U.S. patent application number 15/729860 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 Susan M. Freier, Shuling Guo, Brett P. Monia, Andrew M. Siwkowski.
Application Number | 20180273946 15/729860 |
Document ID | / |
Family ID | 44903998 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180273946 |
Kind Code |
A1 |
Monia; Brett P. ; et
al. |
September 27, 2018 |
MODULATION OF TRANSTHYRETIN EXPRESSION
Abstract
Provided herein are methods, compounds, and compositions for
reducing expression of transthyretin mRNA and protein in an animal.
Such methods, compounds, and compositions are useful to treat,
prevent, delay, or ameliorate transthyretin amyloidosis, or a
symptom thereof.
Inventors: |
Monia; Brett P.; (Encinitas,
CA) ; Freier; Susan M.; (San Diego, CA) ;
Siwkowski; Andrew M.; (Carlsbad, CA) ; Guo;
Shuling; (Carlsbad, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
44903998 |
Appl. No.: |
15/729860 |
Filed: |
October 11, 2017 |
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15190533 |
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9816092 |
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15729860 |
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14717746 |
May 20, 2015 |
9399774 |
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14184984 |
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9061044 |
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14717746 |
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13944786 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/00 20180101; A61P
1/10 20180101; A61P 3/00 20180101; C12N 2310/14 20130101; A61P
15/10 20180101; A61P 15/00 20180101; A61P 25/08 20180101; A61P 1/16
20180101; A61P 1/12 20180101; A61P 13/00 20180101; C12N 15/113
20130101; C12N 2320/30 20130101; A61P 9/04 20180101; A61P 21/00
20180101; A61P 25/00 20180101; A61P 1/08 20180101; A61P 25/22
20180101; A61P 27/02 20180101; C12N 2310/52 20130101; A61P 9/06
20180101; C12N 2310/321 20130101; C12N 2310/3341 20130101; A61K
31/712 20130101; A61P 25/28 20180101; C12N 2310/11 20130101; A61P
7/04 20180101; A61P 7/10 20180101; A61P 25/24 20180101; A61P 25/20
20180101; A61P 19/10 20180101; A61P 25/02 20180101; A61P 11/00
20180101; A61P 9/00 20180101; A61P 3/10 20180101; A61P 25/18
20180101; A61P 27/06 20180101; C12N 2310/315 20130101; C12N
2310/341 20130101; C12N 2310/321 20130101; C12N 2310/3521
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/712 20060101 A61K031/712 |
Claims
1-40. (canceled)
41. A compound consisting of a conjugate group and a sodium salt of
a single-stranded modified oligonucleotide consisting of 20 linked
nucleosides having a nucleobase sequence consisting of SEQ ID NO:
80, wherein the modified oligonucleotide comprises: a gap segment
consisting of ten linked deoxynucleosides; a 5' wing segment
consisting of five linked nucleosides; and a 3' wing segment
consisting of five linked nucleosides; wherein the gap segment is
positioned between the 5' wing segment and the 3' wing segment,
wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar, wherein each internucleoside linkage is a
phosphorothioate linkage, and wherein each cytosine of the modified
oligonucleotide is a 5-methylcytosine.
42. A composition comprising the compound of claim 41.
43. A method of treating transthyretin amyloidosis in an individual
comprising administering to the individual the compound of claim
41.
44. The method of claim 43, wherein the transthyretin amyloidosis
is familial amyloid polyneuropathy (FAP).
45. The method of claim 43, wherein the transthyretin amyloidosis
is familial amyloid cardiopathy (FAC).
46. A method of treating transthyretin amyloidosis in an individual
comprising administering to the individual the composition of claim
42.
47. The method of claim 46, wherein the transthyretin amyloidosis
is familial amyloid polyneuropathy (FAP).
48. The method of claim 46, wherein the transthyretin amyloidosis
is familial amyloid cardiopathy (FAC).
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 BIOL0123USC3SEQ_ST25.txt created Oct. 6, 2017 which
is 64 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] Provided herein are methods, compounds, and compositions for
reducing expression of transthyretin mRNA and protein in an animal.
Such methods, compounds, and compositions are useful, for example,
to treat, prevent, or ameliorate transthyretin amyloidosis.
BACKGROUND OF THE INVENTION
[0003] Transthyretin (TTR), (also known as prealbumin,
hyperthytoxinemia, dysprealbuminemic, thyroxine; senile systemic
amyloidosis, amyloid polyneuropathy, amyloidosis I, PALB;
dystransthyretinemic, HST2651; TBPA; dysprealbuminemic euthyroidal
hyperthyroxinemia) is a serum/plasma and cerebrospinal fluid
protein responsible for the transport of thyroxine and retinol
(Sakaki et al, Mol Biol Med. 1989, 6:161-8). Structurally, TTR is a
homotetramer; point mutations and misfolding of the protein leads
to deposition of amyloid fibrils and is associated with disorders,
such as senile systemic amyloidosis (SSA), familial amyloid
polyneuropathy (FAP), and familial amyloid cardiopathy (FAC).
[0004] TTR is synthesized primarily by the liver and the choroid
plexus of the brain and, to a lesser degree, by the retina in
humans (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300).
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).
[0005] With the availability of genetic and immunohistochemical
diagnostic tests, patients with TTR amyloidosis have been found in
many nations worldwide. Recent studies indicate that TTR
amyloidosis is not a rare endemic disease as previously thought,
and may affect as much as 25% of the elderly population (Tanskanen
et al, Ann Med. 2008; 40(3):232-9).
[0006] At the biochemical level, TTR was identified as the major
protein component in the amyloid deposits of FAP patients (Costa et
al, Proc. Natl. Acad. Sci. USA 1978, 75:4499-4503) and later, a
substitution of methionine for valine at position 30 of the protein
was found to be the most common molecular defect causing the
disease (Saraiva et al, J. Clin. Invest. 1984, 74: 104-119). In
FAP, widespread systemic extracellular deposition of TTR aggregates
and amyloid fibrils occurs throughout the connective tissue,
particularly in the peripheral nervous system (Sousa and Saraiva,
Prog. Neurobiol. 2003, 71: 385-400). Following TTR deposition,
axonal degeneration occurs, starting in the unmyelinated and
myelinated fibers of low diameter, and ultimately leading to
neuronal loss at ganglionic sites.
[0007] The compounds and treatment methods described herein provide
significant advantages over the treatments options currently
available for TTR related disorders. TTR amyloidosis typically lead
to death within ten years, and until recently, was considered
incurable. Liver transplantation is an effective means of replacing
the disease-associated allele by a wild type (WT) allele in
familial cases because the liver is typically the source of
amyloidogenic TTR. While liver transplantation is effective as a
form of gene therapy it is not without its problems.
Transplantation is complicated by the need for invasive surgery for
the recipient and the donor, long-term post-transplantation
immunosuppressive therapy, a shortage of donors, its high cost, and
the large number of TTR amyloidosis patients that are not good
candidates because of their disease progression. Unfortunately,
cardiac amyloidosis progresses in some familial patients even after
liver transplantation because WT TTR often continues to deposit.
Central nervous system (CNS) deposition of TTR is also not relieved
by transplantation owing to its synthesis by the choroid plexus.
Transplantation is not a viable option for the most prevalent TTR
disease, senile systemic amyloidosis (SSA), affecting approximately
25% of those over 80 due to the deposition of WT TTR.
[0008] Antisense technology is emerging as an effective means for
reducing the expression of specific gene products and may therefore
prove to be uniquely useful in a number of therapeutic, diagnostic,
and research applications for the modulation of TTR expression (See
U.S. Patent Publication Nos. 2008/0039418 and 2007/0299027.
[0009] The present invention provides compositions and methods for
modulating transthyretin expression. Antisense compounds for
modulating expression of transthyretin are disclosed in the
aforementioned published patent applications. However, there
remains a need for additional such compounds.
SUMMARY OF THE INVENTION
[0010] Provided herein are methods, compounds, and compositions for
modulating expression of transthyretin (TTR) mRNA and protein. In
certain embodiments, compounds useful for modulating expression of
TTR mRNA and protein are antisense compounds. In certain
embodiments, the antisense compounds are antisense
oligonucleotides.
[0011] In certain embodiments, modulation can occur in a cell or
tissue. In certain embodiments, the cell or tissue is in an animal.
In certain embodiments, the animal is a human. In certain
embodiments, TTR mRNA levels are reduced. In certain embodiments,
TTR protein levels are reduced. Such reduction can occur in a
time-dependent manner or in a dose-dependent manner.
[0012] Provided herein are methods, compounds, and compositions for
modulating expression of transthyretin and treating, preventing,
delaying or ameliorating transthyretin amyloidosis and or a symptom
thereof. In certain embodiments are methods, compounds, and
compositions for modulating expression of transthyretin and
treating, preventing, delaying or ameliorating transthyretin
amyloid disease or transthyretin amyloidosis or transthyretin
related amyloidosis (e.g., hereditary TTR amyloidosis,
leptomeningeal amyloidosis, transthyretin amyloid polyneuropathy,
familial amyloid polyneuropathy, familial amyloid cardiomyopathy,
or senile systemic amyloidosis).
[0013] In certain embodiments, an animal at risk for transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 8 to 80 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 8 to 80 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0014] In certain embodiments, an animal at risk for transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 50 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 50 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0015] In certain embodiments, an animal at risk for transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0016] In certain embodiments, an animal at risk for transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 15 to 25 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 15 to 25 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0017] In certain embodiments, an animal at risk for transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 18 to 21 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 18 to 21 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0018] In certain embodiments, an animal at risk for transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 20 to 30 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 20 to 30 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0019] In certain embodiments, an animal having transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 8 to 80 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2, or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 8 to 80 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0020] In certain embodiments, an animal having transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 50 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2, or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 50 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0021] In certain embodiments, an animal having transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2, or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0022] In certain embodiments, an animal having transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 15 to 25 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 15 to 25 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0023] In certain embodiments, an animal having transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 18 to 21 linked nucleosides,
wherein the modified oligonucleotide is complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 18 to 21 linked nucleosides
and having a nucleobase sequence comprising at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of a
nucleobase sequence selected from any one of nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.
[0024] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 8 to
80 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 8 to
80 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of a nucleobase sequence selected from any
one of nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, or
87.
[0025] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 12 to
50 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 12 to
50 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of a nucleobase sequence selected from any
one of nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, or
87.
[0026] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 12 to
30 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 12 to
30 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of a nucleobase sequence selected from any
one of nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, or
87.
[0027] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 15 to
25 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO:
[0028] 2; or a therapeutically effective amount of a compound
comprising a modified oligonucleotide consisting of 15 to 25 linked
nucleosides and having a nucleobase sequence comprising at least 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous
nucleobases of a nucleobase sequence selected from any one of
nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, or 87.
[0029] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 18 to
21 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 18 to
21 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of a nucleobase sequence selected from any
one of nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, or
87.
[0030] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 20 to
30 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 20 to
30 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of a nucleobase sequence selected from any
one of nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, or
87.
[0031] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 8 to
80 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 8 to
80 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of the nucleobase sequence recited in SEQ ID
NO: 80.
[0032] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 12 to
50 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 12 to
50 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of the nucleobase sequence recited in SEQ ID
NO: 80.
[0033] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 12 to
30 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 12 to
30 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of the nucleobase sequence recited in SEQ ID
NO: 80.
[0034] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 15 to
25 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 15 to
25 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of the nucleobase sequence recited in SEQ ID
NO: 80.
[0035] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 18 to
21 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 18 to
21 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of the nucleobase sequence recited in SEQ ID
NO: 80.
[0036] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 20 to
30 linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 20 to
30 linked nucleosides and having a nucleobase sequence comprising
at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases of the nucleobase sequence recited in SEQ ID
NO: 80.
[0037] In certain embodiments, an animal at risk for transthyretin
amyloidosis or having transthyretin amyloidosis is treated by
administering to the animal a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 20
linked nucleosides, wherein the modified oligonucleotide is
complementary to a transthyretin nucleic acid as shown in SEQ ID
NO: 1 or SEQ ID NO: 2; or a therapeutically effective amount of a
compound comprising a modified oligonucleotide consisting of 20
linked nucleosides and having a nucleobase sequence comprising at
least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
contiguous nucleobases of the nucleobase sequence recited in SEQ ID
NO: 80.
[0038] In certain embodiments, an animal having transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 20 linked nucleosides,
wherein the modified oligonucleotide is 100% complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO:
2; or a therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 20 linked nucleosides and
having the nucleobase sequence recited in SEQ ID NO: 80.
[0039] In certain embodiments, an animal having transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 20 linked nucleosides,
wherein the modified oligonucleotide is 100% complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1; and wherein
the compound comprises a modified oligonucleotide consisting of 20
linked nucleosides having the nucleobase sequence recited in SEQ ID
NO: 80.
[0040] In certain embodiments, an animal having transthyretin
amyloidosis is treated by administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 20 linked nucleosides,
wherein the modified oligonucleotide is 100% complementary to a
transthyretin nucleic acid as shown in SEQ ID NO: 1; wherein the
compound comprises a modified oligonucleotide consisting of 20
linked nucleosides having the nucleobase sequence recited in SEQ ID
NO: 80; and wherein the modified oligonucleotides has a gap segment
of 10 linked deoxynucleosides between two wing segments that
independently have 5 linked modified nucleosides. In certain
embodiments, one or more modified nucleosides in the wing segment
have a modified sugar. In certain embodiments, the modified
nucleoside is a 2'-substituted nucleoside. In certain embodiments,
the modified nucleoside is a 2'-MOE nucleoside.
[0041] In certain embodiments, modulation can occur in a cell,
tissue, organ or organism. In certain embodiments, the cell, tissue
or organ is in an animal. In certain embodiments, the animal is a
human. In certain embodiments, transthyretin mRNA levels are
reduced. In certain embodiments, transthyretin protein levels are
reduced. Such reduction can occur in a time-dependent manner or in
a dose-dependent manner.
[0042] Also provided are methods, compounds, and compositions
useful for preventing, treating, and ameliorating diseases,
disorders, and conditions related to transthyretin amyloidosis. In
certain embodiments, such diseases, disorders, and conditions are
transthyretin amyloidosis related diseases disorders or
conditions.
[0043] In certain embodiments, methods of treatment include
administering a TTR antisense compound to an individual in need
thereof. In certain embodiments, methods of treatment include
administering a TTR antisense oligonucleotide to an individual in
need thereof.
[0044] In certain embodiments, methods of treatment include
administering a transthyretin antisense oligonucleotide and an
additional therapy to an individual in need thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0045] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0046] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference for the portions of the document
discussed herein, as well as in their entirety.
Definitions
[0047] Unless specific definitions are provided, the nomenclature
utilized in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Where permitted, all
patents, applications, published applications and other
publications, GENBANK Accession Numbers and associated sequence
information obtainable through databases such as National Center
for Biotechnology Information (NCBI) and other data referred to
throughout in the disclosure herein are incorporated by reference
for the portions of the document discussed herein, as well as in
their entirety.
[0048] Unless otherwise indicated, the following terms have the
following meanings: "2'-O-methoxyethyl" (also 2'-MOE and
2'-O(CH.sub.2).sub.2--OCH.sub.3) refers to an O-methoxy-ethyl
modification of the 2' position of a furosyl ring. A
2'-O-methoxyethyl modified sugar is a modified sugar.
[0049] "2'-O-methoxyethyl nucleotide" means a nucleotide comprising
a 2'-O-methoxyethyl modified sugar moiety.
[0050] "5-methylcytosine" means a cytosine modified with a methyl
group attached to the 5' position. A 5-methylcytosine is a modified
nucleobase.
[0051] "Active pharmaceutical agent" means the substance or
substances in a pharmaceutical composition that provide a
therapeutic benefit when administered to an individual. For
example, in certain embodiments an antisense oligonucleotide
targeted to transthyretin is an active pharmaceutical agent.
[0052] "Active target region" or "target region" means a region to
which one or more active antisense compounds is targeted. "Active
antisense compounds" means antisense compounds that reduce target
nucleic acid levels or protein levels.
[0053] "Administered concomitantly" refers to the co-administration
of two agents in any manner in which the pharmacological effects of
both are manifest in the patient at the same time. Concomitant
administration does not require that both agents be administered in
a single pharmaceutical composition, in the same dosage form, or by
the same route of administration. The effects of both agents need
not manifest themselves at the same time. The effects need only be
overlapping for a period of time and need not be coextensive.
[0054] "Administering" means providing a pharmaceutical agent to an
individual, and includes, but is not limited to administering by a
medical professional and self-administering.
[0055] "Amelioration" refers to a lessening of at least one
indicator, sign, or symptom of an associated disease, disorder, or
condition. The severity of indicators may be determined by
subjective or objective measures, which are known to those skilled
in the art.
[0056] "Amyloidosis" is a group of diseases or disorders resulting
from abnormal protein (amyloid or amyloid fibril) deposits in
various body tissues. The amyloid proteins may either be deposited
in one particular area of the body (localized amyloidosis) or they
may be deposited throughout the body (systemic amyloidosis). There
are three types of systemic amyloidosis: primary (AL), secondary
(AA), and familial (ATTR). Primary amyloidosis is not associated
with any other diseases and is considered a disease entity of its
own. Secondary amyloidosis occurs as a result of another illness.
Familial Mediterranean Fever is a form of familial (inherited)
amyloidosis.
[0057] "Animal" refers to a human or non-human animal, including,
but not limited to, mice, rats, rabbits, dogs, cats, pigs, and
non-human primates, including, but not limited to, monkeys and
chimpanzees.
[0058] "Antisense activity" means any detectable or measurable
activity attributable to the hybridization of an antisense compound
to its target nucleic acid. In certain embodiments, antisense
activity is a decrease in the amount or expression of a target
nucleic acid or protein encoded by such target nucleic acid.
[0059] "Antisense compound" means an oligomeric compound that is
capable of undergoing hybridization to a target nucleic acid
through hydrogen bonding.
[0060] "Antisense inhibition" means reduction of target nucleic
acid levels or target protein levels in the presence of an
antisense compound complementary to a target nucleic acid compared
to target nucleic acid levels or target protein levels in the
absence of the antisense compound.
[0061] "Antisense oligonucleotide" means a single-stranded
oligonucleotide having a nucleobase sequence that permits
hybridization to a corresponding region or segment of a target
nucleic acid.
[0062] "Bicyclic sugar" means a furosyl ring modified by the
bridging of two non-geminal ring atoms. A bicyclic sugar is a
modified sugar.
[0063] "Bicyclic nucleic acid" or "BNA" refers to a nucleoside or
nucleotide wherein the furanose portion of the nucleoside or
nucleotide includes a bridge connecting two carbon atoms on the
furanose ring, thereby forming a bicyclic ring system.
