'UTR 4 532
actgctttagtaaaaatgga 57 77 304303 3'UTR 4 539 tgaaaacactgctttagtaa
54 78 304304 3'UTR 4 546 tatgaggtgaaaacactgct 48 79 304305 3'UTR 4
551 tagcatatgaggtgaaaaca 68 80 304306 3'UTR 4 559
ttctaacatagcatatgagg 72 81 304307 3'UTR 4 564 tggacttctaacatagcata
79 82 304308 3'UTR 4 572 tctctgcctggacttctaac 75 83 304309 3'UTR 4
578 ttattgtctctgcctggact 83 84 304310 3'UTR 4 595
cctttcacaggaatgtttta 46 85 304311 3'UTR 4 597 tgcctttcacaggaatgttt
79 86 304312 3'UTR 4 598 gtgcctttcacaggaatgtt 80 87 304313 3'UTR 4
600 aagtgcctttcacaggaatg 68 88 304314 3'UTR 4 604
tgaaaagtgcctttcacagg 8 89
[0159] As shown in Table 1, SEQ ID NOs 15, 20, 24, 29, 30, 34, 35,
37, 39, 42, 44, 47, 49, 50, 53, 54, 59, 60, 61, 62, 63, 64, 65, 66,
67, 69, 70, 71, 72, 73, 74, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87 and 88 demonstrated at least 27% inhibition of human
transthyretin expression in this assay and are therefore preferred.
More preferred are SEQ ID NOs 84, 87, and 86. The target regions to
which these preferred sequences are complementary are herein
referred to as "preferred target segments" and are therefore
preferred for targeting by compounds of the present invention.
These preferred target segments are shown in Table 2. These
sequences are shown to contain thymine (T) but one of skill in the
art will appreciate that thymine (T) is generally replaced by
uracil (U) in RNA sequences. The sequences represent the reverse
complement of the preferred antisense compounds shown in Table 1.
"Target site" indicates the first (5'-most) nucleotide number on
the particular target nucleic acid to which the oligonucleotide
binds. Also shown in Table 2 is the species in which each of the
preferred target segments was found.
TABLE-US-00004 TABLE 2 Sequence and position of preferred target
segments identified in transthyretin. REV TARGET COMP SEQ SITE SEQ
ID TARGET OF SEQ ID ID NO SITE SEQUENCE ID ACTIVE IN NO 220029 11
3880 gcatgcagaggtgagtatac 15 H. sapiens 90 220034 4 6
agtccactcattcttggcag 20 H. sapiens 91 220038 4 59
tgctggactggtatttgtgt 24 H. sapiens 92 220043 4 91
ctacgggcaccggtgaatcc 29 H. sapiens 93 220044 4 100
ccggtgaatccaagtgtcct 30 H. sapiens 94 220048 4 126
gtcaaagttctagatgctgt 34 H. sapiens 95 220049 4 133
ttctagatgctgtccgaggc 35 H. sapiens 96 220051 4 170
ggccgtgcatgtgttcagaa 37 H. sapiens 97 220053 4 197
tgatgacacctgggagccat 39 H. sapiens 98 220056 4 217
ttgcctctgggaaaaccagt 42 H. sapiens 99 220058 4 232
ccagtgagtctggagagctg 44 H. sapiens 100 220061 4 250
tgcatgggctcacaactgag 47 H. sapiens 101 220063 4 264
actgaggaggaatttgtaga 49 H. sapiens 102 220064 4 278
tgtagaagggatatacaaag 50 H. sapiens 103 220067 4 323
gaaggcacttggcatctccc 53 H. sapiens 104 220068 4 342
ccattccatgagcatgcaga 54 H. sapiens 105 220073 4 425
ctactcctattccaccacgg 59 H. sapiens 106 220074 4 427
actcctattccaccacggct 60 H. sapiens 107 220075 4 431
ctattccaccacggctgtcg 61 H. sapiens 108 220076 4 438
accacggctgtcgtcaccaa 62 H. sapiens 109 220077 4 440
cacggctgtcgtcaccaatc 63 H. sapiens 110 220078 4 442
cggctgtcgtcaccaatccc 64 H. sapiens 111 220079 4 443
ggctgtcgtcaccaatccca 65 H. sapiens 112 220080 4 449
cgtcaccaatcccaaggaat 66 H. sapiens 113 220081 4 450
gtcaccaatcccaaggaatg 67 H. sapiens 114 220083 4 460
ccaaggaatgagggacttct 69 H. sapiens 115 220084 4 472
ggacttctcctccagtggac 70 H. sapiens 116 220085 4 481