[0064] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
[0065] "Central nervous system (CNS)" refers to the vertebrate
nervous system which is enclosed in meninges. It contains the
majority of the nervous system, and consists of the brain (in
vertebrates which have brains), and the spinal cord. The CNS is
contained within the dorsal cavity, with the brain within the
cranial cavity, and the spinal cord in the spinal cavity. The brain
is also protected by the skull, and the spinal cord is, in
vertebrates, also protected by the vertebrae.
[0066] "Chemically distinct region" refers to a region of an
antisense compound that is in some way chemically different than
another region of the same antisense compound. For example, a
region having 2'-O-methoxyethyl nucleotides is chemically distinct
from a region having nucleotides without 2'-O-- methoxyethyl
modifications.
[0067] "Chimeric antisense compound" means an antisense compound
that has at least two chemically distinct regions.
[0068] "Choroid plexus" is the area on the ventricles of the brain
where cerebrospinal fluid (CSF) is produced.
[0069] "Co-administration" means administration of two or more
pharmaceutical agents to an individual. The two or more
pharmaceutical agents may be in a single pharmaceutical
composition, or may be in separate pharmaceutical compositions.
Each of the two or more pharmaceutical agents may be administered
through the same or different routes of administration.
Co-administration encompasses parallel or sequential
administration.
[0070] "Complementarity" means the capacity for pairing between
nucleobases of a first nucleic acid and a second nucleic acid.
[0071] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other.
[0072] "Diluent" means an ingredient in a composition that lacks
pharmacological activity, but is pharmaceutically necessary or
desirable. For example, the diluent in an injected composition may
be a liquid, e.g. saline solution.
[0073] "Dose" means a specified quantity of a pharmaceutical agent
provided in a single administration, or in a specified time period.
In certain embodiments, a dose may be administered in one, two, or
more boluses, tablets, or injections. For example, in certain
embodiments where subcutaneous administration is desired, the
desired dose requires a volume not easily accommodated by a single
injection, therefore, two or more injections may be used to achieve
the desired dose. In certain embodiments, the pharmaceutical agent
is administered by infusion over an extended period of time or
continuously. Doses may be stated as the amount of pharmaceutical
agent per hour, day, week, or month.
[0074] "Effective amount" means the amount of active pharmaceutical
agent sufficient to effectuate a desired physiological outcome in
an individual in need of the agent. The effective amount may vary
among individuals depending on the health and physical condition of
the individual to be treated, the taxonomic group of the
individuals to be treated, the formulation of the composition,
assessment of the individual's medical condition, and other
relevant factors.
[0075] "Familial amyloidosis" or "hereditary amyloidosis" is a form
of inherited amyloidosis.
[0076] "Familial amyloid polyneuropathy" or "FAP" is a
neurodegenerative genetically transmitted disorder, characterized
by systemic depositions of amyloid variants of transthyretin
proteins, causing progressive sensory and motorial
polyneuropathy.
[0077] "Fully complementary" or "100% complementary" means each
nucleobase of a nucleobase sequence of a first nucleic acid has a
complementary nucleobase in a second nucleobase sequence of a
second nucleic acid. In certain embodiments, a first nucleic acid
is an antisense compound and a target nucleic acid is a second
nucleic acid.
[0078] "Gapmer" means a chimeric antisense compound in which an
internal region having a plurality of nucleosides that support
RNase H cleavage is positioned between external regions having one
or more nucleosides, wherein the nucleosides comprising the
internal region are chemically distinct from the nucleoside or
nucleosides comprising the external regions. The internal region
may be referred to as a "gap segment" and the external regions may
be referred to as "wing segments."
[0079] "Gap-widened" means a chimeric antisense compound having a
gap segment of 12 or more contiguous 2'-deoxyribonucleosides
positioned between and immediately adjacent to 5' and 3' wing
segments having from one to six nucleosides.
[0080] "Hereditary transthyretin (TTR) amyloidosis" is a systemic
disease caused by mutations in transthyretin, a plasma transport
protein for thyroxine and vitamin A. It is most frequently
associated with peripheral neuropathy and restrictive
cardiomyopathy, but amyloid deposits in blood vessel walls and
connective tissue structures throughout the body often cause
dysfunction of other organ systems. Gastrointestinal motility
abnormalities are common in this disease with constipation,
diarrhea and early satiety from delayed gastric-emptying.
Connective tissue deposits of amyloid in the wrist may cause carpal
tunnel syndrome. Amyloid deposits in spinal blood vessels and
surrounding structures cause spinal stenosis with symptoms of
claudication.
[0081] "Hybridization" means the annealing of complementary nucleic
acid molecules. In certain embodiments, complementary nucleic acid
molecules include an antisense compound and a target nucleic
acid.
[0082] "Immediately adjacent" means there are no intervening
elements between the immediately adjacent elements.
[0083] "Individual" means a human or non-human animal selected for
treatment or therapy.
[0084] "Intracerebroventricular administration" or "cerebral
intraventricular administration" or "cerebral ventricular
administration" means administration through injection or infusion
into the ventricular system of the brain.
[0085] "Intraperitoneal administration" means administration to the
peritoneal cavity.
[0086] "Intrathecal administration" means administration through
injection or infusion into the cerebrospinal fluid bathing the
spinal cord and brain.
[0087] "Intravenous administration" means administration into a
vein.
[0088] "Intraventricular administration" means administration into
the ventricles of either the brain or heart.
[0089] "Internucleoside linkage" refers to the chemical bond
between nucleosides.
[0090] "Leptomeningeal" means having to do with the leptomeninges,
the two innermost layers of tissues that cover the brain and spinal
cord. "Leptomeningeal amyloidosis" refers to amyloidosis of the
leptomeninges resulting from transthyretin amyloid deposition
within the leptomeninges.
[0091] "Linked nucleosides" means adjacent nucleosides which are
bonded together.
[0092] "Mismatch" or "non-complementary nucleobase" refers to the
case when a nucleobase of a first nucleic acid is not capable of
pairing with the corresponding nucleobase of a second or target
nucleic acid.
[0093] "Modified internucleoside linkage" refers to a substitution
or any change from a naturally occurring internucleoside bond (i.e.
a phosphodiester internucleoside bond).
[0094] "Modified nucleobase" refers to any nucleobase other than
adenine, cytosine, guanine, thymidine, or uracil. An "unmodified
nucleobase" means the purine bases adenine (A) and guanine (G), and
the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
[0095] "Modified nucleotide" means a nucleotide having,
independently, a modified sugar moiety, modified internucleoside
linkage, or modified nucleobase. A "modified nucleoside" means a
nucleoside having, independently, a modified sugar moiety or
modified nucleobase.
[0096] "Modified oligonucleotide" means an oligonucleotide
comprising at least one modified nucleotide.
[0097] "Modified sugar" refers to a substitution or change from a
natural sugar.
[0098] "Motif" means the pattern of chemically distinct regions in
an antisense compound.
[0099] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0100] "Natural sugar moiety" means a sugar found in DNA (2'-H) or
RNA (2'-OH).
[0101] "Nucleic acid" refers to molecules composed of monomeric
nucleotides. A nucleic acid includes ribonucleic acids (RNA),
deoxyribonucleic acids (DNA), single-stranded nucleic acids,
double-stranded nucleic acids, small interfering ribonucleic acids
(siRNA), and microRNAs (miRNA). A nucleic acid may also comprise a
combination of these elements in a single molecule.
[0102] "Nucleobase" means a heterocyclic moiety capable of pairing
with a base of another nucleic acid.
[0103] "Nucleobase sequence" means the order of contiguous
nucleobases independent of any sugar, linkage, or nucleobase
modification.
[0104] "Nucleoside" means a nucleobase linked to a sugar.
[0105] "Nucleotide" means a nucleoside having a phosphate group
covalently linked to the sugar portion of the nucleoside.
[0106] "Oligomeric compound" or "oligomer" means a polymer of
linked monomeric subunits which is capable of hybridizing to at
least a region of a nucleic acid molecule.
[0107] "Oligonucleotide" means a polymer of linked nucleosides each
of which can be modified or unmodified, independent one from
another.
[0108] "Parenteral administration" means administration through
injection or infusion. Parenteral administration includes
subcutaneous administration, intravenous administration,
intramuscular administration, intraarterial administration,
intraperitoneal administration, or intracranial administration,
e.g.
[0109] intracerebral administration, intrathecal administration,
intraventricular administration, ventricular administration,
intracerebroventricular administration, cerebral intraventricular
administration or cerebral ventricular administration.
Administration can be continuous, or chronic, or short or
intermittent.
[0110] "Peptide" means a molecule formed by linking at least two
amino acids by amide bonds. Peptide refers to polypeptides and
proteins.
[0111] "Pharmaceutical composition" means a mixture of substances
suitable for administering to an individual. For example, a
pharmaceutical composition may comprise one or more active
pharmaceutical agents and a sterile aqueous solution.
[0112] "Pharmaceutically acceptable salts" means physiologically
and pharmaceutically acceptable salts of antisense compounds, i.e.,
salts that retain the desired biological activity of the parent
oligonucleotide and do not impart undesired toxicological effects
thereto.
[0113] "Phosphorothioate linkage" means a linkage between
nucleosides where the phosphodiester bond is modified by replacing
one of the non-bridging oxygen atoms with a sulfur atom. A
phosphorothioate linkage is a modified internucleoside linkage.
[0114] "Portion" means a defined number of contiguous (i.e. linked)
nucleobases of a nucleic acid. In certain embodiments, a portion is
a defined number of contiguous nucleobases of a target nucleic
acid. In certain embodiments, a portion is a defined number of
contiguous nucleobases of an antisense compound.
[0115] "Prevent" refers to delaying or forestalling the onset or
development of a disease, disorder, or condition for a period of
time from minutes to indefinitely. Prevent also means reducing risk
of developing a disease, disorder, or condition.
[0116] "Prodrug" means a therapeutic agent that is prepared in an
inactive form that is converted to an active form within the body
or cells thereof by the action of endogenous enzymes or other
chemicals or conditions.
[0117] "Side effects" means physiological responses attributable to
a treatment other than the desired effects. In certain embodiments,
side effects include injection site reactions, liver function test
abnormalities, renal function abnormalities, liver toxicity, renal
toxicity, central nervous system abnormalities, myopathies, and
malaise. For example, increased aminotransferase levels in serum
may indicate liver toxicity or liver function abnormality. For
example, increased bilirubin may indicate liver toxicity or liver
function abnormality.
[0118] "Single-stranded oligonucleotide" means an oligonucleotide
which is not hybridized to a complementary strand.
[0119] "Specifically hybridizable" refers to an antisense compound
having a sufficient degree of complementarity between an antisense
oligonucleotide and a target nucleic acid to induce a desired
effect, while exhibiting minimal or no effects on non-target
nucleic acids under conditions in which specific binding is
desired, i.e. under physiological conditions in the case of in vivo
assays and therapeutic treatments.
[0120] "Subcutaneous administration" means administration just
below the skin.
[0121] "Targeting" or "targeted" means the process of design and
selection of an antisense compound that will specifically hybridize
to a target nucleic acid and induce a desired effect.
[0122] "Target nucleic acid," "target RNA," and "target RNA
transcript" all refer to a nucleic acid capable of being targeted
by antisense compounds.
[0123] "Target segment" means the sequence of nucleotides of a
target nucleic acid to which an antisense compound is targeted. "5'
target site" refers to the 5'-most nucleotide of a target segment.
"3' target site" refers to the 3'-most nucleotide of a target
segment.
[0124] "Therapeutically effective amount" means an amount of a
pharmaceutical agent that provides a therapeutic benefit to an
individual.
[0125] "Transthyretin-specific inhibitor" or "Transthyretin
inhibitor" means any compound capable of decreasing transthyretin
mRNA or protein expression. Examples of such compounds include a
nucleic acid, a peptide, an antibody, or a histone deacetylase
inhibitor.
[0126] "Transthyretin specific modulator" or "transthyretin
modulator" means any compound capable of increasing or decreasing
transthyretin mRNA or protein expression.
[0127] "Transthyretin-related amyloidosis" or "transthyretin
amyloidosis" or "Transthyretin amyloid disease", as used herein, is
any pathology or disease associated with dysfunction or
dysregulation of transthyretin that result in formation of
transthyretin-containing amyloid fibrils. Transthyretin amyloidosis
includes, but is not limited to, hereditary TTR amyloidosis,
leptomeningeal amyloidosis, familial amyloid polyneuropathy (FAP),
familial amyloid cardiomyopathy, familial oculoleptomeningeal
amyloidosis, senile cardiac amyloidosis, or senile systemic
amyloidosis.
[0128] "Treat" refers to administering a pharmaceutical composition
to effect an alteration or improvement of a disease, disorder, or
condition.
[0129] "Unmodified nucleotide" means a nucleotide composed of
naturally occurring nucleobases, sugar moieties, and
internucleoside linkages. In certain embodiments, an unmodified
nucleotide is an RNA nucleotide (i.e. .beta.-D-ribonucleosides) or
a DNA nucleotide (i.e. .beta.-D-deoxyribonucleoside).
Certain Embodiments
[0130] Certain embodiments provide methods, compounds, and
compositions for inhibiting transthyretin expression.
[0131] Certain embodiments provide antisense compounds targeted to
a transthyretin nucleic acid. In certain embodiments, the
transthyretin nucleic acid is any of the sequences set forth in
GENBANK Accession No. NM 000371.2 (incorporated herein as SEQ ID
NO: 1), GENBANK Accession No. NT_010966.10 truncated from
nucleotides 2009236 to U.S. Pat. No. 2,017,289 (incorporated herein
as SEQ ID NO: 2); exons 1-4 extracted from the rhesus monkey
genomic sequence GENBANK Accession No. NW_001105671.1, based on
similarity to human exons; and GENBANK Accession No. NW_001105671.1
truncated from nucleotides 628000 to 638000 (incorporated herein as
SEQ ID NO: 4).
[0132] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 8 to 80 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 contiguous nucleobases of a sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, 87,
115, 120, 122, and 124.
[0133] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 50 linked nucleosides wherein
the linked nucleosides comprise at least 8, at least 9, at least
10, at least 11, at least 12, at least 13, at least 14, at least
15, at least 16, at least 17, at least 18, at least 19, or at least
20 contiguous nucleobases of a sequence selected from among the
nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, 87, 115,
120, 122, and 124. In certain embodiments, the modified
oligonucleotide comprises at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, or at least 20 contiguous
nucleobases of a sequence selected from among the nucleobase
sequences recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and
124.
[0134] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 contiguous nucleobases of a sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, 87,
115, 120, 122, and 124.
[0135] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 15 to 25 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124.
[0136] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 18 to 21 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 contiguous nucleobases of a sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, 87,
115, 120, 122, and 124.
[0137] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 8 to 80 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, and 87. In certain embodiments, the
modified oligonucleotide comprises at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19, or at least 20
contiguous nucleobases of a sequence selected from among the
nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, and 87.
[0138] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 50 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, and 87. In certain embodiments, the
modified oligonucleotide comprises at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19, or at least 20
contiguous nucleobases of a sequence selected from among the
nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, and 87.
[0139] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, and 87. In certain embodiments, the
modified oligonucleotide comprises at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19, or at least 20
contiguous nucleobases of a sequence selected from among the
nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, and 87.
[0140] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 15 to 25 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, and 87. In certain embodiments, the
modified oligonucleotide comprises at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19, or at least 20
contiguous nucleobases of a sequence selected from among the
nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, and 87.
[0141] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 18 to 21 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of a sequence selected from among the nucleobase sequences recited
in SEQ ID NOs: 25, 80, 86, and 87. In certain embodiments, the
modified oligonucleotide comprises at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19 or at least 20
contiguous nucleobases of a sequence selected from among the
nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, and 87.
[0142] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 8 to 80 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of the nucleobase sequence recited in SEQ ID NO: 80. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 contiguous nucleobases of a sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 80.
[0143] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 50 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of the nucleobase sequence recited in SEQ ID NO: 80. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 contiguous nucleobases of a sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 80.
[0144] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of the nucleobase sequence recited in SEQ ID NO: 80. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 contiguous nucleobases of a sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 80.
[0145] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 15 to 25 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of the nucleobase sequence recited in SEQ ID NO: 80. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 contiguous nucleobases of a sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 80.
[0146] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 18 to 21 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of the nucleobase sequence recited in SEQ ID NO: 80. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, or at
least 20 contiguous nucleobases of a sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 80.
[0147] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 to 30 linked nucleosides wherein
the linked nucleosides comprise at least 8 contiguous nucleobases
of the nucleobase sequence recited in SEQ ID NO: 80. In certain
embodiments, the modified oligonucleotide comprises at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, or at least 19
contiguous nucleobases of the nucleobase sequence recited in SEQ ID
NO: 80.
[0148] In certain embodiments, the compound comprises a modified
oligonucleotide consisting of 20 linked nucleosides recited in SEQ
ID NO: 80.
[0149] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides wherein
the linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 120-139, 212-236,
226-245, 293-468, 293-326, 347-381, 425-468, 425-467, 452-478,
452-474, 459-478, 461-519, 462-500, 500-519, 501-535, 502-531,
505-524, 507-526, 508-527, 514-540, 514-539, 515-534, 516-535,
523-542, 544-606, 544-564, 564-583, 578-601, 580-608, 580-599,
584-606, 585-604, 587-606, or 597-617 of SEQ ID NO: 1. In certain
embodiments the region is selected from 507-526, 508-527, 515-534,
516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1. In
certain embodiments the region is selected from 501-535 or 580-608
of SEQ ID NO: 1. In certain embodiments, the modified
oligonucleotide has at least a 9, at least a 10, at least an 11, at
least an 12, at least an 13, at least an 14, at least an 15, at
least an 16, at least an 17, at least an 18, at least an 19 or at
least a 20 contiguous nucleobase portion of which is complementary
within a region described herein.
[0150] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides wherein
the linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 501-535 or 580-608 of
SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide
has at least a 9, at least a 10, at least an 11, at least an 12, at
least an 13, at least an 14, at least an 15, at least an 16, at
least an 17, at least an 18, at least an 19 or at least a 20
contiguous nucleobase portion of which is complementary within a
region described herein.
[0151] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides wherein
the linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 508-527 of SEQ ID NO:
1. In certain embodiments, the modified oligonucleotide has at
least a 9, at least a 10, at least an 11, at least an 12, at least
an 13, at least an 14, at least an 15, at least an 16, at least an
17, at least an 18, at least an 19 or at least a 20 contiguous
nucleobase portion of which is complementary within a region
described herein.
[0152] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 15 to 25 linked nucleosides wherein
the linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 507-526, 508-527,
515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID
NO: 1. In certain embodiments, the modified oligonucleotide has at
least a 9, at least a 10, at least an 11, at least an 12, at least
an 13, at least an 14, at least an 15, at least an 16, at least an
17, at least an 18, at least an 19 or at least a 20 contiguous
nucleobase portion of which is complementary within a region
described herein.