ctccagtggacctgaaggac 71 H. sapiens 117 220086 4 489
gacctgaaggacgagggatg 72 H. sapiens 118 220087 4 501
gagggatgggatttcatgta 73 H. sapiens 119 220088 4 507
tgggatttcatgtaaccaag 74 H. sapiens 120 220089 4 513
ttcatgtaaccaagagtatt 75 H. sapiens 121 220091 4 532
tccatttttactaaagcagt 77 H. sapiens 122 220092 4 539
ttactaaagcagtgttttca 78 H. sapiens 123 220093 4 546
agcagtgttttcacctcata 79 H. sapiens 124 220094 4 551
tgttttcacctcatatgcta 80 H. sapiens 125 220095 4 559
cctcatatgctatgttagaa 81 H. sapiens 126 220096 4 564
tatgctatgttagaagtcca 82 H. sapiens 127 220097 4 572
gttagaagtccaggcagaga 83 H. sapiens 128 220098 4 578
agtccaggcagagacaataa 84 H. sapiens 129 220099 4 595
taaaacattcctgtgaaagg 85 H. sapiens 130 220100 4 597
aaacattcctgtgaaaggca 86 H. sapiens 131 220101 4 598
aacattcctgtgaaaggcac 87 H. sapiens 132 220102 4 600
cattcctgtgaaaggcactt 88 H. sapiens 133
[0160] As these "preferred target segments" have been found by
experimentation to be open to, and accessible for, hybridization
with the antisense compounds of the present invention, one of skill
in the art will recognize or be able to ascertain, using no more
than routine experimentation, further embodiments of the invention
that encompass other compounds that specifically hybridize to these
preferred target segments and consequently inhibit the expression
of transthyretin.
[0161] According to the present invention, antisense compounds
include antisense oligomeric compounds, antisense oligonucleotides,
ribozymes, external guide sequence (EGS) oligonucleotides,
alternate splicers, primers, probes, and other short oligomeric
compounds which hybridize to at least a portion of the target
nucleic acid.
Example 16--Western Blot Analysis of Transthyretin Protein
Levels
[0162] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a
16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to transthyretin is used, with a radiolabeled or
fluorescently labeled secondary antibody directed against the
primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale Calif.).
[0163] All of the applications, patents and references cited are
hereby incorporated herein by reference.
[0164] It will be apparent to those skilled in the art that various
modifications and variations can be made in the methods and
compositions of the present invention without departing from the
spirit or scope of the invention. Thus it is intended that the
present invention cover modifications and variations of thi
invention. The invention is limited only by the claims below.
Sequence CWU 1
1
137120DNAArtificial sequenceSynthetic oligonucleotide 1tccgtcatcg
ctcctcaggg 20220DNAArtificial sequenceSynthetic oligonucleotide
2gtgcgcgcga gcccgaaatc 20320DNAArtificial sequenceSynthetic
oligonucleotide 3atgcattctg cccccaagga 204650DNAHomo sapiens
4acagaagtcc actcattctt ggcaggatgg cttctcatcg tctgctcctc ctctgccttg
60ctggactggt atttgtgtct gaggctggcc ctacgggcac cggtgaatcc aagtgtcctc
120tgatggtcaa agttctagat gctgtccgag gcagtcctgc catcaatgtg
gccgtgcatg 180tgttcagaaa ggctgctgat gacacctggg agccatttgc
ctctgggaaa accagtgagt 240ctggagagct gcatgggctc acaactgagg
aggaatttgt agaagggata tacaaagtgg 300aaatagacac caaatcttac
tggaaggcac ttggcatctc cccattccat gagcatgcag 360aggtggtatt
cacagccaac gactccggcc cccgccgcta caccattgcc gccctgctga
420gcccctactc ctattccacc acggctgtcg tcaccaatcc caaggaatga
gggacttctc 480ctccagtgga cctgaaggac gagggatggg atttcatgta
accaagagta ttccattttt 540actaaagcag tgttttcacc tcatatgcta
tgttagaagt ccaggcagag acaataaaac 600attcctgtga aaggcacttt
tcattccaaa aaaaaaaaaa aaaaaaaaaa 650519DNAArtificial sequencePrimer
5ccctgctgag cccctactc 19620DNAArtificial sequencePrimer 6tccctcattc
cttgggattg 20721DNAArtificial sequenceProbe 7attccaccac ggctgtcgtc
a 21819DNAArtificial sequencePrimer 8gaaggtgaag gtcggagtc
19920DNAArtificial sequencePrimer 9gaagatggtg atgggatttc
201020DNAArtificial sequenceProbe 10caagcttccc gttctcagcc
20118054DNAHomo sapiens 11ttgttgaccc atggatccat caagtgcaaa
cattttctaa tgcactatat ttaagcctgt 60gcagctagat gtcattcaac atgaaataca
ttattacaac ttgcatctgt ctaaaatctt 120gcatctaaaa tgagagacaa
aaaatctata aaaatggaaa acatgcatag aaatatgtga 180gggaggaaaa
aattaccccc aagaatgtta gtgcacgcag tcacacaggg agaagactat
240ttttgttttg ttttgattgt tttgttttgt tttggttgtt ttgttttggt
gacctaactg 300gtcaaatgac ctattaagaa tatttcatag aacgaatgtt
ccgatgctct aatctctcta 360gacaaggttc atatttgtat gggttactta
ttctctcttt gttgactaag tcaataatca 420gaatcagcag gtttgcagtc
agattggcag ggataagcag cctagctcag gagaagtgag 480tataaaagcc
ccaggctggg agcagccatc acagaagtcc actcattctt ggcaggatgg
540cttctcatcg tctgctcctc ctctgccttg ctggactggt atttgtgtct
gaggctggcc 600ctacggtgag tgtttctgtg acatcccatt cctacattta
agattcacgc taaatgaagt 660agaagtgact ccttccagct ttgccaacca
gcttttatta ctagggcaag ggtacccagc 720atctattttt aatataatta
attcaaactt caaaaagaat gaagttccac tgagcttact 780gagctgggac
ttgaactctg agcattctac ctcattgctt tggtgcatta ggtttgtaat
840atctggtacc tctgtttcct cagatagatg atagaaataa agatatgata
ttaaggaagc 900tgttaatact gaattttcag aaaagtatcc ctccataaaa
tgtatttggg ggacaaactg 960caggagatta tattctggcc ctatagttat
tcaaaacgta tttattgatt aatctttaaa 1020aggcttagtg aacaatattc
tagtcagata tctaattctt aaatcctcta gaagaattaa 1080ctaatactat
aaaatgggtc tggatgtagt tctgacatta ttttataaca actggtaaga
1140gggagtgact atagcaacaa ctaaaatgat ctcaggaaaa cctgtttggc
cctatgtatg 1200gtacattaca tcttttcagt aattccactc aaatggagac
ttttaacaaa gcaactgttc 1260tcaggggacc tattttctcc cttaaaattc
attatacaca tccctggttg atagcagtgt 1320gtctggaggc agaaaccatt
cttgctttgg aaacaattac gtctgtgtta tactgagtag 1380ggaagctcat
taattgtcga cacttacgtt cctgataatg ggatcagtgt gtaattcttg
1440tttcgctcca gatttctaat accacaaaga ataaatcctt tcactctgat
caattttgtt 1500aacttctcac gtgtcttctc tacacccagg gcaccggtga
atccaagtgt cctctgatgg 1560tcaaagttct agatgctgtc cgaggcagtc
ctgccatcaa tgtggccgtg catgtgttca 1620gaaaggctgc tgatgacacc
tgggagccat ttgcctctgg gtaagttgcc aaagaaccct 1680cccacaggac
ttggttttat cttcccgttt gcccctcact tggtagagag aggctcacat
1740catctgctaa agaatttaca agtagattga aaaacgtagg cagaggtcaa
gtatgccctc 1800tgaaggatgc cctctttttg ttttgcttag ctaggaagtg
accaggaacc tgagcatcat 1860ttaggggcag acagtagaga aaagaaggaa
tcagaactcc tctcctctag ctgtggtttg 1920caaccctttt gggtcacaga
acactttatg taggtgatga aaagtaaaca ttctatgccc 1980agaaaaaatg
cacagataca cacacataca aaatcatata tgtgatttta ggagtttcac
2040agattccctg gtgtccctgg gtaacaccaa agctaagtgt ccttgtctta
gaattttagg 2100aaaaggtata atgtgtatta acccattaac aaaaggaaag
gaattcagaa atattattaa 2160ccaggcatct gtctgtagtt aatatggatc
acccaaaacc caaggctttt gcctaatgaa 2220cactttgggg cacctactgt
gtgcaaggct gggggctgtc aagctcagtt aaaaaaaaaa 2280agatagaaga
gatggatcca tgaggcaaag tacagcccca ggctaatccc acgatcaccc