[0153] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 18 to 21 linked nucleosides wherein
the linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 507-526, 508-527,
515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID
NO: 1. In certain embodiments, the modified oligonucleotide has at
least a 9, at least a 10, at least an 11, at least an 12, at least
an 13, at least an 14, at least an 15, at least an 16, at least an
17, at least an 18, at least an 19 or at least a 20 contiguous
nucleobase portion of which is complementary within a region
described herein.
[0154] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides wherein the
linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 507-526, 508-527,
515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID
NO: 1. In certain embodiments, the modified oligonucleotide has at
least a 9, at least a 10, at least an 11, at least a 12, at least a
13, at least a 14, at least a 15, at least a 16, at least a 17, at
least an 18, at least 19 or at least a 20 contiguous nucleobase
portion of which is complementary within a region described herein.
In certain embodiments, the modified oligonucleotide is 90%, 95%,
99%, or 100% complementary to a nucleic acid encoding human
transthyretin (TTR), eg. SEQ ID No: 1.
[0155] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides wherein the
linked nucleosides comprise at least an 8 contiguous nucleobase
portion that is complementary to an equal length nucleobase portion
within the region selected from nucleotides 508-527 of SEQ ID NO:
1. In certain embodiments, the modified oligonucleotide has at
least a 9, at least a 10, at least an 11, at least a 12, at least a
13, at least a 14, at least a 15, at least a 16, at least a 17, at
least an 18, at least 19 or at least a 20 contiguous nucleobase
portion of which is complementary to an equal length portion within
the region selected from nucleotides 508-527 of SEQ ID NO: 1. In
certain embodiments, the modified oligonucleotide is 90%, 95%, 99%,
or 100% complementary to a nucleic acid encoding human
transthyretin (TTR), eg. SEQ ID No: 1
[0156] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 60%
complementary within the region selected from nucleotides 507-526,
508-527, 515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of
SEQ ID NO: 1.
[0157] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 70%
complementary within the region selected from nucleotides 507-526,
508-527, 515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of
SEQ ID NO: 1.
[0158] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 80%
complementary within the region selected from nucleotides 507-526,
508-527, 515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of
SEQ ID NO: 1.
[0159] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 90%
complementary within the region selected from nucleotides 507-526,
508-527, 515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of
SEQ ID NO: 1.
[0160] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 95%
complementary within the region selected from nucleotides 507-526,
508-527, 515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of
SEQ ID NO: 1.
[0161] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within the region selected from nucleotides 507-526,
508-527, 515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of
SEQ ID NO: 1.
[0162] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 100%
complementary within the region selected from nucleotides 507-526,
508-527, 515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of
SEQ ID NO: 1.
[0163] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 60%
complementary within nucleotides 508-527 of SEQ ID NO: 1.
[0164] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 70%
complementary within nucleotides 508-527 of SEQ ID NO: 1.
[0165] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 80%
complementary within nucleotides 508-527 of SEQ ID NO: 1.
[0166] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 90%
complementary within nucleotides 508-527 of SEQ ID NO: 1.
[0167] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 95%
complementary within nucleotides 508-527 of SEQ ID NO: 1.
[0168] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 99%
complementary within nucleotides 508-527 of SEQ ID NO: 1.
[0169] Certain embodiments provide compounds comprising a modified
oligonucleotide consisting of 20 linked nucleosides 100%
complementary within nucleotides 508-527 of SEQ ID NO: 1.
[0170] In certain embodiments, an antisense compound or modified
oligonucleotide targeted to a transthyretin nucleic acid targets
the following nucleotide regions of SEQ ID NO: 1: 120-139, 212-236,
226-245, 293-468, 293-326, 347-381, 425-468, 425-467, 452-478,
452-474, 459-478, 461-519, 462-500, 500-519, 502-531, 507-526,
505-524, 508-527, 514-540, 514-539, 515-534, 516-535, 523-542,
544-606, 544-564, 564-583, 578-601, 580-599, 584-606, 585-604,
587-606, or 597-617.
[0171] In certain embodiments, antisense compounds or modified
oligonucleotides targets a region of a transthyretin nucleic acid.
In certain embodiments, such compounds or oligonucleotides targeted
to a region of a transthyretin nucleic acid have a contiguous
nucleobase portion that is complementary to an equal length
nucleobase portion of the region. For example, the portion can be
at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
contiguous nucleobases portion complementary to an equal length
portion of a region recited herein. In certain embodiments, such
compounds or oligonucleotide target the following nucleotide
regions of SEQ ID NO: 1: 120-139, 212-236, 226-245, 293-381,
293-366, 353-381, 293-468, 425-468, 425-467, 452-476, 461-481,
461-500, 500-519, 461-519, 502-531, 502-539, 504-536, 505-525,
506-530, 507-527, 508-527, 508-536, 514-540, 523-542, 544-606,
544-564, 544-583, or 597-617.
[0172] In certain embodiments, such compounds or oligonucleotides
targeted to a region of a transthyretin nucleic acid have a
contiguous nucleobase portion that is complementary to an equal
length nucleobase portion of the region 501-535 or 580-608 of SEQ
ID NO: 1.
[0173] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 60% inhibition: 226-245,
293-366, 357-467, 452-474, 457-476, 459-478, 462-500, 500-519,
502-531, 504-536, 505-525, 506-530, 507-527, 508-527, 508-536,
514-539, 544-564, 564-583, 578-601, 584-606, or 597-617.
[0174] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 65% inhibition: 293-366,
357-376, 425-449, 432-467, 452-474, 459-478, 462-500, 500-519,
502-531, 504-536, 505-525, 506-530, 507-527, 508-527, 508-536,
514-539, 544-563, 564-583, 578-601,585-606, or 597-617.
[0175] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 70% inhibition: 293-366,
425-449, 432-467, 452-474, 459-478, 462-500, 500-519, 502-531,
504-536, 505-525, 506-530, 507-527, 508-527, 508-536, 514-539,
564-583, 578-598, 581-600, or 597-617.
[0176] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 75% inhibition: 293-322,
347-366, 425-449, 432-467, 452-474, 459-478, 462-500, 500-519,
503-531, 504-536, 505-525, 506-530, 507-527, 508-527, 508-536,
514-539, 578-598, 581-600, or 597-616.
[0177] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 80% inhibition: 303-322,
425-449, 432-460, 443-467, 452-473, 481-500, 500-519, 503-531,
504-536, 505-525, 506-530, 507-527, 508-527, 508-536, 514-536,
519-539, 579-598, 581-600, or 597-616.
[0178] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 85% inhibition: 427-449,
432-458, 441-460, 443-467, 452-473, 504-531, 504-536, 505-525,
506-530, 507-527, 508-527, 508-536, 514-536, 519-539, or
581-600.
[0179] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 90% inhibition: 428-449,
432-456, 439-458, 441-460, 445-466, 452-473, 504-525, 508-527, or
515-536.
[0180] In certain embodiments, the following nucleotide regions of
SEQ ID NO: 1, when targeted by antisense compounds or
oligonucleotides, displays at least 95% inhibition: 434-453,
436-456, 441-460, 445-465, 505-524, or 516-535.
[0181] In certain embodiments, the following antisense compounds
target a region of a SEQ ID NO: 1, a nucleic acid encoding human
transthyretin, and demonstrate at least 60% inhibition of a
transthyretin mRNA: ISIS NOs:420954, 420904, 304286, 420874,
420948, 420883, 420955, 420952, 420956, 420957, 420882, 420947,
420950, 304312, 304307, 420879, 420910, 420902, 420908, 420924,
420877, 420880, 304309, 304289, 420906, 304311, 420878, 420911,
304284, 304288, 420909, 304296, 420949, 304290, 304299, 420898,
420920, 420925, 420951, 304287, 420894, 420916, 420918, 420926,
304285, 420919, 420923, 420886, 420900, 420912, 420915, 420917,
420921, 420884, 420885, 420887, 420889, 420892, 420901, 420914,
420897, 420899, 420888, 420895, 420896, 420913, 420922, 420893,
420890, or 420891.
[0182] In certain embodiments, the following antisense compounds
target a region of a SEQ ID NO: 1, a nucleic acid encoding human
transthyretin and demonstrate at least 65% inhibition of a
transthyretin mRNA: ISIS NOs: 420955, 420952, 420956, 420957,
420882, 420947, 420950, 304312, 304307, 420879, 420910, 420902,
420908, 420924, 420877, 420880, 304309, 304289, 420906, 304311,
420878, 420911, 304284, 304288, 420909, 304296, 420949, 304290,
304299, 420898, 420920, 420925, 420951, 304287, 420894, 420916,
420918, 420926, 304285, 420919, 420923, 420886, 420900, 420912,
420915, 420917, 420921, 420884, 420885, 420887, 420889, 420892,
420901, 420914, 420897, 420899, 420888, 420895, 420896, 420913,
420922, 420893, 420890, or 420891.
[0183] In certain embodiments, the following antisense compounds
target a region of a SEQ ID NO: 1, a nucleic acid encoding human
transthyretin and demonstrate at least 70% inhibition of a
transthyretin mRNA: ISIS NOs: 304312, 304307, 420879, 420910,
420902, 420908, 420924, 420877, 420880, 304309, 304289, 420906,
304311, 420878, 420911, 304284, 304288, 420909, 304296, 420949,
304290, 304299, 420898, 420920, 420925, 420951, 304287, 420894,
420916, 420918, 420926, 304285, 420919, 420923, 420886, 420900,
420912, 420915, 420917, 420921, 420884, 420885, 420887, 420889,
420892, 420901, 420914, 420897, 420899, 420888, 420895, 420896,
420913, 420922, 420893, 420890, or 420891.
[0184] In certain embodiments, the following antisense compounds
target a region of a SEQ ID NO: 1, a nucleic acid encoding human
transthyretin and demonstrate at least 75% inhibition of a
transthyretin mRNA: ISIS NOs: 420877, 420878, 420880, 304284,
304285, 420884, 420885, 420886, 420887, 420888, 420889, 420890,
420891, 304287, 420892, 304288, 420893, 304289, 304290, 420894,
420895, 420896, 420897, 420898, 420899, 420900, 420901, 420902,
420906, 420908, 304296, 420909, 420911, 420912, 420913, 420914,
304299, 420915, 420916, 420917, 420918, 420919, 420920, 420921,
420922, 420923, 420924, 420925, 420926, 304309, 420949, 420951, or
304311.
[0185] In certain embodiments, the following antisense compounds
target a region of a SEQ ID NO: 1, a nucleic acid encoding human
transthyretin and demonstrate at least 80% inhibition of a
transthyretin mRNA: ISIS NOs: 304311, 420878, 420911, 304284,
304288, 420909, 304296, 420949, 304290, 304299, 420898, 420920,
420925, 420951, 304287, 420894, 420916, 420918, 420926, 304285,
420919, 420923, 420886, 420900, 420912, 420915, 420917, 420921,
420884, 420885, 420887, 420889, 420892, 420901, 420914, 420897,
420899, 420888, 420895, 420896, 420913, 420922, 420893, 420890, or
42089.
[0186] In certain embodiments, the following antisense compounds
target a region of a SEQ ID NO: 1, a nucleic acid encoding human
transthyretin and demonstrate at least 85% inhibition of a
transthyretin mRNA: ISIS NOs: 304290, 304299, 420898, 420920,
420925, 420951, 304287, 420894, 420916, 420918, 420926, 304285,
420919, 420923, 420886, 420900, 420912, 420915, 420917, 420921,
420884, 420885, 420887, 420889, 420892, 420901, 420914, 420897,
420899, 420888, 420895, 420896, 420913, 420922, 420893, 420890, or
420891.
[0187] In certain embodiments, the following antisense compounds
target a region of a SEQ ID NO: 1, a nucleic acid encoding human
transthyretin and demonstrate at least 90% inhibition of a
transthyretin mRNA: ISIS NOs: 420923, 420886, 420900, 420912,
420915, 420917, 420921, 420884, 420885, 420887, 420889, 420892,
420901, 420914, 420897, 420899, 420888, 420895, 420896, 420913,
420922, 420893, 420890, or 420891.
[0188] In certain embodiments, the following antisense compounds
target a region of a SEQ ID NO: 1, a nucleic acid encoding human
transthyretin and demonstrate at least 95% inhibition of a
transthyretin mRNA: ISIS NOs: 420888, 420895, 420896, 420913,
420922, 420893, 420890, or 420891.
[0189] In certain embodiments, a target region is nucleotides
120-139 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 120-139 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NO: 37. In certain such embodiments, an antisense
compound targeted to nucleotides 120-139 of SEQ ID NO: 1 is
selected from ISIS NO: 420872.
[0190] In certain embodiments, a target region is nucleotides
212-236 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 212-236 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 8 and 38. In certain such embodiments, an
antisense compound targeted to nucleotides 212-236 of SEQ ID NO: 1
is selected from ISIS NOs: 420873 or 304267.
[0191] In certain embodiments, a target region is nucleotides
226-245 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 226-245 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NO: 39. In certain such embodiments, an antisense
compound targeted to nucleotides 226-245 of SEQ ID NO: 1 is
selected from ISIS NO: 420874.
[0192] In certain embodiments, a target region is nucleotides
293-381 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 293-381 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 10, 42-48. In certain such embodiments, an
antisense compound targeted to nucleotides 293-381 of SEQ ID NO: 1
is selected from ISIS NOs: 420877, 420878, 420879, 420880, 304280,
420881, 420882, or 420883.
[0193] In certain embodiments, a target region is nucleotides
293-366 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 293-366 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 42-45. In certain such embodiments, an antisense
compound targeted to nucleotides 293-366 of SEQ ID NO: 1 is
selected from ISIS NOs: 420877, 420878, 420879, or 420880.
[0194] In certain embodiments, a target region is nucleotides
353-381 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 353-381 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 10, 46-48. In certain such embodiments, an
antisense compound targeted to nucleotides 353-381 of SEQ ID NO: 1
is selected from ISIS NOs: 304280, 420881, 420882, or 420883.
[0195] In certain embodiments, a target region is nucleotides
293-468 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 293-468 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 10-18, 42-63. In certain such embodiments, an
antisense compound targeted to nucleotides 293-468 of SEQ ID NO: 1
is selected from ISIS NOs: 420877, 420878, 420879, 420880, 304280,
420881, 420882, 420883, 304284, 304285, 420884, 420885, 304286,
420886, 420887, 420888, 420889, 420890, 420891, 304287, 420892,
304288, 420893, 304289, 304290, 420894, 420895, 420896, 420897,
420898, or 304291.
[0196] In certain embodiments, a target region is nucleotides
425-468 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 425-468 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 11-18, 49-63. In certain such embodiments, an
antisense compound targeted to nucleotides 425-468 of SEQ ID NO: 1
is selected from ISIS NOs: 304284, 304285, 420884, 420885, 304286,
420886, 420887, 420888, 420889, 420890, 420891, 304287, 420892,
304288, 420893, 304289, 304290, 420894, 420895, 420896, 420897,
420898, or 304291.
[0197] In certain embodiments, a target region is nucleotides
425-467 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 425-468 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 11-17, 49-63. In certain such embodiments, an
antisense compound targeted to nucleotides 425-468 of SEQ ID NO: 1
is selected from ISIS NOs: 304284, 304285, 420884, 420885, 304286,
420886, 420887, 420888, 420889, 420890, 420891, 304287, 420892,
304288, 420893, 304289, 304290, 420894, 420895, 420896, 420897, or
420898.
[0198] In certain embodiments, a target region is nucleotides
452-476 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 452-476 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 64-69. In certain such embodiments, an antisense
compound targeted to nucleotides 452-476 of SEQ ID NO: 1 is
selected from ISIS NOs: 420889, 420890, 420891, 304287, 420892,
304288, 420893, 304289, 304290, 420894, 420895, 420896, 420897,
420898, 304291, 304292, 304293, 420899, 420900, 420901, 420902,
420903, or 420904.
[0199] In certain embodiments, a target region is nucleotides
461-481 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 461-481 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 72-73. In certain such embodiments, an antisense
compound targeted to nucleotides 461-481 of SEQ ID NO: 1 is
selected from ISIS NOs: 420907 or 420908.
[0200] In certain embodiments, a target region is nucleotides
461-500 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 461-500 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 22, 72 and 73. In certain such embodiments, an
antisense compound targeted to nucleotides 461-500 of SEQ ID NO: 1
is selected from ISIS NOs: 420907, 420908 or 304296.
[0201] In certain embodiments, a target region is nucleotides
500-519 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 500-519 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NO: 74. In certain such embodiments, an antisense
compound targeted to nucleotides 500-519 of SEQ ID NO: 1 is
selected from ISIS NO: 420909.
[0202] In certain embodiments, a target region is nucleotides
461-519 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 461-519 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 22, 23, 72-74. In certain such embodiments, an
antisense compound targeted to nucleotides 461-519 of SEQ ID NO: 1
is selected from ISIS NOs: 420907, 420908, 304296, or 420909.
[0203] In certain embodiments, a target region is nucleotides
502-531 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 502-531 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 25, 75-84. In certain such embodiments, an
antisense compound targeted to nucleotides 502-531 of SEQ ID NO: 1
is selected from ISIS NOs: 420910, 420911, 420912, 420913, 420914,
304299, 420915, 420916, 420917, 420918, or 420919.
[0204] In certain embodiments, a target region is nucleotides
502-539 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 502-539 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 25, 26, 75-91. In certain such embodiments, an
antisense compound targeted to nucleotides 502-539 of SEQ ID NO: 1
is selected from ISIS NOs: 420910, 420911, 420912, 420913, 420914,
304299, 420915, 420916, 420917, 420918, 420919, 304300, 420920,
420921, 420922, 420923, 420924, 420925, or 420926.
[0205] In certain embodiments, a target region is nucleotides
504-536 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 504-536 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 25, 26, 77-88. In certain such embodiments, an
antisense compound targeted to nucleotides 504-536 of SEQ ID NO: 1
is selected from ISIS NOs: 420912, 420913, 420914, 304299, 420915,
420916, 420917, 420918, 420919, 304300, 420920, 420921, 420922, or
420923.
[0206] In certain embodiments, a target region is nucleotides
505-535 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 505-535 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 25, 26, 78-87. In certain such embodiments, an
antisense compound targeted to nucleotides 505-535 of SEQ ID NO: 1
is selected from ISIS NOs: 420913, 420914, 304299, 420915, 420916,
420917, 420918, 420919, 304300, 420920, 420921, or 420922.
[0207] In certain embodiments, a target region is nucleotides
506-530 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 506-530 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 25, 79-83. In certain such embodiments, an
antisense compound targeted to nucleotides 506-530 of SEQ ID NO: 1
is selected from ISIS NOs: 420913, 420914, 304299, 420915, 420916,
420917, 420918, or 420919.
[0208] In certain embodiments, a target region is nucleotides
507-527 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 507-527 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 25 or 80. In certain such embodiments, an
antisense compound targeted to nucleotides 507-527 of SEQ ID NO: 1
is selected from ISIS NO: 304299 or 420915.
[0209] In certain embodiments, a target region is nucleotides
508-527 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 508-527 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NO: 80. In certain such embodiments, an antisense
compound targeted to nucleotides 508-527 of SEQ ID NO: 1 is
selected from ISIS NO: 420915.
[0210] In certain embodiments, a target region is nucleotides
514-540 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 514-540 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 85-92. In certain such embodiments, an antisense
compound targeted to nucleotides 514-540 of SEQ ID NO: 1 is
selected from ISIS NOs: 420920, 420921, 420922, 420923, 420924,
420925, 420926, or 420927.
[0211] In certain embodiments, a target region is nucleotides
523-542 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 523-542 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NO: 94. In certain such embodiments, an antisense
compound targeted to nucleotides 523-542 of SEQ ID NO: 1 is
selected from ISIS NO: 420929.
[0212] In certain embodiments, a target region is nucleotides
544-606 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 544-606 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 30-33, 112-122. In certain such embodiments, an
antisense compound targeted to nucleotides 544-606 of SEQ ID NO: 1
is selected from ISIS NOs: 420947, 420948, 304304, 304307, 304308,
304309, 420949, 420950, 420951, 420952, 420953, 420954, 420955,
420956, or 420957.
[0213] In certain embodiments, a target region is nucleotides
544-564 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 544-564 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 112-113. In certain such embodiments, an antisense
compound targeted to nucleotides 544-564 of SEQ ID NO: 1 is
selected from ISIS NOs: 420947 or 420948.
[0214] In certain embodiments, a target region is nucleotides
544-583 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 544-583 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 30, 31, 112, and 113. In certain such embodiments,
an antisense compound targeted to nucleotides 544-583 of SEQ ID NO:
1 is selected from ISIS NOs: 420947, 420948, 304304, or 304307.
[0215] In certain embodiments, a target region is nucleotides
597-617 of SEQ ID NO: 1. In certain embodiments, an antisense
compound is targeted to nucleotides 597-617 of SEQ ID NO: 1. In
certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid comprises a nucleobase sequence selected
from SEQ ID NOs: 34-35. In certain such embodiments, an antisense
compound targeted to nucleotides 597-617 of SEQ ID NO: 1 is
selected from ISIS NOs: 304311 or 304312.
[0216] In certain embodiments, the modified oligonucleotide
consists of a single-stranded modified oligonucleotide.
[0217] In certain embodiments, the modified oligonucleotide
consists of 20 linked nucleosides.
[0218] In certain embodiments, the nucleobase sequence of the
modified oligonucleotide is at least 90% complementary over its
entire length to a nucleobase sequence of SEQ ID NO: 1, 2, or 4. In
certain embodiments, the nucleobase sequence of the modified
oligonucleotide is at least 95% complementary over its entire
length to a nucleobase sequence of SEQ ID NO: 1, 2, or 4. In
certain embodiments, the modified oligonucleotide is at least 99%
complementary over its entire length to SEQ ID NO: 1, 2, or 4. In
certain embodiments, the nucleobase sequence of the modified
oligonucleotide is 100% complementary over its entire length to a
nucleobase sequence of SEQ ID NO: 1, 2, or 4.
[0219] In certain embodiments, the compound has at least one
modified internucleoside linkage. In certain embodiments, the
internucleoside linkage is a phosphorothioate internucleoside
linkage.
[0220] In certain embodiments, the compound has at least one
nucleoside comprising a modified sugar. In certain embodiments, the
at least one modified sugar is a bicyclic sugar. In certain
embodiments, the at least one bicyclic sugar comprises a
4'-CH(CH.sub.3)--O-2' bridge. In certain embodiments, the at least
one modified sugar comprises a 2'-O-methoxyethyl.
[0221] In certain embodiments, the compound comprises at least one
at least one tetrahydropyran modified nucleoside wherein a
tetrahydropyran ring replaces the furanose ring. In certain
embodiments, the at least one tetrahydropyran modified nucleoside
has the structure:
##STR00001##
wherein Bx is an optionally protected heterocyclic base moiety.
[0222] In certain embodiments, the compound has at least one
nucleoside comprising a modified nucleobase.
[0223] In certain embodiments, the modified nucleobase is a
5-methylcytosine.
[0224] In certain embodiments, the modified oligonucleotide of the
compound comprises:
(i) a gap segment consisting of linked deoxynucleosides; (ii) a 5'
wing segment consisting of linked nucleosides; (iii) a 3' wing
segment consisting of linked nucleosides, wherein the gap segment
is positioned between the 5' wing segment and the 3' wing segment
and wherein each nucleoside of each wing segment comprises a
modified sugar.
[0225] In certain embodiments, the modified oligonucleotide of the
compound comprises:
(i) a gap segment consisting of ten linked deoxynucleosides; (ii) a
5' wing segment consisting of five linked nucleosides; (iii) a 3'
wing segment consisting of five linked nucleosides, wherein the gap
segment is positioned immediately adjacent to and between the 5'
wing segment and the 3' wing segment, wherein each nucleoside of
each wing segment comprises a 2'-O-methoxyethyl sugar; and wherein
each internucleoside linkage is a phosphorothioate linkage.
[0226] In certain embodiments, the modified oligonucleotide of the
compound comprises:
(i) a gap segment consisting of eight linked deoxynucleosides; (ii)
a 5' wing segment consisting of six linked nucleosides; (iii) a 3'
wing segment consisting of six linked nucleosides, wherein the gap
segment is positioned immediately adjacent to and between the 5'
wing segment and the 3' wing segment, wherein each nucleoside of
each wing segment comprises a 2'-O-methoxyethyl sugar; and wherein
each internucleoside linkage is a phosphorothioate linkage.
[0227] In certain embodiments, the modified oligonucleotide of the
compound comprises:
(i) a gap segment consisting of eight linked deoxynucleosides; (ii)
a 5' wing segment consisting of five linked nucleosides; (iii) a 3'
wing segment consisting of five linked nucleosides, wherein the gap
segment is positioned immediately adjacent to and between the 5'
wing segment and the 3' wing segment, wherein each nucleoside of
each wing segment comprises a 2'-O-methoxyethyl sugar; and wherein
each internucleoside linkage is a phosphorothioate linkage.
[0228] In certain embodiments, the modified oligonucleotide of the
compound comprises:
(i) a gap segment consisting of ten linked deoxynucleosides; (ii) a
5' wing segment consisting of five linked nucleosides; (iii) a 3'
wing segment consisting of five linked nucleosides, wherein the gap
segment is positioned immediately adjacent to and between the 5'
wing segment and the 3' wing segment, wherein each nucleoside of
each wing segment comprises a 2'-O-methoxyethyl sugar; and wherein
each internucleoside linkage is a phosphorothioate linkage; and
wherein the nucleobase sequence comprises at least 8 contiguous
nucleobases of the nucleobase sequence recited in SEQ ID NO:
80.
[0229] In certain embodiments, the modified oligonucleotide of the
compound comprises:
(i) a gap segment consisting of ten linked deoxynucleosides; (ii) a
5' wing segment consisting of five linked nucleosides; (iii) a 3'
wing segment consisting of five linked nucleosides, wherein the gap
segment is positioned immediately adjacent to and between the 5'
wing segment and the 3' wing segment, wherein each nucleoside of
each wing segment comprises a 2'-O-methoxyethyl sugar; and wherein
each internucleoside linkage is a phosphorothioate linkage; and
wherein the nucleobase sequence is recited in SEQ ID NO: 80.
[0230] Certain embodiments provide a composition comprising a
compound as described herein, or a salt thereof, and a
pharmaceutically acceptable carrier or diluent. In certain
embodiments, the composition comprises a modified oligonucleotide
consisting of 12 to 30 linked nucleosides and having a nucleobase
sequence comprising at least 12 contiguous nucleobases of a
nucleobase sequence selected from among the nucleobase sequences
recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124 or a
salt thereof and a pharmaceutically acceptable carrier or
diluent.
[0231] Certain embodiments provide a composition comprising a
compound as described herein, or a salt thereof, and a
pharmaceutically acceptable carrier or diluent. In certain
embodiments, the composition comprises a modified oligonucleotide
consisting of 12 to 30 linked nucleosides and having a nucleobase
sequence comprising at least 12 contiguous nucleobases of the
nucleobase sequences recited in SEQ ID NO: 80 or a salt thereof and
a pharmaceutically acceptable carrier or diluent.
[0232] Certain embodiments provide a composition comprising a
compound as described herein, wherein the viscosity level is less
than 40 cP. In certain embodiments, the composition has a viscosity
level less than 15 cP. In certain embodiments, the composition has
a viscosity level less than 12 cP. In certain embodiments, the
composition has a viscosity level less than 10 cP.
[0233] Certain embodiments provide methods of treating, preventing,
or ameliorating transthyretin amyloidosis.
[0234] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a modified oligonucleotide consisting of 12 to 30 linked
nucleosides and having a nucleobase sequence comprising at least 8
contiguous nucleobases of a nucleobase sequence selected from among
the nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, 87,
115, 120, 122, and 124.
[0235] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a compound or modified oligonucleotide consisting 12 to 30
linked nucleosides, wherein the linked nucleosides comprise at
least an 8 contiguous nucleobase portion complementary to an equal
length portion within the region selected from nucleotides 501-535
or 580-608 of SEQ ID NO: 1.
[0236] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a modified oligonucleotide consisting of 12 to 30 linked
nucleosides and having a nucleobase sequence comprising at least 8
contiguous nucleobases of a nucleobase sequence recited in SEQ ID
NO: 80.
[0237] Certain embodiments provide methods comprising administering
to an animal a compound as described herein to an animal. In
certain embodiments, the method comprises administering to an
animal a compound or modified oligonucleotide consisting 12 to 30
linked nucleosides, wherein the linked nucleosides comprise at
least an 8 contiguous nucleobase portion complementary to an equal
length portion within the region selected from nucleotides 508-527
of SEQ ID NO: 1.
[0238] In certain embodiments, the animal is a human.
[0239] In certain embodiments, the administering prevents, treats,
ameliorates, or slows progression of transthyretin amyloidosis as
described herein.
[0240] In certain embodiments, the compound is co-administered with
a second agent.
[0241] In certain embodiments, the compound and the second agent
are administered concomitantly.
[0242] In certain embodiments, the administering is parenteral
administration. In certain embodiments, the parenteral
administration is subcutaneous administration. In certain
embodiments, the formulation for administering is the compound in
saline. In certain embodiments, the compound comprises a modified
oligonucleotide consisting of 12 to 30 linked nucleosides and
having a nucleobase sequence comprising at least 12 contiguous
nucleobases of a nucleobase sequence selected from among the
nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, 87, 115,
120, 122, and 124 or a salt thereof and saline. In certain
embodiments, the formulation does not include any stabilizing
agents or additional stabilizing agents including lipid agents.
[0243] In certain embodiments, the administering is parenteral
administration. In certain embodiments, the parenteral
administration is intracranial administration. In certain
embodiments, the intracranial administration is intracerebral,
intrathecal, intraventricular, ventricular,
intracerebroventricular, cerebral intraventricular or cerebral
ventricular administration.
[0244] Certain embodiments further provide a method to reduce
transthyretin mRNA or protein expression in an animal comprising
administering to the animal a compound or composition as described
herein to reduce transthyretin mRNA or protein expression in the
animal. In certain embodiments, the animal is a human. In certain
embodiments, reducing transthyretin mRNA or protein expression
prevents, treats, ameliorates, or slows progression of
transthyretin amyloidosis.
[0245] Certain embodiments provide a method for treating a human
with a transthyretin related disease comprising identifying the
human with the disease and administering to the human a
therapeutically effective amount of a compound or composition as
described herein. In certain embodiments, the treatment reduces a
symptom selected from the group consisting of restlessness, lack of
coordination, nystagmus, spastic paraparesis, lack of muscle
coordination, impaired vision, insomnia, unusual sensations,
myoclonus, blindness, loss of speech, Carpal tunnel syndrome,
seizures, subarachnoid hemorrhages, stroke and bleeding in the
brain, hydrocephalus, ataxia, and spastic paralysis, coma, sensory
neuropathy, parathesia, hypesthesia, motor neuropathy, autonomic
neuropathy, orthostatic hypotension, cyclic constipation, cyclic
diarrhea, nausea, vomiting, reduced sweating, impotence, delayed
gastric emptying, urinary retention, urinary incontinence,
progressive cardiopathy, fatigue, shortness of breath, weight loss,
lack of appetite, numbness, tingling, weakness, enlarged tongue,
nephrotic syndrome, congestive heart failure, dyspnea on exertion,
peripheral edema, arrhythmias, palpitations, light-headedness,
syncope, postural hypotension, peripheral nerve problems, sensory
motor impairment, lower limb neuropathy, upper limb neuropathy,
hyperalgesia, altered temperature sensation, lower extremity
weakness, cachexia, peripheral edema, hepatomegaly, purpura,
diastolic dysfunction, premature ventricular contractions, cranial
neuropathy, diminished deep tendon reflexes, amyloid deposits in
the corpus vitreum, vitreous opacity, dry eyes, glaucoma, scalloped
appearance in the pupils, swelling of the feet due to water
retention. In certain embodiments, the symptom is a cognitive
symptom selected from the group consisting of impaired memory,
impaired judgment, and thinking, impaired planning, impaired
flexibility, impaired abstract thinking, impaired rule acquisition,
impaired initiation of appropriate actions, impaired inhibition of
inappropriate actions, impaired short-term memory, impaired
long-term memory, paranoia, disorientation, confusion,
hallucination and dementia. In certain embodiments, the symptom is
a psychiatric symptom selected from the group consisting of
dementia; anxiety, depression, blunted affect, egocentrisms,
aggression, compulsive behavior, irritability, personality changes,
including, impaired memory, judgment, and thinking and suicidal
ideation.
[0246] Further embodiments provide a method of treating a human
with transthyretin amyloidosis leading to cardiac amyloidosis and
administering to the human a therapeutically effective amount of a
compound or composition as described herein. In certain
embodiments, the treatment reduces a symptom selected from the
group consisting of congestive heart failure, cardiomegaly, dyspnea
on exertion, peripheral edema, arrhythmias, palpitations,
lightheadedness, syncope, deposition in the subendothelium of the
peripheral vasculature can lead to severe postural hypotension,
diastolic dysfunction, heart block, premature ventricular
contractions, and various tachyarrhythmias.
[0247] Further embodiments provide a method of treating a human
with transthyretin amyloidosis leading to peripheral neuropathic
disorders and administering to the human a therapeutically
effective amount of a compound or composition as described herein.
In certain embodiments, the treatment reduces a symptom selected
from the group consisting of peripheral nerve problems,
sensorimotor impairment, lower-limb neuropathy, upper-limb
neuropathy, hyperalgesia, altered temperature sensation, lower
extremity weakness, pain, autonomic dysfunction, often manifested
as sexual or urinary dysfunction, symmetric sensory impairment and
weakness, orthostatic hypotension, diarrhea, and/or impotence.
[0248] Further embodiments provide a method of treating a human
with transthyretin amyloidosis leading to gastrointestinal
disorders and administering to the human a therapeutically
effective amount of a compound or composition a described herein.
In certain embodiments, the treatment reduces a symptom selected
from the group consisting of diarrhea, constipation, nausea,
vomiting, and related kidney and liver disorders.
[0249] Further provided is a method for reducing or preventing
transthyretin amyloidosis comprising administering to a human a
therapeutically effective amount compound or composition as
described herein, thereby reducing or preventing transthyretin
amyloidosis.
[0250] Further provided is a method for reducing or preventing a
cardiac disease comprising administering to a human a
therapeutically effective amount compound or composition as
described herein, thereby reducing or preventing a cardiac disease.
Further provided is a method for reducing or preventing a
neuropathic disease comprising administering to a human a
therapeutically effective amount compound or composition as
described herein, thereby reducing or preventing a neuropathic
disease. Further provided is a method for reducing or preventing a
gastrointestinal disease comprising administering to a human a
therapeutically effective amount compound or composition as
described herein, thereby reducing or preventing a gastrointestinal
disease.
[0251] Further provided is a method for ameliorating a symptom of
transthyretin amyloidosis, comprising administering to a human in
need thereof a compound comprising a modified oligonucleotide
consisting of 12 to 30 linked nucleosides, wherein said modified
oligonucleotide specifically hybridizes to SEQ ID NO: 1, 2, or 4,
thereby ameliorating a symptom of transthyretin amyloidosis in the
human.
[0252] Further provided is a method for reducing the rate of
progression of a symptom associated with transthyretin amyloidosis,
comprising administering to a human in need thereof a compound
comprising a modified oligonucleotide consisting of 12 to 30 linked
nucleosides, wherein said modified oligonucleotide specifically
hybridizes to SEQ ID NO: 1, 2, or 4, thereby reducing the rate of
progression a symptom of transthyretin amyloidosis in the
human.
[0253] Further provided is a method for reversing degeneration
indicated by a symptom associated with a transthyretin amyloidosis,
administering to a human in need thereof a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides,
wherein said modified oligonucleotide specifically hybridizes to
SEQ ID NO: 1, 2, or 4, thereby reversing degeneration indicated by
a symptom of transthyretin amyloid disease in the human.
[0254] Further provided is a method for ameliorating a symptom of
transthyretin amyloidosis, comprising administering to a human in
need thereof a compound comprising a modified oligonucleotide
consisting of 12 to 30 linked nucleosides and having a nucleobase
sequence comprising at least 8 contiguous nucleobases of a
nucleobase sequence recited in SEQ ID NO: 80, thereby ameliorating
a symptom of transthyretin amyloidosis in the human.
[0255] Further embodiments provide a method of treating a human
with transthyretin amyloidosis, administering to a human in need
thereof a compound comprising a modified oligonucleotide consisting
of 12 to 30 linked nucleosides and having a nucleobase sequence
comprising at least 8 contiguous nucleobases of a nucleobase
sequence recited in SEQ ID NO: 80, thereby treating transthyretin
amyloidosis in a human.