2340gacttcatgt ccaagagtgg cttctcacct tcattagcca gttcacaatt
ttcatggagt 2400ttttctacct gcactagcaa aaacttcaag gaaaatacat
attaataaat ctaagcaaag 2460tgaccagaag acagagcaat caggagaccc
tttgcatcca gcagaagagg aactgctaag 2520tatttacatc tccacagaga
agaatttctg ttgggtttta attgaacccc aagaaccaca 2580tgattcttca
accattattg ggaagatcat tttcttaggt ctggttttaa ctggcttttt
2640atttgggaat tcatttatgt ttatataaaa tgccaagcat aacatgaaaa
gtggttacag 2700gactattcta agggagagac agaatggaca ccaaaaatat
tccaatgttc ttgtgaatct 2760tttccttgca ccaggacaaa aaaaaaaaga
agtgaaaaga agaaaggagg aggggcataa 2820tcagagtcag taaagacaac
tgctattttt atctatcgta gctgttgcag tcaaatggga 2880agcaatttcc
aacattcaac tatggagctg gtacttacat ggaaatagaa gttgcctagt
2940gtttgttgct ggcaaagagt tatcagagag gttaaatata taaaagggaa
aagagtcaga 3000tacaggttct tcttcctact ttaggttttc cactgtgtgt
gcaaatgata ctccctggtg 3060gtgtgcagat gcctcaaagc tatcctcaca
ccacaaggga gaggagcgag atcctgctgt 3120cctggagaag tgcagagtta
gaacagctgt ggccacttgc atccaatcat caatcttgaa 3180tcacagggac
tctttcttaa gtaaacatta tacctggccg ggcacggtgg ctcacgcctg
3240taatcccagc actttgggat gccaaagtgg gcatatcatc tgaggtcagg
agttcaagac 3300cagcctggcc aacatggcaa aactccgtct ttatgaaaaa
tacaaaaatt agccaggcat 3360ggtggcaggc gcctgtaatc ccagctaatt
gggaggctga ggctggagaa tcccttgaat 3420ctaggaggca gaggttgcag
tgagctgaga tcgtgccatt gcactccagc ctgggtgaca 3480agagtaaaac
tctgtctcaa aaaaaaaaaa ttatacctac attctcttct tatcagagaa
3540aaaaatctac agtgagcttt tcaaaaagtt tttacaaact ttttgccatt
taatttcagt 3600taggagtttt ccctacttct gacttagttg aggggaaatg
ttcataacat gtttataaca 3660tgtttatgtg tgttagttgg tgggggtgta
ttactttgcc atgccatttg tttcctccat 3720gcgtaactta atccagactt
tcacacctta taggaaaacc agtgagtctg gagagctgca 3780tgggctcaca
actgaggagg aatttgtaga agggatatac aaagtggaaa tagacaccaa
3840atcttactgg aaggcacttg gcatctcccc attccatgag catgcagagg
tgagtataca 3900gaccttcgag ggttgttttg gttttggttt ttgcttttgg
cattccagga aatgcacagt 3960tttactcagt gtaccacaga aatgtcctaa
ggaaggtgat gaatgaccaa aggttccctt 4020tcctattata caagaaaaaa
ttcacaacac tctgagaagc aaatttcttt ttgactttga 4080tgaaaatcca
cttagtaaca tgacttgaac ttacatgaaa ctactcatag tctattcatt
4140ccactttata tgaatattga tgtatctgct gttgaaataa tagtttatga
ggcagccctc 4200cagaccccac gtagagtgta tgtaacaaga gatgcaccat
tttatttctc gaaaacccgt 4260aacattcttc attccaaaac acatctggct
tctcggaggt ctggacaagt gattcttggc 4320aacacatacc tatagagaca
ataaaatcaa agtaataatg gcaacacaat agataacatt 4380taccaagcat
acaccatgtg gcagacacaa ttataagtgt tttccatatt taacctactt
4440aatcctcagg aataagccac tgaggtcagt cctattatta tccccatctt
atagatgaag 4500aaaatgaggc accaggaagt caaataactt gtcaaaggtc
acaagactag gaaatacaca 4560agtagaaatg tttacaatta aggcccaggc
tgggtttgcc ctcagttctg ctatgcctcg 4620cattatgccc caggaaactt
tttcccttgt gaaagccaag cttaaaaaaa gaaaagccac 4680atttgtaacg
tgctctgttc ccctgcctat ggtgaggatc ttcaaacagt tatacatgga
4740cccagtcccc ctgccttctc cttaatttct taagtcattt gaaacagatg
gctgtcatgg 4800aaatagaatc cagacatgtt ggtcagagtt aaagatcaac
taattccatc aaaaatagct 4860cggcatgaaa gggaactatt ctctggctta
gtcatggatg agactttcaa ttgctataaa 4920gtggttcctt tattagacaa
tgttaccagg gaaacaacag gggtttgttt gacttctggg 4980gcccacaagt
caacaagaga gccccatcta ccaaggagca tgtccctgac tacccctcag
5040ccagcagcaa gacatggacc ccagtcaggg caggagcagg gtttcggcgg
cgcccagcac 5100aagacattgc ccctagagtc tcagccccta ccctcgagta
atagatctgc ctacctgaga 5160ctgttgtttg cccaagagct gggtctcagc
ctgatgggaa ccatataaaa aggttcactg 5220acatactgcc cacatgttgt
tctctttcat tagatcttag cttccttgtc tgctcttcat 5280tcttgcagta
ttcattcaac aaacattaaa aaaaaaaaaa agcattctat gtgtggaaca
5340ctctgctaga tgctgtggat ttagaaatga aaatacatcc cgacccttgg
aatggaaggg 5400aaaggactga agtaagacag attaagcagg accgtcagcc
cagcttgaag cccagataaa 5460tacggagaac aagagagagc gagtagtgag
agatgagtcc caatgcctca ctttggtgac 5520gggtgcgtgg tgggcttcat
gcagcttctt ctgataaatg cctccttcag aactggtcaa 5580ctctaccttg
gccagtgacc caggtggtca tagtagattt accaagggaa aatggaaact
5640tttattagga gctcttaggc ctcttcactt catggatttt tttttccttt
ttttttgaga 5700tggagttttg ccctgtcacc caggctggaa tgcagtggtg
caatctcagc tcactgcaac 5760ctccgcctcc caggttcaag caattctcct
gcctcagcct cccgagtagc tgggactaca 5820ggtgtgcgcc accacaccag
gctaattttt gtattttttg taaagacagg ttttcaccac 5880gttggccagg
ctggtctgaa ctccagacct caggtgattc acctgtctca gcctcccaaa
5940gtgctgggat tacaggtgtg agccaccgtg cccggctact tcatggattt
ttgattacag 6000attatgcctc ttacaatttt taagaagaat caagtgggct
gaaggtcaat gtcaccataa 6060gacaaaagac atttttatta gttgattcta
gggaattggc cttaagggga gccctttctt 6120cctaagagat tcttaggtga
ttctcacttc ctcttgcccc agtattattt ttgtttttgg 6180tatggctcac
tcagatcctt ttttcctcct atccctaagt aatccgggtt tctttttccc
6240atatttagaa caaaatgtat ttatgcagag tgtgtccaaa cctcaaccca
aggcctgtat 6300acaaaataaa tcaaattaaa cacatcttta ctgtcttcta
cctctttcct gacctcaata 6360tatcccaact tgcctcactc tgagaaccaa
ggctgtccca gcacctgagt cgcagatatt 6420ctactgattt gacagaactg
tgtgactatc tggaacagca ttttgatcca caatttgccc 6480agttacaaag
cttaaatgag ctctagtgca tgcatatata tttcaaaatt ccaccatgat
6540cttccacact ctgtattgta aatagagccc tgtaatgctt ttacttcgta
tttcattgct 6600tgttatacat aaaaatatac ttttcttctt catgttagaa
aatgcaaaga ataggagggt 6660gggggaatct ctgggcttgg agacaggaga
cttgccttcc tactatggtt ccatcagaat 6720gtagactggg acaatacaat
aattcaagtc tggtttgctc atctgtaaat tgggaagaat 6780gtttccagct
ccagaatgct aaatctctaa gtctgtggtt ggcagccact attgcagcag
6840ctcttcaatg actcaatgca gttttgcatt ctccctacct tttttttcta
aaaccaataa 6900aatagataca gcctttaggc tttctgggat ttcccttagt
caagctaggg tcatcctgac 6960tttcggcgtg aatttgcaaa acaagacctg
actctgtact cctgctctaa ggactgtgca 7020tggttccaaa ggcttagctt
gccagcatat ttgagctttt tccttctgtt caaactgttc 7080caaaatataa
aagaataaaa ttaattaagt tggcactgga cttccggtgg tcagtcatgt
7140gtgtcatctg tcacgttttt cgggctctgg tggaaatgga tctgtctgtc
ttctctcata 7200ggtggtattc acagccaacg actccggccc ccgccgctac
accattgccg ccctgctgag 7260cccctactcc tattccacca cggctgtcgt
caccaatccc aaggaatgag ggacttctcc 7320tccagtggac ctgaaggacg
agggatggga tttcatgtaa ccaagagtat tccattttta 7380ctaaagcagt
gttttcacct catatgctat gttagaagtc caggcagaga caataaaaca
7440ttcctgtgaa aggcactttt cattccactt taacttgatt ttttaaattc
ccttattgtc 7500ccttccaaaa aaaagagaat caaaatttta caaagaatca
aaggaattct agaaagtatc 7560tgggcagaac gctaggagag atccaaattt
ccattgtctt gcaagcaaag cacgtattaa 7620atatgatctg cagccattaa
aaagacacat tctgtaaatg agagagcctt attttcctgt 7680aaccttcagc
aaatagcaaa agacacattc caagggccca cttctttact gtgggcattt
7740cttttttttt ctttttttct tttttccttt tttgagacaa agtctcactc
tgttgcccag 7800gctagaatgc agtggtgtaa tctcagctca ctgcaacctc
tgcttcctgg gttcaagcga 7860ttctcctgcc tcagcctccc aagtaactgg
gattacaggc gcatgccacc acgcctagct 7920catttttgta tttttagtag
agatgggatt ttgccatgtt ggctaggctg gtctacgaac 7980tcctgacctc
aggtgatcca cctgcctcag cctcccaaag tgctgggatt acaggcatga