[0256] In certain embodiments, the symptom is a physical,
cognitive, psychiatric, or peripheral symptom. In certain
embodiments, the symptom is a physical symptom selected from the
group consisting of restlessness, lack of coordination, nystagmus,
spastic paraparesis, lack of muscle coordination, impaired vision,
insomnia, unusual sensations, myoclonus, blindness, loss of speech,
Carpal tunnel syndrome, seizures, subarachnoid hemorrhages, stroke
and bleeding in the brain, hydrocephalus, ataxia, and spastic
paralysis, coma, sensory neuropathy, parathesia, hypesthesia, motor
neuropathy, autonomic neuropathy, orthostatic hypotension, cyclic
constipation, cyclic diarrhea, nausea, vomiting, reduced sweating,
impotence, delayed gastric emptying, urinary retention, urinary
incontinence, progressive cardiopathy, fatigue, shortness of
breath, weight loss, lack of appetite, numbness, tingling,
weakness, enlarged tongue, nephrotic syndrome, congestive heart
failure, dyspnea on exertion, peripheral edema, arrhythmias,
palpitations, light-headedness, syncope, postural hypotension,
peripheral nerve problems, sensory motor impairment, lower limb
neuropathy, upper limb neuropathy, hyperalgesia, altered
temperature sensation, lower extremity weakness, cachexia,
peripheral edema, hepatomegaly, purpura, diastolic dysfunction,
premature ventricular contractions, cranial neuropathy, diminished
deep tendon reflexes, amyloid deposits in the corpus vitreum,
vitreous opacity, dry eyes, glaucoma, scalloped appearance in the
pupils, swelling of the feet due to water retention. In certain
embodiments, the symptom is a cognitive symptom selected from the
group consisting of impaired memory, impaired judgment, and
thinking, impaired planning, impaired flexibility, impaired
abstract thinking, impaired rule acquisition, impaired initiation
of appropriate actions, impaired inhibition of inappropriate
actions, impaired short-term memory, impaired long-term memory,
paranoia, disorientation, confusion, hallucination and dementia. In
certain embodiments, the symptom is a psychiatric symptom selected
from the group consisting of dementia; anxiety, depression, blunted
affect, egocentrisms, aggression, compulsive behavior,
irritability, personality changes, including, impaired memory,
judgment, and thinking and suicidal ideation.
[0257] In certain embodiments the symptom is at least one of at
least one physical symptom, at least one cognitive symptom, at
least one psychiatric symptom, and at least one peripheral
symptom.
[0258] In certain embodiments the physical symptom is selected from
the group consisting of restlessness, lack of coordination,
unintentionally initiated motions, unintentionally uncompleted
motions, unsteady gait, chorea, rigidity, writhing motions,
abnormal posturing, instability, abnormal facial expressions,
difficulty chewing, difficulty swallowing, difficulty speaking,
seizure, and sleep disturbances.
[0259] In certain embodiments the cognitive symptom is selected
from the group consisting of impaired memory, impaired judgment,
and thinking, impaired planning, impaired flexibility, impaired
abstract thinking, impaired rule acquisition, impaired initiation
of appropriate actions, impaired inhibition of inappropriate
actions, impaired short-term memory, impaired long-term memory,
paranoia, disorientation, confusion, hallucination and
dementia.
[0260] In certain embodiments the psychiatric symptom is selected
from the group consisting of dementia; anxiety, depression, blunted
affect, egocentrisms, aggression, compulsive behavior,
irritability, personality changes, including, impaired memory,
judgment, and thinking and suicidal ideation.
[0261] In certain embodiments the peripheral symptom is selected
from the group consisting of reduced brain mass, muscle atrophy,
cardiac failure, impaired glucose tolerance, weight loss,
osteoporosis, and testicular atrophy.
[0262] Also provided are methods and compounds for the preparation
of a medicament for the treatment, prevention, or amelioration of a
central nervous system related disease.
[0263] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
ameliorating, or preventing a transthyretin amyloidosis.
[0264] Certain embodiments provide a compound as described herein
for use in treating, preventing, or ameliorating transthyretin
amyloidosis as described herein by combination therapy with an
additional agent or therapy as described herein. Agents or
therapies can be co-administered or administered concomitantly.
[0265] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating transthyretin amyloidosis as described
herein by combination therapy with an additional agent or therapy
as described herein. Agents or therapies can be co-administered or
administered concomitantly.
[0266] Certain embodiments provide the use of a compound as
described herein in the manufacture of a medicament for treating,
preventing, or ameliorating transthyretin amyloidosis as described
herein in a patient who is subsequently administered an additional
agent or therapy as described herein.
[0267] Certain embodiments provide a kit for treating, preventing,
or ameliorating transthyretin amyloidosis as described herein
wherein the kit comprises:
(i) a compound as described herein; and alternatively (ii) an
additional agent or therapy as described herein.
[0268] A kit as described herein may further include instructions
for using the kit to treat, prevent, or ameliorate transthyretin
amyloidosis as described herein by combination therapy as described
herein.
Antisense Compounds
[0269] Oligomeric compounds include, but are not limited to,
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics, antisense compounds, antisense
oligonucleotides, and siRNAs. An oligomeric compound may be
"antisense" to a target nucleic acid, meaning that is capable of
undergoing hybridization to a target nucleic acid through hydrogen
bonding.
[0270] In certain embodiments, an antisense compound has a
nucleobase sequence that, when written in the 5' to 3' direction,
comprises the reverse complement of the target segment of a target
nucleic acid to which it is targeted. In certain such embodiments,
an antisense oligonucleotide has a nucleobase sequence that, when
written in the 5' to 3' direction, comprises the reverse complement
of the target segment of a target nucleic acid to which it is
targeted.
[0271] In certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid is 12 to 30 nucleotides in length. In
other words, antisense compounds are from 12 to 30 linked
nucleobases. In other embodiments, the antisense compound comprises
a modified oligonucleotide consisting of 8 to 80, 12 to 50, 12 to
30, 15 to 30, 18 to 24, 18 to 21, 19 to 22, or 20 linked
nucleobases. In certain such embodiments, the antisense compound
comprises a modified oligonucleotide consisting 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 linked nucleobases in length, or a range defined by any two
of the above values.
[0272] In certain embodiments, the antisense compound comprises a
shortened or truncated modified oligonucleotide. The shortened or
truncated modified oligonucleotide can have a single nucleoside
deleted from the 5' end (5' truncation), or alternatively from the
3' end (3' truncation). A shortened or truncated oligonucleotide
may have two nucleosides deleted from the 5' end, or alternatively
may have two subunits deleted from the 3' end. Alternatively, the
deleted nucleosides may be dispersed throughout the modified
oligonucleotide, for example, in an antisense compound having one
nucleoside deleted from the 5' end and one nucleoside deleted from
the 3' end.
[0273] When a single additional nucleoside is present in a
lengthened oligonucleotide, the additional nucleoside may be
located at the 5' or 3' end of the oligonucleotide. When two or
more additional nucleosides are present, the added nucleosides may
be adjacent to each other, for example, in an oligonucleotide
having two nucleosides added to the 5' end (5' addition), or
alternatively to the 3' end (3' addition), of the oligonucleotide.
Alternatively, the added nucleoside may be dispersed throughout the
antisense compound, for example, in an oligonucleotide having one
nucleoside added to the 5' end and one subunit added to the 3'
end.
[0274] It is possible to increase or decrease the length of an
antisense compound, such as an antisense oligonucleotide, and/or
introduce mismatch bases without eliminating activity. For example,
in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a
series of antisense oligonucleotides 13-25 nucleobases in length
were tested for their ability to induce cleavage of a target RNA in
an oocyte injection model. Antisense oligonucleotides 25
nucleobases in length with 8 or 11 mismatch bases near the ends of
the antisense oligonucleotides were able to direct specific
cleavage of the target mRNA, albeit to a lesser extent than the
antisense oligonucleotides that contained no mismatches. Similarly,
target specific cleavage was achieved using 13 nucleobase antisense
oligonucleotides, including those with 1 or 3 mismatches.
[0275] Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March
2001) demonstrated the ability of an oligonucleotide having 100%
complementarity to the bcl-2 mRNA and having 3 mismatches to the
bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in
vitro and in vivo. Furthermore, this oligonucleotide demonstrated
potent anti-tumor activity in vivo.
[0276] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988)
tested a series of tandem 14 nucleobase antisense oligonucleotides,
and a 28 and 42 nucleobase antisense oligonucleotides comprised of
the sequence of two or three of the tandem antisense
oligonucleotides, respectively, for their ability to arrest
translation of human DHFR in a rabbit reticulocyte assay. Each of
the three 14 nucleobase antisense oligonucleotides alone was able
to inhibit translation, albeit at a more modest level than the 28
or 42 nucleobase antisense oligonucleotides.
Antisense Compound Motifs
[0277] In certain embodiments, antisense compounds targeted to a
transthyretin nucleic acid have chemically modified subunits
arranged in patterns, or motifs, to confer to the antisense
compounds properties such as enhanced the inhibitory activity,
increased binding affinity for a target nucleic acid, or resistance
to degradation by in vivo nucleases.
[0278] Chimeric antisense compounds typically contain at least one
region modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, increased binding affinity
for the target nucleic acid, and/or increased inhibitory activity.
A second region of a chimeric antisense compound may optionally
serve as a substrate for the cellular endonuclease RNase H, which
cleaves the RNA strand of an RNA:DNA duplex.
[0279] Antisense compounds having a gapmer motif are considered
chimeric antisense compounds. In a gapmer an internal region having
a plurality of nucleotides or linked nucleosides that supports
RNaseH cleavage is positioned between external regions having a
plurality of nucleotides or linked nucleosides that are chemically
distinct from the nucleotides or linked nucleosides of the internal
region. In the case of an antisense oligonucleotide having a gapmer
motif, the gap segment generally serves as the substrate for
endonuclease cleavage, while the wing segments comprise modified
nucleosides. In certain embodiments, the regions of a gapmer are
differentiated by the types of sugar moieties comprising each
distinct region. The types of sugar moieties that are used to
differentiate the regions of a gapmer may in some embodiments
include .beta.-D-ribonucleosides, .beta.-D-deoxyribonucleosides,
2'-modified nucleosides (such 2'-modified nucleosides may include
2'-MOE, and 2'-O--CH.sub.3, among others), and bicyclic sugar
modified nucleosides (such bicyclic sugar modified nucleosides may
include those having a 4'-(CH.sub.2)n-O-2' bridge, where n=1 or
n=2). Preferably, each distinct region comprises uniform sugar
moieties. The wing-gap-wing motif is frequently described as
"X-Y-Z", where "X" represents the length of the 5' wing region, "Y"
represents the length of the gap region, and "Z" represents the
length of the 3' wing region. As used herein, a gapmer described as
"X-Y-Z" has a configuration such that the gap segment is positioned
immediately adjacent each of the 5' wing segment and the 3' wing
segment. Thus, no intervening nucleotides exist between the 5' wing
segment and gap segment, or the gap segment and the 3' wing
segment. Any of the antisense compounds described herein can have a
gapmer motif. In some embodiments, X and Z are the same, in other
embodiments they are different. In a preferred embodiment, Y is
between 8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30
or more nucleotides. Thus, gapmers include, but are not limited to,
for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2,
1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6 or 5-8-5.
[0280] In certain embodiments, the antisense compound as a
"wingmer" motif, having a wing-gap or gap-wing configuration, i.e.
an X-Y or Y-Z configuration as described above for the gapmer
configuration. Thus, wingmer configurations include, but are not
limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1,
10-3, 2-10, 1-10, 8-2, 2-13, or 5-13.
[0281] In certain embodiments, antisense compounds targeted to a
transthyretin nucleic acid possess a 5-10-5 gapmer motif.
[0282] In certain embodiments, antisense compounds targeted to a
transthyretin nucleic acid possess a 6-8-6 gapmer motif.
[0283] In certain embodiments, antisense compounds targeted to a
transthyretin nucleic acid possess a 5-8-5 gapmer motif.
[0284] In certain embodiments, an antisense compound targeted to a
transthyretin nucleic acid has a gap-widened motif.
[0285] In certain embodiments, a gap-widened antisense
oligonucleotide targeted to a transthyretin nucleic acid has a gap
segment of ten 2'-deoxyribonucleotides positioned immediately
adjacent to and between wing segments of five chemically modified
nucleosides. In certain embodiments, the chemical modification
comprises a 2'-sugar modification. In another embodiment, the
chemical modification comprises a 2'-MOE sugar modification.
[0286] In certain embodiments, a gap-widened antisense
oligonucleotide targeted to a transthyretin nucleic acid has a gap
segment of eight 2'-deoxyribonucleotides positioned immediately
adjacent to and between wing segments of five chemically modified
nucleosides. In certain embodiments, the chemical modification
comprises a 2'-sugar modification. In another embodiment, the
chemical modification comprises a 2'-MOE sugar modification.
[0287] In certain embodiments, a gap-widened antisense
oligonucleotide targeted to a transthyretin nucleic acid has a gap
segment of eight 2'-deoxyribonucleotides positioned immediately
adjacent to and between wing segments of six chemically modified
nucleosides. In certain embodiments, the chemical modification
comprises a 2'-sugar modification. In another embodiment, the
chemical modification comprises a 2'-MOE sugar modification.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
[0288] In certain embodiments, the transthyretin nucleic acid is
any of the sequences set forth in GENBANK Accession No. NM
000371.2, first deposited with GENBANK.RTM. on Feb. 13, 2008
(incorporated herein as SEQ ID NO: 1), GENBANK Accession No.
NT_010966.10 truncated from nucleotides 2009236 to 2017289, first
deposited with GENBANK.RTM. on August 1.sup.st, 2002 (incorporated
herein as SEQ ID NO: 2); exons 1-4 extracted from the rhesus monkey
genomic sequence GENBANK Accession No. NW 001105671.1, based on
similarity to human exons; and GENBANK Accession No. NW 001105671.1
truncated from nucleotides 628000 to 638000 (incorporated herein as
SEQ ID NO: 4), first deposited with GENBANK.RTM. on Mar. 28,
2006.
[0289] It is understood that the sequence set forth in each SEQ ID
NO in the Examples contained herein is independent of any
modification to a sugar moiety, an internucleoside linkage, or a
nucleobase. As such, antisense compounds defined by a SEQ ID NO may
comprise, independently, one or more modifications to a sugar
moiety, an internucleoside linkage, or a nucleobase. Antisense
compounds described by Isis Number (Isis No) or ISIS NO indicate a
combination of nucleobase sequence and motif.
[0290] In certain embodiments, a target region is a structurally
defined region of the target nucleic acid. For example, a target
region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an
exon/intron junction, a coding region, a translation initiation
region, translation termination region, or other defined nucleic
acid region. The structurally defined regions for transthyretin can
be obtained by accession number from sequence databases such as
NCBI and such information is incorporated herein by reference. In
certain embodiments, a target region may encompass the sequence
from a 5' target site of one target segment within the target
region to a 3' target site of another target segment within the
target region.
[0291] Targeting includes determination of at least one target
segment to which an antisense compound hybridizes, such that a
desired effect occurs. In certain embodiments, the desired effect
is a reduction in mRNA target nucleic acid levels. In certain
embodiments, the desired effect is reduction of levels of protein
encoded by the target nucleic acid or a phenotypic change
associated with the target nucleic acid.
[0292] A target region may contain one or more target segments.
Multiple target segments within a target region may be overlapping.
Alternatively, they may be non-overlapping. In certain embodiments,
target segments within a target region are separated by no more
than about 300 nucleotides. In certain embodiments, target segments
within a target region are separated by a number of nucleotides
that is, is about, is no more than, is no more than about, 250,
200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on
the target nucleic acid, or is a range defined by any two of the
preceding values. In certain embodiments, target segments within a
target region are separated by no more than, or no more than about,
5 nucleotides on the target nucleic acid. In certain embodiments,
target segments are contiguous. Contemplated are target regions
defined by a range having a starting nucleic acid that is any of
the 5' target sites or 3' target sites listed herein.
[0293] Suitable target segments may be found within a 5' UTR, a
coding region, a 3' UTR, an intron, an exon, or an exon/intron
junction. Target segments containing a start codon or a stop codon
are also suitable target segments. A suitable target segment may
specifically exclude a certain structurally defined region such as
the start codon or stop codon.
[0294] The determination of suitable target segments may include a
comparison of the sequence of a target nucleic acid to other
sequences throughout the genome. For example, the BLAST algorithm
may be used to identify regions of similarity amongst different
nucleic acids. This comparison can prevent the selection of
antisense compound sequences that may hybridize in a non-specific
manner to sequences other than a selected target nucleic acid
(i.e., non-target or off-target sequences).
[0295] There may be variation in activity (e.g., as defined by
percent reduction of target nucleic acid levels) of the antisense
compounds within an active target region. In certain embodiments,
reductions in transthyretin mRNA levels are indicative of
inhibition of transthyretin expression. Reductions in levels of a
transthyretin protein are also indicative of inhibition of target
mRNA expression. Further, phenotypic changes are indicative of
inhibition of transthyretin expression. For example, increase in
brain size to normal, improvement in motor coordination, decrease
in continual muscular spasms (dystonia), decrease in irritability
and/or anxiety, improvement of memory, or an increase in energy,
among other phenotypic changes that may be assayed. Other
phenotypic indications, e.g., symptoms associated with
transthyretin amyloidosis, may also be assessed as described
below.
Hybridization
[0296] In some embodiments, hybridization occurs between an
antisense compound disclosed herein and a transthyretin nucleic
acid. The most common mechanism of hybridization involves hydrogen
bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding) between complementary nucleobases of the nucleic
acid molecules.
[0297] Hybridization can occur under varying conditions. Stringent
conditions are sequence-dependent and are determined by the nature
and composition of the nucleic acid molecules to be hybridized.
[0298] Methods of determining whether a sequence is specifically
hybridizable to a target nucleic acid are well known in the art. In
certain embodiments, the antisense compounds provided herein are
specifically hybridizable with a transthyretin nucleic acid.
Complementarity
[0299] An antisense compound and a target nucleic acid are
complementary to each other when a sufficient number of nucleobases
of the antisense compound can hydrogen bond with the corresponding
nucleobases of the target nucleic acid, such that a desired effect
will occur (e.g., antisense inhibition of a target nucleic acid,
such as a transthyretin nucleic acid).
[0300] An antisense compound may hybridize over one or more
segments of a transthyretin nucleic acid such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure, mismatch or hairpin structure).
[0301] In certain embodiments, the antisense compounds provided
herein, or a specified portion thereof, are, or are at least, 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% complementary to a transthyretin nucleic
acid, a target region, target segment, or specified portion
thereof. Percent complementarity of an antisense compound with a
target nucleic acid can be determined using routine methods.
[0302] 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. As such, an antisense compound
which is 18 nucleobases in length having 4 (four) noncomplementary
nucleobases which are flanked by two regions of complete
complementarity with the target nucleic acid would have 77.8%
overall complementarity with the target nucleic acid and would thus
fall within the scope of the present invention. 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). 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).