8040gccactacac ccgg 80541220DNAArtificial sequenceSynthetic
oligonucleotide 12aaacactcac cgtagggcca 201320DNAArtificial
sequenceSynthetic oligonucleotide 13caccggtgcc ctgggtgtag
201420DNAArtificial sequenceSynthetic oligonucleotide 14tgagcctctc
tctaccaagt 201520DNAArtificial sequenceSynthetic oligonucleotide
15gtatactcac ctctgcatgc 201620DNAArtificial sequenceSynthetic
oligonucleotide 16ttctcagagt gttgtgaatt 201720DNAArtificial
sequenceSynthetic oligonucleotide 17actctgcata aatacatttt
201820DNAArtificial sequenceSynthetic oligonucleotide 18tcttgttttg
caaattcacg 201920DNAArtificial sequenceSynthetic oligonucleotide
19tgaataccac ctatgagaga 202020DNAArtificial sequenceSynthetic
oligonucleotide 20ctgccaagaa tgagtggact 202120DNAArtificial
sequenceSynthetic oligonucleotide 21tgagaagcca tcctgccaag
202220DNAArtificial sequenceSynthetic oligonucleotide 22cagacgatga
gaagccatcc 202320DNAArtificial sequenceSynthetic oligonucleotide
23aggagcagac gatgagaagc 202420DNAArtificial sequenceSynthetic
oligonucleotide 24acacaaatac cagtccagca 202520DNAArtificial
sequenceSynthetic oligonucleotide 25gacacaaata ccagtccagc
202620DNAArtificial sequenceSynthetic oligonucleotide 26gcctcagaca
caaataccag 202720DNAArtificial sequenceSynthetic oligonucleotide
27gtagggccag cctcagacac 202820DNAArtificial sequenceSynthetic
oligonucleotide 28caccggtgcc cgtagggcca 202920DNAArtificial
sequenceSynthetic oligonucleotide 29ggattcaccg gtgcccgtag
203020DNAArtificial sequenceSynthetic oligonucleotide 30aggacacttg
gattcaccgg 203120DNAArtificial sequenceSynthetic oligonucleotide
31atcagaggac acttggattc 203220DNAArtificial sequenceSynthetic
oligonucleotide 32tgaccatcag aggacacttg 203320DNAArtificial
sequenceSynthetic oligonucleotide 33actttgacca tcagaggaca
203420DNAArtificial sequenceSynthetic oligonucleotide 34acagcatcta
gaactttgac 203520DNAArtificial sequenceSynthetic oligonucleotide
35gcctcggaca gcatctagaa 203620DNAArtificial sequenceSynthetic
oligonucleotide 36tgatggcagg actgcctcgg 203720DNAArtificial
sequenceSynthetic oligonucleotide 37ttctgaacac atgcacggcc
203820DNAArtificial sequenceSynthetic oligonucleotide 38tgtcatcagc
agcctttctg 203920DNAArtificial sequenceSynthetic oligonucleotide
39atggctccca ggtgtcatca 204020DNAArtificial sequenceSynthetic
oligonucleotide 40aggcaaatgg ctcccaggtg 204120DNAArtificial
sequenceSynthetic oligonucleotide 41ttcccagagg caaatggctc
204220DNAArtificial sequenceSynthetic oligonucleotide 42actggttttc
ccagaggcaa 204320DNAArtificial sequenceSynthetic oligonucleotide
43gactcactgg ttttcccaga 204420DNAArtificial sequenceSynthetic
oligonucleotide 44cagctctcca gactcactgg 204520DNAArtificial
sequenceSynthetic oligonucleotide 45gcccatgcag ctctccagac
204620DNAArtificial sequenceSynthetic oligonucleotide 46tgtgagccca
tgcagctctc 204720DNAArtificial sequenceSynthetic oligonucleotide
47ctcagttgtg agcccatgca 204820DNAArtificial sequenceSynthetic
oligonucleotide 48attcctcctc agttgtgagc 204920DNAArtificial
sequenceSynthetic oligonucleotide 49tctacaaatt cctcctcagt
205020DNAArtificial sequenceSynthetic oligonucleotide 50ctttgtatat
cccttctaca 205120DNAArtificial sequenceSynthetic oligonucleotide
51agatttggtg tctatttcca 205220DNAArtificial sequenceSynthetic
oligonucleotide 52caagtgcctt ccagtaagat 205320DNAArtificial
sequenceSynthetic oligonucleotide 53gggagatgcc aagtgccttc
205420DNAArtificial sequenceSynthetic oligonucleotide 54tctgcatgct