[0303] In certain embodiments, the antisense compounds provided
herein, or specified portions thereof, are fully complementary
(i.e. 100% complementary) to a target nucleic acid, or specified
portion thereof. For example, antisense compound may be fully
complementary to a transthyretin nucleic acid, or a target region,
or a target segment or target sequence thereof. As used herein,
"fully complementary" means each nucleobase of an antisense
compound is capable of precise base pairing with the corresponding
nucleobases of a target nucleic acid. For example, a 20 nucleobase
antisense compound is fully complementary to a target sequence that
is 400 nucleobases long, so long as there is a corresponding 20
nucleobase portion of the target nucleic acid that is fully
complementary to the antisense compound. Fully complementary can
also be used in reference to a specified portion of the first
and/or the second nucleic acid. For example, a 20 nucleobase
portion of a 30 nucleobase antisense compound can be "fully
complementary" to a target sequence that is 400 nucleobases long.
The 20 nucleobase portion of the 30 nucleobase oligonucleotide is
fully complementary to the target sequence if the target sequence
has a corresponding 20 nucleobase portion wherein each nucleobase
is complementary to the 20 nucleobase portion of the antisense
compound. At the same time, the entire 30 nucleobase antisense
compound may or may not be fully complementary to the target
sequence, depending on whether the remaining 10 nucleobases of the
antisense compound are also complementary to the target
sequence.
[0304] The location of a non-complementary nucleobase may be at the
5' end or 3' end of the antisense compound. Alternatively, the
non-complementary nucleobase or nucleobases may be at an internal
position of the antisense compound. When two or more
non-complementary nucleobases are present, they may be contiguous
(i.e. linked) or non-contiguous. In one embodiment, a
non-complementary nucleobase is located in the wing segment of a
gapmer antisense oligonucleotide.
[0305] In certain embodiments, antisense compounds that are, or are
up to 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length
comprise no more than 4, no more than 3, no more than 2, or no more
than 1 non-complementary nucleobase(s) relative to a target nucleic
acid, such as a transthyretin nucleic acid, or specified portion
thereof.
[0306] In certain embodiments, antisense compounds that are, or are
up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, or 30 nucleobases in length comprise no more than 6, no
more than 5, no more than 4, no more than 3, no more than 2, or no
more than 1 non-complementary nucleobase(s) relative to a target
nucleic acid, such as a transthyretin nucleic acid, or specified
portion thereof.
[0307] The antisense compounds provided herein also include those
which are complementary to a portion of a target nucleic acid. As
used herein, "portion" refers to a defined number of contiguous
(i.e. linked) nucleobases within a region or segment of a target
nucleic acid. A "portion" can also refer to a defined number of
contiguous nucleobases of an antisense compound. In certain
embodiments, the antisense compounds, are complementary to at least
an 8 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 12 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 15 nucleobase portion of a target segment. Also contemplated are
antisense compounds that are complementary to at least a 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a
target segment, or a range defined by any two of these values.
Identity
[0308] The antisense compounds provided herein may also have a
defined percent identity to a particular nucleotide sequence, SEQ
ID NO, or compound represented by a specific Isis number, or
portion thereof. As used herein, an antisense compound is identical
to the sequence disclosed herein if it has the same nucleobase
pairing ability. For example, a RNA which contains uracil in place
of thymidine in a disclosed DNA sequence would be considered
identical to the DNA sequence since both uracil and thymidine pair
with adenine. Shortened and lengthened versions of the antisense
compounds described herein as well as compounds having
non-identical bases relative to the antisense compounds provided
herein also are contemplated. The non-identical bases may be
adjacent to each other or dispersed throughout the antisense
compound. Percent identity of an antisense compound is calculated
according to the number of bases that have identical base pairing
relative to the sequence to which it is being compared.
[0309] In certain embodiments, the antisense compounds, or portions
thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to one or more of the antisense compounds or
SEQ ID NOs, or a portion thereof, disclosed herein.
Modifications
[0310] A nucleoside is a base-sugar combination. The nucleobase
(also known as base) portion of the nucleoside is normally a
heterocyclic base moiety. 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 the 2', 3' or 5'
hydroxyl moiety of the sugar. Oligonucleotides are formed through
the covalent linkage of adjacent nucleosides to one another, to
form a linear polymeric oligonucleotide. Within the oligonucleotide
structure, the phosphate groups are commonly referred to as forming
the internucleoside linkages of the oligonucleotide.
[0311] Modifications to antisense compounds encompass substitutions
or changes to internucleoside linkages, sugar moieties, or
nucleobases. Modified antisense compounds are often preferred over
native forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced affinity for nucleic acid
target, increased stability in the presence of nucleases, or
increased inhibitory activity.
[0312] Chemically modified nucleosides may also be employed to
increase the binding affinity of a shortened or truncated antisense
oligonucleotide for its target nucleic acid. Consequently,
comparable results can often be obtained with shorter antisense
compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
[0313] The naturally occurring internucleoside linkage of RNA and
DNA is a 3' to 5' phosphodiester linkage. Antisense compounds
having one or more modified, i.e. non-naturally occurring,
internucleoside linkages are often selected over antisense
compounds having naturally occurring internucleoside linkages
because of desirable properties such as, for example, enhanced
cellular uptake, enhanced affinity for target nucleic acids, and
increased stability in the presence of nucleases.
[0314] Oligonucleotides having modified internucleoside linkages
include internucleoside linkages that retain a phosphorus atom as
well as internucleoside linkages that do not have a phosphorus
atom. Representative phosphorus containing internucleoside linkages
include, but are not limited to, phosphodiesters, phosphotriesters,
methylphosphonates, phosphoramidate, and phosphorothioates. Methods
of preparation of phosphorous-containing and
non-phosphorous-containing linkages are well known.
[0315] In certain embodiments, antisense compounds targeted to a
transthyretin nucleic acid comprise one or more modified
internucleoside linkages. In certain embodiments, the modified
internucleoside linkages are phosphorothioate linkages. In certain
embodiments, each internucleoside linkage of an antisense compound
is a phosphorothioate internucleoside linkage.
Modified Sugar Moieties
[0316] Antisense compounds of the invention can optionally contain
one or more nucleosides wherein the sugar group has been modified.
Such sugar modified nucleosides may impart enhanced nuclease
stability, increased binding affinity, or some other beneficial
biological property to the antisense compounds. In certain
embodiments, nucleosides comprise chemically modified ribofuranose
ring moieties. Examples of chemically modified ribofuranose rings
include without limitation, addition of substitutent groups
(including 5' and 2' substituent groups, bridging of non-geminal
ring atoms to form bicyclic nucleic acids (BNA), replacement of the
ribosyl ring oxygen atom with S, N(R), or C(R.sub.1)(R.sub.2) (R,
R.sub.1 and R.sub.2 are each independently H, C.sub.1-C.sub.12
alkyl or a protecting group) and combinations thereof. Examples of
chemically modified sugars include 2'-F-5'-methyl substituted
nucleoside (see PCT International Application WO 2008/101157
Published on 8/21/08 for other disclosed 5',2'-bis substituted
nucleosides) or replacement of the ribosyl ring oxygen atom with S
with further substitution at the 2'-position (see published U.S.
Patent Application US2005-0130923, published on Jun. 16, 2005) or
alternatively 5'-substitution of a BNA (see PCT International
Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA
is substituted with for example a 5'-methyl or a 5'-vinyl
group).
[0317] Examples of nucleosides having modified sugar moieties
include without limitation nucleosides comprising 5'-vinyl,
5'-methyl (R or 5), 4'-S, 2'-F, 2'-OCH.sub.3,
2'--OCH.sub.2CH.sub.3, 2'--OCH.sub.2CH.sub.2F and
2'-O(CH.sub.2).sub.20CH.sub.3 substituent groups. The substituent
at the 2' position can also be selected from allyl, amino, azido,
thio, O-allyl, O--C.sub.1-C.sub.10 alkyl, OCF.sub.3, OCH.sub.2F,
O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n),
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), and
O--CH.sub.2--C(.dbd.O)--N(R.sub.l)--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n)-
, where each R.sub.l, R.sub.m and R.sub.n is, independently, H or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl.
[0318] As used herein, "bicyclic nucleosides" refer to modified
nucleosides comprising a bicyclic sugar moiety. Examples of
bicyclic nucleosides include without limitation nucleosides
comprising a bridge between the 4' and the 2' ribosyl ring atoms.
In certain embodiments, antisense compounds provided herein include
one or more bicyclic nucleosides comprising a 4' to 2' bridge.
Examples of such 4' to 2' bridged bicyclic nucleosides, include but
are not limited to one of the formulae: 4'-(CH.sub.2)--O-2' (LNA);
4'-(CH.sub.2)--S-2'; 4'--(CH.sub.2).sub.2--O-2' (ENA);
4'-CH(CH.sub.3)--O-2' and 4'-CH(CH.sub.2OCH.sub.3)--O-2' (and
analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15,
2008); 4'-C(CH.sub.3)(CH.sub.3)--O-2' (and analogs thereof see
published International Application WO/2009/006478, published Jan.
8, 2009); 4'-CH.sub.2--N(OCH.sub.3)-2' (and analogs thereof see
published International Application WO/2008/150729, published Dec.
11, 2008); 4'-CH.sub.2--O--N(CH.sub.3)-2' (see published U.S.
Patent Application US2004-0171570, published Sep. 2, 2004);
4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or
a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23,
2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see Chattopadhyaya et al.,
J. Org. Chem., 2009, 74, 118-134); and
4'-CH.sub.2--C--(.dbd.CH.sub.2)-2' (and analogs thereof see
published International Application WO 2008/154401, published on
Dec. 8, 2008).
[0319] Further reports related to bicyclic nucleosides can also be
found in published literature (see for example: Singh et al., Chem.
Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,
3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U S. A., 2000,
97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,
2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;
Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379;
Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561;
Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr.
Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490;
6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207;
7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No.
US2008-0039618; US2009-0012281; U.S. Patent Serial Nos. 60/989,574;
61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and
61/099,844; Published PCT International applications WO
1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO
2008/150729; WO 2008/154401; and WO 2009/006478. Each of the
foregoing bicyclic nucleosides can be prepared having one or more
stereochemical sugar configurations including for example
.alpha.-L-ribofuranose and .beta.-D-ribofuranose (see PCT
international application PCT/DK98/00393, published on Mar. 25,
1999 as WO 99/14226).
[0320] In certain embodiments, bicyclic sugar moieties of BNA
nucleosides include, but are not limited to, compounds having at
least one bridge between the 4' and the 2' position of the
pentofuranosyl sugar moiety wherein such bridges independently
comprises 1 or from 2 to 4 linked groups independently selected
from --[C(R.sub.n)(R.sub.b)].sub.n--,
--C(R.sub.n).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--, --C(.dbd.O)--,
--C(.dbd.NR.sub.a)--, --C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--,
--S(.dbd.O).sub.x--, and --N(R.sub.a)--;
[0321] wherein:
[0322] x is 0, 1, or 2;
[0323] n is 1, 2, 3, or 4;
[0324] each R.sub.a and R.sub.b is, independently, H, a protecting
group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted
C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle
radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7
alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical,
halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1,
acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0325] each J.sub.1 and J.sub.2 is, independently, H,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl
(C(.dbd.O)--H), substituted acyl, a heterocycle radical, a
substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl,
substituted C.sub.1-C.sub.12 aminoalkyl or a protecting group.
[0326] In certain embodiments, the bridge of a bicyclic sugar
moiety is --[C(R.sub.n)(R.sub.b)].sub.n--,
--[C(R.sub.n)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O--
or --C(R.sub.aR.sub.b)--O--N(R)--. In certain embodiments, the
bridge is 4'-CH.sub.2-2', 4'--(CH.sub.2).sub.2-2',
(CH.sub.2).sub.3-2', 4'--CH.sub.2--O-2',
4'--(CH.sub.2).sub.2--O-2', 4'--CH.sub.2--O--N(R)-2' and
4'-CH.sub.2--N(R)--O-2'- wherein each R is, independently, H, a
protecting group or C.sub.1-C.sub.12 alkyl.
[0327] In certain embodiments, bicyclic nucleosides are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L
configuration or in the 13-D configuration. Previously,
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') BNA's have been
incorporated into antisense oligonucleotides that showed antisense
activity (Frieden et al., Nucleic Acids Research, 2003, 21,
6365-6372).
[0328] In certain embodiments, bicyclic nucleosides include, but
are not limited to, (A) .alpha.-L-methyleneoxy (4'-CH.sub.2--O-2')
BNA, (B) .beta.-D-methyleneoxy (4'-CH.sub.2--O-2') BNA, (C)
ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) aminooxy
(4'-CH.sub.2--O--N(R)-2') BNA, (E) oxyamino
(4'-CH.sub.2--N(R)--O-2') BNA, and (F) methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA, (G) methylene-thio (4'-CH.sub.2--S-2')
BNA, (H) methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl
carbocyclic (4'-CH.sub.2--CH(CH.sub.3)-2') BNA, and (J) propylene
carbocyclic (4'-(CH.sub.2).sub.3-2') BNA as depicted below.
##STR00002## ##STR00003##
wherein Bx is the base moiety and R is independently H, a
protecting group or C.sub.1-C.sub.12 alkyl.
[0329] In certain embodiments, bicyclic nucleosides are provided
having Formula I:
##STR00004##
wherein:
[0330] Bx is a heterocyclic base moiety;
[0331] -Q.sub.a-Q.sub.b-Q.sub.c- is --CH.sub.2--N(RO--CH.sub.2--,
--C(.dbd.O)--N(RO--CH.sub.2--, --CH.sub.2--O--N(R.sub.c)--,
--CH.sub.2--N(R.sub.c)--O-- or --N(R.sub.c)--O--CH.sub.2;
[0332] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting
group; and
[0333] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium.
[0334] In certain embodiments, bicyclic nucleosides are provided
having Formula II:
##STR00005##
wherein:
[0335] Bx is a heterocyclic base moiety;
[0336] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0337] Z.sub.a is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl, acyl, substituted acyl, substituted amide, thiol or
substituted thio.
[0338] In one embodiment, each of the substituted groups is,
independently, mono or poly substituted with substituent groups
independently selected from halogen, oxo, hydroxyl, OJ.sub.c,
NJ.sub.cJ.sub.d, SJ.sub.c, N.sub.3, OC(.dbd.X)J.sub.c, and
NJ.sub.cC(.dbd.X)NJ.sub.eJ.sub.d, wherein each J.sub.e, J.sub.d and
J.sub.e is, independently, H, C.sub.1-C.sub.6 alkyl, or substituted
C.sub.1-C.sub.6 alkyl and X is O or NJ.sub.e.
[0339] In certain embodiments, bicyclic nucleosides are provided
having Formula III:
##STR00006##
wherein:
[0340] Bx is a heterocyclic base moiety;
[0341] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0342] Z.sub.b is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl or substituted acyl (C(.dbd.O)--).
[0343] In certain embodiments, bicyclic nucleosides are provided
having Formula IV:
##STR00007##
wherein:
[0344] Bx is a heterocyclic base moiety;
[0345] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0346] R.sub.d is C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl;
[0347] each q.sub.a, q.sub.b, q.sub.e and q.sub.d is,
independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, substituted
C.sub.1-C.sub.6 alkoxyl, acyl, substituted acyl, C.sub.1-C.sub.6
aminoalkyl or substituted C.sub.1-C.sub.6 aminoalkyl;
[0348] In certain embodiments, bicyclic nucleosides are provided
having Formula V:
##STR00008##
wherein:
[0349] Bx is a heterocyclic base moiety;
[0350] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0351] q.sub.a, q.sub.b, q.sub.e and of are each, independently,
hydrogen, halogen, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxy, substituted
C.sub.1-C.sub.12 alkoxy, OJ.sub.j, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j,
C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j,
O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k,
N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;
[0352] or q.sub.e and q.sub.f together are
.dbd.C(q.sub.g)(q.sub.h);
[0353] q.sub.g and q.sub.h are each, independently, H, halogen,
C.sub.1-C.sub.12 alkyl or substituted C.sub.1-C.sub.12 alkyl.
[0354] The synthesis and preparation of the methyleneoxy
(4'-CH.sub.2--O-2') BNA monomers adenine, cytosine, guanine,
5-methyl-cytosine, thymine and uracil, along with their
oligomerization, and nucleic acid recognition properties have been
described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs
and preparation thereof are also described in WO 98/39352 and WO
99/14226.
[0355] Analogs of methyleneoxy (4'-CH.sub.2--O-2') BNA and
2'-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med.
Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside
analogs comprising oligodeoxyribonucleotide duplexes as substrates
for nucleic acid polymerases has also been described (Wengel et
al., WO 99/14226). Furthermore, synthesis of 2'-amino-BNA, a novel
comformationally restricted high-affinity oligonucleotide analog
has been described in the art (Singh et al., J. Org. Chem., 1998,
63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's
have been prepared and the thermal stability of their duplexes with
complementary RNA and DNA strands has been previously reported.
[0356] In certain embodiments, bicyclic nucleosides are provided
having Formula VI:
##STR00009##
wherein:
[0357] Bx is a heterocyclic base moiety;
[0358] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0359] each q.sub.i, q.sub.j, q.sub.k and q.sub.l is,
independently, H, halogen, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxyl, substituted
C.sub.1-C.sub.12 alkoxyl, OJ.sub.j, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j,
C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j,
O--C(.dbd.O)NJ.sub.jJ.sub.k; and N(H)C(.dbd.NH)NJ.sub.jJ.sub.k,
N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;
and
[0360] q.sub.i and q.sub.j or q.sub.l and q.sub.k together are
.dbd.C(q.sub.g)(q.sub.h), wherein q.sub.g and q.sub.h are each,
independently, H, halogen, C.sub.1-C.sub.12 alkyl or substituted
C.sub.1-C.sub.12 alkyl.
[0361] One carbocyclic bicyclic nucleoside having a
4'-(CH.sub.2).sub.3-2' bridge and the alkenyl analog bridge
4'-CH.dbd.CH--CH.sub.2-2' have been described (Freier et al.,
Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,
J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation
of carbocyclic bicyclic nucleosides along with their
oligomerization and biochemical studies have also been described
(Srivastava et al., J. Am. Chem. Soc., 2007, 129(26),
8362-8379).
[0362] As used herein, "4'-2' bicyclic nucleoside" or "4' to 2'
bicyclic nucleoside" refers to a bicyclic nucleoside comprising a
furanose ring comprising a bridge connecting two carbon atoms of
the furanose ring connects the 2' carbon atom and the 4' carbon
atom of the sugar ring.
[0363] As used herein, "monocylic nucleosides" refer to nucleosides
comprising modified sugar moieties that are not bicyclic sugar
moieties. In certain embodiments, the sugar moiety, or sugar moiety
analogue, of a nucleoside may be modified or substituted at any
position.
[0364] As used herein, "2'-modified sugar" means a furanosyl sugar
modified at the 2' position. In certain embodiments, such
modifications include substituents selected from: a halide,
including, but not limited to substituted and unsubstituted alkoxy,
substituted and unsubstituted thioalkyl, substituted and
unsubstituted amino alkyl, substituted and unsubstituted alkyl,
substituted and unsubstituted allyl, and substituted and
unsubstituted alkynyl. In certain embodiments, 2' modifications are
selected from substituents including, but not limited to:
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nF,
O(CH.sub.2).sub.nONH.sub.2, OCH.sub.2C(.dbd.O)N(H)CH.sub.3, 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 2'-substituent groups can also be
selected from: C.sub.1-C.sub.12 alkyl, substituted alkyl, alkenyl,
alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3,
OCN, Cl, Br, CN, F, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving
pharmacokinetic properties, or a group for improving the
pharmacodynamic properties of an antisense compound, and other
substituents having similar properties. In certain embodiments,
modified nucleosides comprise a 2'-MOE side chain (Baker et al., J.
Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have
been described as having improved binding affinity compared to
unmodified nucleosides and to other modified nucleosides, such as
2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having
the 2'-MOE substituent also have been shown to be antisense
inhibitors of gene expression with promising features for in vivo
use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al.,
Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans.,
1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,
1997, 16, 917-926).
[0365] As used herein, a "modified tetrahydropyran nucleoside" or
"modified THP nucleoside" means a nucleoside having a six-membered
tetrahydropyran "sugar" substituted in for the pentofuranosyl
residue in normal nucleosides (a sugar surrogate). Modified THP
nucleosides include, but are not limited to, what is referred to in
the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),
manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002,
10, 841-854), fluoro HNA (F-HNA) or those compounds having Formula
VII:
##STR00010##
wherein independently for each of said at least one tetrahydropyran
nucleoside analog of Formula VII:
[0366] Bx is a heterocyclic base moiety;
[0367] T.sub.a and T.sub.b are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.a and
T.sub.b is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.a and T.sub.b is H, a hydroxyl protecting group, a
linked conjugate group or a 5' or 3'-terminal group;
[0368] q.sub.i, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and
q.sub.7 are each independently, H, C.sub.1-C.sub.6 alkyl,
substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or
substituted C.sub.2-C.sub.6 alkynyl; and each of R.sub.1 and
R.sub.2 is selected from hydrogen, hydroxyl, halogen, substituted
or unsubstituted alkoxy, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3,
OC(.dbd.X)J.sub.1, OC(.dbd.X)NJ.sub.1J.sub.2,
NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2 and CN, wherein X is O, S or
NJ.sub.1 and each 71, J.sub.2 and J.sub.3 is, independently, H or
C.sub.1-C.sub.6 alkyl.
[0369] In certain embodiments, the modified THP nucleosides of
Formula VII are provided wherein q.sub.i, q.sub.2, q.sub.3,
q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain
embodiments, at least one of q.sub.i, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain
embodiments, at least one of q.sub.i, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP
nucleosides of Formula VII are provided wherein one of R.sub.1 and
R.sub.2 is fluoro. In certain embodiments, R.sub.1 is fluoro and
R.sub.2 is H; R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is H
and R.sub.2 is methoxyethoxy.
[0370] As used herein, "2'-modified" or "2'-substituted" refers to
a nucleoside comprising a sugar comprising a substituent at the 2'
position other than H or OH. 2'-modified nucleosides, include, but
are not limited to, bicyclic nucleosides wherein the bridge
connecting two carbon atoms of the sugar ring connects the 2'
carbon and another carbon of the sugar ring; and nucleosides with
non-bridging 2' substituents, such as allyl, amino, azido, thio,
O-allyl, O--C.sub.1-C.sub.10 alkyl, --OCF.sub.3,
O--(CH.sub.2).sub.2--O--CH.sub.3, 2'--O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl. 2'-modified nucleosides may further
comprise other modifications, for example at other positions of the
sugar and/or at the nucleobase.
[0371] As used herein, "2'-F" refers to a nucleoside comprising a
sugar comprising a fluoro group at the 2' position.
[0372] As used herein, "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl"
each refers to a nucleoside comprising a sugar comprising an
--OCH.sub.3 group at the 2' position of the sugar ring.
[0373] As used herein, "MOE" or "2'-MOE" or
"2'-OCH.sub.2CH.sub.2OCH.sub.3" or "2'-O-methoxyethyl" each refers
to a nucleoside comprising a sugar comprising a
--OCH.sub.2CH.sub.2OCH.sub.3 group at the 2' position of the sugar
ring.
[0374] As used herein, "oligonucleotide" refers to a compound
comprising a plurality of linked nucleosides. In certain
embodiments, one or more of the plurality of nucleosides is
modified. In certain embodiments, an oligonucleotide comprises one
or more ribonucleosides (RNA) and/or deoxyribonucleosides
(DNA).
[0375] Many other bicyclo and tricyclo sugar surrogate ring systems
are also known in the art that can be used to modify nucleosides
for incorporation into antisense compounds (see for example review
article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854).
[0376] Such ring systems can undergo various additional
substitutions to enhance activity.
[0377] Methods for the preparations of modified sugars are well
known to those skilled in the art. In nucleotides having modified
sugar moieties, the nucleobase moieties (natural, modified or a
combination thereof) are maintained for hybridization with an
appropriate nucleic acid target.
[0378] In certain embodiments, antisense compounds comprise one or
more nucleosides having modified sugar moieties. In certain
embodiments, the modified sugar moiety is 2'-MOE. In certain
embodiments, the 2'-MOE modified nucleosides are arranged in a
gapmer motif. In certain embodiments, the modified sugar moiety is
a bicyclic nucleoside having a (4'-CH(CH.sub.3)--O-2') bridging
group. In certain embodiments, the (4'-CH(CH.sub.3)--O-2') modified
nucleosides are arranged throughout the wings of a gapmer
motif.
Modified Nucleobases
[0379] Nucleobase (or base) modifications or substitutions are
structurally distinguishable from, yet functionally interchangeable
with, naturally occurring or synthetic unmodified nucleobases. Both
natural and modified nucleobases are capable of participating in
hydrogen bonding. Such nucleobase modifications may impart nuclease
stability, binding affinity or some other beneficial biological
property to antisense compounds. Modified nucleobases include
synthetic and natural nucleobases such as, for example,
5-methylcytosine (5-me-C). Certain nucleobase substitutions,
including 5-methylcytosine substitutions, are particularly useful
for increasing the binding affinity of an antisense compound for a
target nucleic acid. For example, 5-methylcytosine substitutions
have been shown to increase nucleic acid duplex stability by
0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B.,
eds., Antisense Research and Applications, CRC Press, Boca Raton,
1993, pp. 276-278).
[0380] Additional unmodified nucleobases include 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.
[0381] Heterocyclic base moieties 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. Nucleobases that are particularly useful for increasing
the binding affinity of antisense compounds include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted
purines, including 2 aminopropyladenine, 5-propynyluracil and
5-propynylcytosine.
[0382] In certain embodiments, antisense compounds targeted to a
transthyretin nucleic acid comprise one or more modified
nucleobases. In certain embodiments, gap-widened antisense
oligonucleotides targeted to a transthyretin nucleic acid comprise
one or more modified nucleobases. In certain embodiments, the
modified nucleobase is 5-methylcytosine. In certain embodiments,
each cytosine is a 5-methylcytosine.
Compositions and Methods for Formulating Pharmaceutical
Compositions
[0383] Antisense oligonucleotides may be admixed with
pharmaceutically acceptable active or inert substance for the
preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical
compositions are dependent upon a number of criteria, including,
but not limited to, route of administration, extent of disease, or
dose to be administered.
[0384] Antisense compound targeted to a transthyretin nucleic acid
can be utilized in pharmaceutical compositions by combining the
antisense compound with a suitable pharmaceutically acceptable
diluent or carrier. A pharmaceutically acceptable diluent includes
phosphate-buffered saline (PBS). PBS is a diluent suitable for use
in compositions to be delivered parenterally. Accordingly, in one
embodiment, employed in the methods described herein is a
pharmaceutical composition comprising an antisense compound
targeted to a transthyretin nucleic acid and a pharmaceutically
acceptable diluent. In certain embodiments, the pharmaceutically
acceptable diluent is PBS. In certain embodiments, the antisense
compound is an antisense oligonucleotide.
[0385] Pharmaceutical compositions comprising antisense compounds
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters, or any other oligonucleotide which, upon
administration to an animal, including a human, is capable of
providing (directly or indirectly) the biologically active
metabolite or residue thereof. Accordingly, for example, the
disclosure is also drawn to pharmaceutically acceptable salts of
antisense compounds, prodrugs, pharmaceutically acceptable salts of
such prodrugs, and other bioequivalents. Suitable pharmaceutically
acceptable salts include, but are not limited to, sodium and
potassium salts.
[0386] A prodrug can include the incorporation of additional
nucleosides at one or both ends of an antisense compound which are
cleaved by endogenous nucleases within the body, to form the active
antisense compound.
Conjugated Antisense Compounds
[0387] Antisense compounds may be covalently linked to one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the resulting antisense
oligonucleotides. Typical conjugate groups include cholesterol
moieties and lipid moieties. Additional conjugate groups include
carbohydrates, phospholipids, biotin, phenazine, folate,
phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,
coumarins, and dyes.
[0388] Antisense compounds can also be modified to have one or more
stabilizing groups that are generally attached to one or both
termini of antisense compounds to enhance properties such as, for
example, nuclease stability. Included in stabilizing groups are cap
structures. These terminal modifications protect the antisense
compound having terminal nucleic acid from exonuclease degradation,
and can help in delivery and/or localization within a cell. The cap
can be present at the 5' terminus (5'-cap), or at the 3'-terminus
(3'-cap), or can be present on both termini. Cap structures are
well known in the art and include, for example, inverted deoxy
abasic caps. Further 3' and 5'-stabilizing groups that can be used
to cap one or both ends of an antisense compound to impart nuclease
stability include those disclosed in WO 03/004602 published on Jan.
16, 2003.
Cell Culture and Antisense Compounds Treatment
[0389] The effects of antisense compounds on the level, activity or
expression of transthyretin nucleic acids can be tested in vitro in
a variety of cell types. Cell types used for such analyses are
available from commercial vendors (e.g. American Type Culture
Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park,
N.C.; Clonetics Corporation, Walkersville, Md.) and cells are
cultured according to the vendor's instructions using commercially
available reagents (e.g. Invitrogen Life Technologies, Carlsbad,
Calif.). Illustrative cell types include, but are not limited to,
HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281
fibroblasts and LLC-MK2 cells.
In Vitro Testing of Antisense Oligonucleotides
[0390] Described herein are methods for treatment of cells with
antisense oligonucleotides, which can be modified appropriately for
treatment with other antisense compounds.
[0391] In general, cells are treated with antisense
oligonucleotides when the cells reach approximately 60-80%
confluence in culture.
[0392] One reagent commonly used to introduce antisense
oligonucleotides into cultured cells includes the cationic lipid
transfection reagent LIPOFECTIN.RTM. (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN.RTM.
in OPTI-MEM.RTM. 1 (Invitrogen, Carlsbad, Calif.) to achieve the
desired final concentration of antisense oligonucleotide and a
LIPOFECTIN.RTM. concentration that typically ranges 2 to 12 ug/mL
per 100 nM antisense oligonucleotide.
[0393] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes LIPOFECTAMINE 2000.RTM. (Invitrogen,
Carlsbad, Calif.). Antisense oligonucleotide is mixed with
LIPOFECTAMINE 2000.RTM. in OPTI-MEM.RTM. 1 reduced serum medium
(Invitrogen, Carlsbad, Calif.) to achieve the desired concentration
of antisense oligonucleotide and a LIPOFECTAMINE.RTM. concentration
that typically ranges 2 to 12 ug/mL per 100 nM antisense
oligonucleotide.
[0394] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes Cytofectin.RTM. (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotide is mixed with Cytofectin.RTM. in
OPTI-MEM.RTM. 1 reduced serum medium (Invitrogen, Carlsbad, Calif.)
to achieve the desired concentration of antisense oligonucleotide
and a Cytofectin.RTM. concentration that typically ranges 2 to 12
ug/mL per 100 nM antisense oligonucleotide.
[0395] Another technique used to introduce antisense
oligonucleotides into cultured cells includes electroporation.
[0396] Cells are treated with antisense oligonucleotides by routine
methods. Cells are typically harvested 16-24 hours after antisense
oligonucleotide treatment, at which time RNA or protein levels of
target nucleic acids are measured by methods known in the art and
described herein. In general, when treatments are performed in
multiple replicates, the data are presented as the average of the
replicate treatments.
[0397] The concentration of antisense oligonucleotide used varies
from cell line to cell line. Methods to determine the optimal
antisense oligonucleotide concentration for a particular cell line
are well known in the art. Antisense oligonucleotides are typically
used at concentrations ranging from 1 nM to 300 nM when transfected
with LIPOFECTAMINE2000.RTM., Lipofectin or Cytofectin. Antisense
oligonucleotides are used at higher concentrations ranging from 625
to 20,000 nM when transfected using electroporation.
RNA Isolation
[0398] RNA analysis can be performed on total cellular RNA or
poly(A)+ mRNA. Methods of RNA isolation are well known in the art.
RNA is prepared using methods well known in the art, for example,
using the TRIZOL.RTM. Reagent (Invitrogen, Carlsbad, Calif.)
according to the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
[0399] Inhibition of levels or expression of a transthyretin
nucleic acid can be assayed in a variety of ways known in the art.
For example, target nucleic acid levels can be quantitated by,
e.g., Northern blot analysis, competitive polymerase chain reaction
(PCR), or quantitative real-time PCR. RNA analysis can be performed
on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are
well known in the art. Northern blot analysis is also routine in
the art. Quantitative real-time PCR can be conveniently
accomplished using the commercially available ABI PRISM.RTM. 7600,
7700, or 7900 Sequence Detection System, available from PE-Applied
Biosystems, Foster City, Calif. and used according to
manufacturer's instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
[0400] Quantitation of target RNA levels may be accomplished by
quantitative real-time PCR using the ABI PRISM.RTM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. Methods of
quantitative real-time PCR are well known in the art.
[0401] Prior to real-time PCR, the isolated RNA is subjected to a
reverse transcriptase (RT) reaction, which produces complementary
DNA (cDNA) that is then used as the substrate for the real-time PCR
amplification. The RT and real-time PCR reactions are performed
sequentially in the same sample well. RT and real-time PCR reagents
are obtained from Invitrogen (Carlsbad, Calif.). RT, real-time-PCR
reactions are carried out by methods well known to those skilled in
the art.
[0402] Gene (or RNA) target quantities obtained by real time PCR
are normalized using either the expression level of a gene whose
expression is constant, such as cyclophilin A, or by quantifying
total RNA using RIBOGREEN.RTM. (Invitrogen, Inc. Carlsbad, Calif.).
Cyclophilin A expression is quantified by real time PCR, by being
run simultaneously with the target, multiplexing, or separately.
Total RNA is quantified using RIBOGREEN.RTM. RNA quantification
reagent (Invitrogen, Inc. Eugene, Oreg.). Methods of RNA
quantification by RIBOGREEN.RTM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR.RTM.
4000 instrument (PE Applied Biosystems) is used to measure
RIBOGREEN.RTM. fluorescence.
[0403] Probes and primers are designed to hybridize to a
transthyretin nucleic acid. Methods for designing real-time PCR
probes and primers are well known in the art, and may include the
use of software such as PRIMER EXPRESS.RTM. Software (Applied
Biosystems, Foster City, Calif.).
Analysis of Protein Levels
[0404] Antisense inhibition of transthyretin nucleic acids can be
assessed by measuring transthyretin protein levels. Protein levels
of transthyretin can be evaluated or quantitated in a variety of
ways well known in the art, such as immunoprecipitation, Western
blot analysis (immunoblotting), enzyme-linked immunosorbent assay
(ELISA), quantitative protein assays, protein activity assays (for
example, caspase activity assays), immunohistochemistry,
immunocytochemistry or fluorescence-activated cell sorting (FACS).
Antibodies directed to a target 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. Antibodies useful for the detection of human
and rat transthyretin are commercially available.
In Vivo Testing of Antisense Compounds
[0405] Antisense compounds, for example, antisense
oligonucleotides, are tested in animals to assess their ability to
inhibit expression of transthyretin and produce phenotypic changes.
Testing may be performed in normal animals, or in experimental
disease models. For administration to animals, antisense
oligonucleotides are formulated in a pharmaceutically acceptable
diluent, such as phosphate-buffered saline. Administration includes
parenteral routes of administration. Following a period of
treatment with antisense oligonucleotides, RNA is isolated from
tissue and changes in transthyretin nucleic acid expression are
measured. Changes in transthyretin protein levels are also
measured.
Certain Compounds
[0406] About two hundred and forty six newly designed antisense
compounds of various lengths, motifs and backbone composition were
tested for their effect on human transthyretin mRNA in vitro in
several cell types. The new compounds were compared with about
seventy nine previously designed compounds including ISIS NOs.
304267, 304268, 304280, 304284, 304285, 304286, 304287, 304288,
304289, 304290, 304291, 304292, 304293, 304294, 304296, 304297,
304298, 304299, 304300, 304301, 304302, 304303, 304304, 304307,
304308, 304309, 304311, and 304312 which have previously been
determined to be some of the most potent antisense compounds in
vitro (see e.g., U.S. Patent Publication Nos. US2005/0244869 and
US2009/0082300). Of the about three hundred and twenty five newly
designed and previously designed antisense compounds, about fifteen
compounds were selected for further study based on in vitro
potency. The selected compounds were tested for in vivo potency and
tolerability in a transgenic mouse model (see Example 10). Of the
fifteen compounds tested, eleven were selected and tested for
systemic tolerability (see Example 11) and half-life measurement in
liver (see Example 12) in CD1 mice, and also for systemic
tolerability (see Example 13) and pharmacokinetic studies of
oligonucleotide concentration in liver (see Example 14) in
Sprague-Dawley rats. From these studies, seven compounds were
tested for dose dependent inhibition and tolerability in transgenic
mice (see Example 15). Furthermore, fifteen additional compounds
were selected from Table 1 and six additional compounds with
various motifs were designed with overlapping sequences to ISIS
420951, which displayed high potency and tolerability in the
above-mentioned assays. These additional compounds were compared
with ISIS 420951 for potency and tolerability in transgenic mice
(see Example 16). Based on all these studies (Examples 10-16),
twenty two compounds were selected and tested for systemic
tolerability in CD1 mice (see Example 17). Seven compounds were
considered tolerable in the mouse model and further tested for
systemic tolerability in Sprague-Dawley rats (see Example 18) and
for pharmacokinetic studies of oligonucleotide concentration in the
liver and kidney (see Example 19). The seven compounds were also
tested for dose-dependent potency in transgenic mice (see Example
20).