catggaatgg 205520DNAArtificial sequenceSynthetic oligonucleotide
55tgaataccac ctctgcatgc 205620DNAArtificial sequenceSynthetic
oligonucleotide 56ttggctgtga ataccacctc
205720DNAArtificial sequenceSynthetic oligonucleotide 57ccggagtcgt
tggctgtgaa 205820DNAArtificial sequenceSynthetic oligonucleotide
58tcagcagggc ggcaatggtg 205920DNAArtificial sequenceSynthetic
oligonucleotide 59ccgtggtgga ataggagtag 206020DNAArtificial
sequenceSynthetic oligonucleotide 60agccgtggtg gaataggagt
206120DNAArtificial sequenceSynthetic oligonucleotide 61cgacagccgt
ggtggaatag 206220DNAArtificial sequenceSynthetic oligonucleotide
62ttggtgacga cagccgtggt 206320DNAArtificial sequenceSynthetic
oligonucleotide 63gattggtgac gacagccgtg 206420DNAArtificial
sequenceSynthetic oligonucleotide 64gggattggtg acgacagccg
206520DNAArtificial sequenceSynthetic oligonucleotide 65tgggattggt
gacgacagcc 206620DNAArtificial sequenceSynthetic oligonucleotide
66attccttggg attggtgacg 206720DNAArtificial sequenceSynthetic
oligonucleotide 67cattccttgg gattggtgac 206820DNAArtificial
sequenceSynthetic oligonucleotide 68tcattccttg ggattggtga
206920DNAArtificial sequenceSynthetic oligonucleotide 69agaagtccct
cattccttgg 207020DNAArtificial sequenceSynthetic oligonucleotide
70gtccactgga ggagaagtcc 207120DNAArtificial sequenceSynthetic
oligonucleotide 71gtccttcagg tccactggag 207220DNAArtificial
sequenceSynthetic oligonucleotide 72catccctcgt ccttcaggtc
207320DNAArtificial sequenceSynthetic oligonucleotide 73tacatgaaat
cccatccctc 207420DNAArtificial sequenceSynthetic oligonucleotide
74cttggttaca tgaaatccca 207520DNAArtificial sequenceSynthetic
oligonucleotide 75aatactcttg gttacatgaa 207620DNAArtificial
sequenceSynthetic oligonucleotide 76ttagtaaaaa tggaatactc
207720DNAArtificial sequenceSynthetic oligonucleotide 77actgctttag
taaaaatgga 207820DNAArtificial sequenceSynthetic oligonucleotide
78tgaaaacact gctttagtaa 207920DNAArtificial sequenceSynthetic
oligonucleotide 79tatgaggtga aaacactgct 208020DNAArtificial
sequenceSynthetic oligonucleotide 80tagcatatga ggtgaaaaca
208120DNAArtificial sequenceSynthetic oligonucleotide 81ttctaacata
gcatatgagg 208220DNAArtificial sequenceSynthetic oligonucleotide
82tggacttcta acatagcata 208320DNAArtificial sequenceSynthetic
oligonucleotide 83tctctgcctg gacttctaac 208420DNAArtificial
sequenceSynthetic oligonucleotide 84ttattgtctc tgcctggact
208520DNAArtificial sequenceSynthetic oligonucleotide 85cctttcacag
gaatgtttta 208620DNAArtificial sequenceSynthetic oligonucleotide
86tgcctttcac aggaatgttt 208720DNAArtificial sequenceSynthetic
oligonucleotide 87gtgcctttca caggaatgtt 208820DNAArtificial
sequenceSynthetic oligonucleotide 88aagtgccttt cacaggaatg
208920DNAArtificial sequenceSynthetic oligonucleotide 89tgaaaagtgc
ctttcacagg 209020DNAArtificial sequenceSynthetic oligonucleotide
90gcatgcagag gtgagtatac 209120DNAArtificial sequenceSynthetic
oligonucleotide 91agtccactca ttcttggcag 209220DNAArtificial
sequenceSynthetic oligonucleotide 92tgctggactg gtatttgtgt
209320DNAArtificial sequenceSynthetic oligonucleotide 93ctacgggcac
cggtgaatcc 209420DNAArtificial sequenceSynthetic oligonucleotide
94ccggtgaatc caagtgtcct 209520DNAArtificial sequenceSynthetic
oligonucleotide 95gtcaaagttc tagatgctgt 209620DNAArtificial
sequenceSynthetic oligonucleotide 96ttctagatgc tgtccgaggc
209720DNAArtificial sequenceSynthetic oligonucleotide 97ggccgtgcat
gtgttcagaa 209820DNAArtificial sequenceSynthetic oligonucleotide
98tgatgacacc tgggagccat 209920DNAArtificial sequenceSynthetic
oligonucleotide 99ttgcctctgg gaaaaccagt 2010020DNAArtificial
sequenceSynthetic oligonucleotide 100ccagtgagtc tggagagctg
2010120DNAArtificial sequenceSynthetic oligonucleotide
101tgcatgggct cacaactgag 2010220DNAArtificial sequenceSynthetic
oligonucleotide 102actgaggagg aatttgtaga 2010320DNAArtificial
sequenceSynthetic oligonucleotide 103tgtagaaggg atatacaaag
2010420DNAArtificial sequenceSynthetic oligonucleotide
104gaaggcactt ggcatctccc 2010520DNAArtificial sequenceSynthetic
oligonucleotide 105ccattccatg agcatgcaga 2010620DNAArtificial
sequenceSynthetic oligonucleotide 106ctactcctat tccaccacgg
2010720DNAArtificial sequenceSynthetic oligonucleotide
107actcctattc caccacggct 2010820DNAArtificial sequenceSynthetic
oligonucleotide 108ctattccacc acggctgtcg 2010920DNAArtificial
sequenceSynthetic oligonucleotide 109accacggctg tcgtcaccaa
2011020DNAArtificial sequenceSynthetic oligonucleotide
110cacggctgtc gtcaccaatc 2011120DNAArtificial sequenceSynthetic
oligonucleotide 111cggctgtcgt caccaatccc 2011220DNAArtificial
sequenceSynthetic oligonucleotide 112ggctgtcgtc accaatccca
2011320DNAArtificial sequenceSynthetic oligonucleotide
113cgtcaccaat cccaaggaat 2011420DNAArtificial sequenceSynthetic
oligonucleotide 114gtcaccaatc ccaaggaatg 2011520DNAArtificial
sequenceSynthetic oligonucleotide 115ccaaggaatg agggacttct
2011620DNAArtificial sequenceSynthetic oligonucleotide
116ggacttctcc tccagtggac 2011720DNAArtificial sequenceSynthetic
oligonucleotide 117ctccagtgga cctgaaggac 2011820DNAArtificial
sequenceSynthetic oligonucleotide 118gacctgaagg acgagggatg
2011920DNAArtificial sequenceSynthetic oligonucleotide
119gagggatggg atttcatgta 2012020DNAArtificial sequenceSynthetic
oligonucleotide 120tgggatttca tgtaaccaag 2012120DNAArtificial
sequenceSynthetic oligonucleotide 121ttcatgtaac caagagtatt
2012220DNAArtificial sequenceSynthetic oligonucleotide
122tccattttta ctaaagcagt 2012320DNAArtificial sequenceSynthetic
oligonucleotide 123ttactaaagc agtgttttca 2012420DNAArtificial
sequenceSynthetic oligonucleotide 124agcagtgttt tcacctcata
2012520DNAArtificial sequenceSynthetic oligonucleotide
125tgttttcacc tcatatgcta 2012620DNAArtificial sequenceSynthetic
oligonucleotide 126cctcatatgc tatgttagaa 2012720DNAArtificial
sequenceSynthetic oligonucleotide 127tatgctatgt tagaagtcca
2012820DNAArtificial sequenceSynthetic oligonucleotide
128gttagaagtc caggcagaga 2012920DNAArtificial sequenceSynthetic
oligonucleotide 129agtccaggca gagacaataa 2013020DNAArtificial
sequenceSynthetic oligonucleotide 130taaaacattc ctgtgaaagg
2013120DNAArtificial sequenceSynthetic oligonucleotide
131aaacattcct gtgaaaggca 2013220DNAArtificial sequenceSynthetic
oligonucleotide 132aacattcctg tgaaaggcac 2013320DNAArtificial
sequenceSynthetic oligonucleotide 133cattcctgtg aaaggcactt
2013419DNAArtificial sequenceSynthetic oligonucleotide
134cgagaggcgg acgggaccg 1913521DNAArtificial sequenceSynthetic
oligonucleotide 135cgagaggcgg acgggaccgt t 2113621DNAArtificial
sequenceSynthetic oligonucleotide 136cggtcccgtc cgcctctcgt t
2113719DNAArtificial sequenceSynthetic oligonucleotide
137cggtcccgtc cgcctctcg 19
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