[0407] Final evaluation of these studies (Examples 16-20), led to
the selection of nine compounds having a nucleobase sequence of a
sequence recited in SEQ ID NO: 25, 78, 80, 86, 87, 115, 120, 122
and 124. By virtue of their complementary sequence, the compounds
are complementary to the regions 505-524, 507-526, 508-527,
513-532, 515-534, 516-535, 580-599, 585-604, 587-606, or 589-608 of
SEQ ID NO: 1. In certain embodiments, the compounds targeting the
listed regions, as further described herein, comprise a modified
oligonucleotide having some nucleobase portion of the sequence
recited in the SEQ ID NOs, as further described herein, In certain
embodiments, the compounds targeting the listed regions or having a
nucleobase portion of a sequence recited in the listed SEQ ID NOs
can be of various length, as further described herein, and can have
one of various motifs, as further described herein. In certain
embodiments, a compound targeting a region or having a nucleobase
portion of a sequence recited in the listed SEQ ID NOs has the
specific length and motif as indicated by the ISIS NOs: ISIS
304299, ISIS 420913, ISIS 420915, ISIS 420921, ISIS 420922, ISIS
420950, ISIS 420955, ISIS 420957, or ISIS 420959.
[0408] The nine compounds having a nucleobase sequence of a
sequence recited in SEQ ID NO: 25, 78, 80, 86, 87, 115, 120, 122
and 124, were further tested for dose dependent inhibition in
primary hepatocytes of cynomolgus monkey (See Example 21). These
compounds were also tested for optimal viscosity (Example 22). The
half life in the liver of CD1 mice of seven of the compounds having
a nucleobase sequence of a sequence recited in SEQ ID NOs: 78, 86,
87. 115, 120 and 124 was also evaluated (Example 23).
[0409] Final evaluation of these studies (Examples 1-23), led to
the selection of eight compounds having a nucleobase sequence of a
sequence recited in SEQ ID NO: 25, 80, 86, 87, 115, 120, 122 and
124. By virtue of their complementary sequence, the compounds are
complementary to the regions 504-523, 505-524, 512-531, 513-532,
577-596, 582-601, 584-603, and 586-605 of SEQ ID NO: 1. In certain
embodiments, the compounds targeting the listed regions, as further
described herein, comprise a modified oligonucleotide having some
nucleobase portion of the sequence recited in the SEQ ID NOs, as
further described herein, In certain embodiments, the compounds
targeting the listed regions or having a nucleobase portion of a
sequence recited in the listed SEQ ID NOs can be of various length,
as further described herein, and can have one of various motifs, as
further described herein. In certain embodiments, a compound
targeting a region or having a nucleobase portion of a sequence
recited in the listed SEQ ID NOs has the specific length and motif
as indicated by the ISIS NOs: ISIS 304299, ISIS 420915, ISIS
420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS 420957, or ISIS
420959.
[0410] These eight compounds were tested for efficacy,
pharmacokinetic profile and tolerability in cynomolgus monkeys
(Example 24). The inhibition studies in these monkeys indicated
that treatment with some of these compounds caused high inhibition
of TTR mRNA in the liver. Specifically, treatment with ISIS 420950,
ISIS 420955 and ISIS 420915 caused 91%, 79% and 78% inhibition,
respectively compared to the PBS control. It was noted that ISIS
420915 caused greater inhibition of TTR (78%) mRNA compared to ISIS
304299 (59%), even though the two oligonucleotides differ from each
other by a single base-pair shift of their target region on SEQ ID
NO: 1. Protein analysis also complemented the RNA analysis data
with treatment with ISIS 420915 causing 76% inhibition and
treatment with ISIS 304299 causing 47% inhibition of TTR protein
compared to the control. RBP4 protein levels, as a protein
associated with transthyretin, was also expected to decrease after
treatment with the antisense compounds. RBP4 protein levels
decreased by 63% after treatment with ISIS 420915. Treatment with
ISIS 304299 decreased RBP4 protein levels by 19%. Additionally,
ISIS 420915 was more tolerable than ISIS 304299, as indicated in
the monkey study (Example 24) Transaminase levels of monkeys
treated with ISIS 304299 (ALT 81 IU/L and AST 101 IU/L) were higher
than those treated with ISIS 420915 (ALT 68 IU/L and AST 62 IU/L).
The complement C.sub.3 levels of monkeys treated with ISIS 304299
(96 mg/dL) were lower than that of monkeys treated with ISIS 420915
(104 mg/dL).
[0411] Accordingly, provided herein are antisense compounds with
any one or more of the improved characteristics. In a certain
embodiments, provided herein are compounds comprising a modified
oligonucleotide as further described herein targeted to or
specifically hybridizable with the region of nucleotides of SEQ ID
NO: 1.
[0412] Accordingly, provided herein are antisense compounds with
any one or more of the improved characteristics. In a certain
embodiments, provided herein are compounds comprising a modified
oligonucleotide as further described herein targeted to or
specifically hybridizable with the region of nucleotides of SEQ ID
NO: 2.
[0413] Accordingly, provided herein are antisense compounds with
any one or more of the improved characteristics. In a certain
embodiments, provided herein are compounds comprising a modified
oligonucleotide as further described herein targeted to or
specifically hybridizable with the region of nucleotides of SEQ ID
NO: 4.
[0414] In certain embodiments, the compounds as described herein
are efficacious by virtue of having at least one of an in vitro
IC.sub.50 of less than 2.9 uM, less than 2.2 uM, less than 2.0 uM,
less than 1.5 uM, less than 1.4 uM, less than 1.3 uM, less than 1.0
uM, less than 0.7 uM, less than 0.6 uM, when delivered to a
cynomolgous monkey hepatocyte cell line using electroporation as
described in Example 67. In certain embodiments, the compounds as
described herein are highly tolerable as demonstrated by having at
least one of an increase in ALT or AST value of no more than 4
fold, 3 fold, or 2 fold over saline treated animals; or an increase
in liver, spleen or kidney weight of no more than 30%, 20%, 15%,
12%, 10%, 5% or 2%.
Certain Indications
[0415] In certain embodiments, provided herein are methods of
treating an individual comprising administering one or more
pharmaceutical compositions as described herein. In certain
embodiments, the individual has central nervous system related
disease.
[0416] As shown in the examples below, compounds targeted to
transthyretin as described herein have been shown to reduce the
severity of physiological symptoms of central nervous system
related diseases. In certain of the experiments, the compounds
reduced rate of amyloid plaque formation, e.g., the animals
continued to experience symptoms, but the symptoms were less severe
compared to untreated animals. In other of the experiments,
however, the compounds appear to result in regeneration of function
over time; e.g., animals treated for a longer period of time
experienced less severe symptoms than those administered the
compounds for a shorter period of time. The ability of the
compounds exemplified below to restore function therefore
demonstrates that symptoms of the disease may be reversed by
treatment with a compound as described herein.
[0417] Accordingly, provided herein are methods for ameliorating a
symptom associated with central nervous system related, cardiac,
neuropathologic or gastrointestinal disease in a subject in need
thereof. In certain embodiments, provided is a method for reducing
the rate of onset of a symptom associated with central nervous
system related, cardiac, neuropathologic or gastrointestinal
disease. In certain embodiments, provided is a method for reducing
the severity of a symptom associated with central nervous system
related, cardiac, neuropathologic or gastrointestinal. In such
embodiments, the methods comprise administering to an individual in
need thereof a therapeutically effective amount of a compound
targeted to a Transthyretin nucleic acid.
[0418] Transthyretin amyloidosis is characterized by numerous
physical, neurological, psychiatric, and/or peripheral symptoms.
Any symptom known to one of skill in the art to be associated with
transthyretin amyloidosis can be ameliorated or otherwise modulated
as set forth above in the methods described above.
[0419] In certain embodiments, the symptom is a physical,
cognitive, psychiatric, or peripheral symptom. In certain
embodiments, the symptom is a physical symptom selected from the
group consisting of restlessness, lack of coordination, nystagmus,
spastic paraparesis, lack of muscle coordination, impaired vision,
insomnia, unusual sensations, myoclonus, blindness, loss of speech,
Carpal tunnel syndrome, seizures, subarachnoid hemorrhages, stroke
and bleeding in the brain, hydrocephalus, ataxia, and spastic
paralysis, coma, sensory neuropathy, parathesia, hypesthesia, motor
neuropathy, autonomic neuropathy, orthostatic hypotension, cyclic
constipation, cyclic diarrhea, nausea, vomiting, reduced sweating,
impotence, delayed gastric emptying, urinary retention, urinary
incontinence, progressive cardiopathy, fatigue, shortness of
breath, weight loss, lack of appetite, numbness, tingling,
weakness, enlarged tongue, nephrotic syndrome, congestive heart
failure, dyspnea on exertion, peripheral edema, arrhythmias,
palpitations, light-headedness, syncope, postural hypotension,
peripheral nerve problems, sensory motor impairment, lower limb
neuropathy, upper limb neuropathy, hyperalgesia, altered
temperature sensation, lower extremity weakness, cachexia,
peripheral edema, hepatomegaly, purpura, diastolic dysfunction,
premature ventricular contractions, cranial neuropathy, diminished
deep tendon reflexes, amyloid deposits in the corpus vitreum,
vitreous opacity, dry eyes, glaucoma, scalloped appearance in the
pupils, swelling of the feet due to water retention. In certain
embodiments, the symptom is a cognitive symptom selected from the
group consisting of impaired memory, impaired judgment, and
thinking, impaired planning, impaired flexibility, impaired
abstract thinking, impaired rule acquisition, impaired initiation
of appropriate actions, impaired inhibition of inappropriate
actions, impaired short-term memory, impaired long-term memory,
paranoia, disorientation, confusion, hallucination and dementia. In
certain embodiments, the symptom is a psychiatric symptom selected
from the group consisting of dementia; anxiety, depression, blunted
affect, egocentrisms, aggression, compulsive behavior,
irritability, personality changes, including, impaired memory,
judgment, and thinking and suicidal ideation.
[0420] In certain embodiments, the symptom is restlessness. In
certain embodiments, the symptom is lack of coordination. In
certain embodiments, the symptom is nystagmus. In certain
embodiments, the symptom is spastic paraparesis. In certain
embodiments, the symptom is lack of muscle coordination. In certain
embodiments, the symptom is impaired vision. In certain
embodiments, the symptom is insomnia. In certain embodiments, the
symptom is unusual sensations. In certain embodiments, the symptom
is myoclonus. In certain embodiments, the symptom is blindness. In
certain embodiments, the symptom is loss of speech. In certain
embodiments, the symptom is Carpal tunnel syndrome. In certain
embodiments, the symptom is seizures. In certain embodiments, the
symptom is subarachnoid hemorrhages. In certain embodiments, the
symptom is stroke. In certain embodiments, the symptom is bleeding
in the brain. In certain embodiments, the symptom is hydrocephalus.
In certain embodiments, the symptom is ataxia. In certain
embodiments, the symptom is spastic paralysis. In certain
embodiments, the symptom is coma. In certain embodiments, the
symptom is sensory neuropathy. In certain embodiments, the symptom
is parathesia. In certain embodiments, the symptom is hypesthesia.
In certain embodiments, the symptom is motor neuropathy. In certain
embodiments, the symptom is autonomic neuropathy. In certain
embodiments, the symptom is orthostatic hypotension. In certain
embodiments, the symptom is cyclic constipation. In certain
embodiments, the symptom is cyclic diarrhea. In certain
embodiments, the symptom is nausea. In certain embodiments, the
symptom is vomiting. In certain embodiments, the symptom is reduced
sweating. In certain embodiments, the symptom is impotence. In
certain embodiments, the symptom is delayed gastric emptying. In
certain embodiments, the symptom is urinary retention. In certain
embodiments, the symptom is urinary incontinence. In certain
embodiments, the symptom is progressive cardiopathy. In certain
embodiments, the symptom is fatigue. In certain embodiments, the
symptom is shortness of breath. In certain embodiments, the symptom
is weight loss. In certain embodiments, the symptom is numbness. In
certain embodiments, the symptom is tingling. In certain
embodiments, the symptom is weakness. In certain embodiments, the
symptom is enlarged tongue. In certain embodiments, the symptom is
nephrotic syndrome. In certain embodiments, the symptom is
congestive heart failure. In certain embodiments, the symptom is
dyspnea on exertion. In certain embodiments, the symptom is
peripheral edema. In certain embodiments, the symptom is
arrhythmias. In certain embodiments, the symptom is palpitations.
In certain embodiments, the symptom is light-headedness. In certain
embodiments, the symptom is syncope. In certain embodiments, the
symptom is postural hypotension. In certain embodiments, the
symptom is peripheral nerve problems. In certain embodiments, the
symptom is sensory motor impairment. In certain embodiments, the
symptom is lower limb neuropathy. In certain embodiments, the
symptom is upper limb neuropathy. In certain embodiments, the
symptom is hyperalgesia. In certain embodiments, the symptom is
altered temperature sensation. In certain embodiments, the symptom
is lower extremity weakness. In certain embodiments, the symptom is
cachexia. In certain embodiments, the symptom is edema. In certain
embodiments, the symptom is hepatomegaly. In certain embodiments,
the symptom is purpura. In certain embodiments, the symptom is
diastolic dysfunction. In certain embodiments, the symptom is
premature ventricular contractions. In certain embodiments, the
symptom is cranial neuropathy. In certain embodiments, the symptom
is diminished deep tendon reflexes. In certain embodiments, the
symptom is amyloid deposits in the corpus vitreum. In certain
embodiments, the symptom is vitreous opacity. In certain
embodiments, the symptom is dry eyes. In certain embodiments, the
symptom is glaucoma. In certain embodiments, the symptom is
scalloped appearance in the pupils. In certain embodiments, the
symptom is swelling of the feet due to water retention.
[0421] In certain embodiments, the symptom is impaired memory. In
certain embodiments, the symptom is impaired judgment, and
thinking. In certain embodiments, the symptom is impaired planning.
In certain embodiments, the symptom is impaired flexibility. In
certain embodiments, the symptom is impaired abstract thinking. In
certain embodiments, the symptom is impaired rule acquisition. In
certain embodiments, the symptom is impaired initiation of
appropriate actions. In certain embodiments, the symptom is
impaired inhibition of inappropriate actions. In certain
embodiments, the symptom is impaired short-term memory. In certain
embodiments, the symptom is impaired long-term memory. In certain
embodiments, the symptom is paranoia. In certain embodiments, the
symptom is disorientation. In certain embodiments, the symptom is
confusion. In certain embodiments, the symptom is hallucination. In
certain embodiments, the symptom is dementia.
[0422] In certain embodiments, the symptom is dementia. In certain
embodiments, the symptom is anxiety. In certain embodiments, the
symptom is depression. In certain embodiments, the symptom is
blunted affect. In certain embodiments, the symptom is
egocentrisms. In certain embodiments, the symptom is aggression. In
certain embodiments, the symptom is compulsive behavior. In certain
embodiments, the symptom is irritability. In certain embodiments,
the symptom is personality changes. In certain embodiments, the
symptom is suicidal ideation.
[0423] In certain embodiments, provided are methods of treating an
individual comprising administering one or more pharmaceutical
compositions as described herein. In certain embodiments, the
individual has central nervous system related disease.
[0424] In certain embodiments, administration of an antisense
compound targeted to a transthyretin nucleic acid results in
reduction of transthyretin expression by at least about 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a
range defined by any two of these values.
[0425] In certain embodiments, pharmaceutical compositions
comprising an antisense compound targeted to transthyretin are used
for the preparation of a medicament for treating a patient
suffering or susceptible to central nervous system related
disease.
[0426] In certain embodiments, the methods described herein include
administering a compound comprising a modified oligonucleotide
having a contiguous nucleobases portion as described herein of a
sequence recited in SEQ ID NO: 25, 78, 80, 86, 87, 115, 120, 122
and 124.
Administration
[0427] In certain embodiments, the compounds and compositions as
described herein 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, pulmonary, e.g.,
by inhalation or insufflation of powders or aerosols, including by
nebulizer; intratracheal, intranasal, epidermal and transdermal,
oral or parenteral. The compounds and compositions as described
herein can be delivered in a manner to target a particular tissue,
such as the liver or brain.
[0428] In certain embodiments, the compounds and compositions as
described herein are administered parenterally. "Parenteral
administration" means administration through injection or infusion.
Parenteral administration includes subcutaneous administration,
intravenous administration, intramuscular administration,
intraarterial administration, intraperitoneal administration, or
intracranial administration, e.g. intracerebral administration,
intrathecal administration, intraventricular administration,
ventricular administration, intracerebroventricular administration,
cerebral intraventricular administration or cerebral ventricular
administration. Administration can be continuous, or chronic, or
short or intermittent.
[0429] In certain embodiments, parenteral administration is by
infusion. Infusion can be chronic or continuous or short or
intermittent. In certain embodiments, infused pharmaceutical agents
are delivered with a pump. In certain embodiments, parenteral
administration is by injection.
[0430] In certain embodiments, parenteral administration is
subcutaneous.
[0431] In further embodiments, the formulation for administration
is the compounds described herein and saline.
[0432] In certain embodiments, compounds and compositions are
delivered to the CNS. In certain embodiments, compounds and
compositions are delivered to the cerebrospinal fluid. In certain
embodiments, compounds and compositions are administered to the
brain parenchyma. In certain embodiments, compounds and
compositions are delivered to an animal into multiple regions of
the central nervous system (e.g., into multiple regions of the
brain, and/or into the spinal cord) by intrathecal administration,
or intracerebroventricular administration. Broad distribution of
compounds and compositions, described herein, within the central
nervous system may be achieved with intraparenchymal
administration, intrathecal administration, or
intracerebroventricular administration.
[0433] In certain embodiments, the present invention includes
pharmaceutical compositions that can be delivered by injection
directly into the brain. The injection can be by stereotactic
injection into a particular region of the brain (e.g., the
substantia nigra, choroid plexus, cortex, hippocampus, striatum,
choroid plexus or globus pallidus). The compound can also be
delivered into diffuse regions of the brain (e.g., diffuse delivery
to the cortex of the brain).
[0434] In certain embodiments, parenteral administration is by
injection. The injection may be delivered with a syringe or a pump.
In certain embodiments, the injection is a bolus injection. In
certain embodiments, the injection is administered directly to a
tissue, such as striatum, caudate, cortex, hippocampus and
cerebellum.
[0435] In certain embodiments, delivery of a compound or
composition described herein can affect the pharmacokinetic profile
of the compound or composition. In certain embodiments, injection
of a compound or composition described herein, to a targeted tissue
improves the pharmacokinetic profile of the compound or composition
as compared to infusion of the compound or composition. In a
certain embodiment, the injection of a compound or composition
improves potency compared to broad diffusion, requiring less of the
compound or composition to achieve similar pharmacology. In certain
embodiments, similar pharmacology refers to the amount of time that
a target mRNA and/or target protein is down-regulated (e.g.
duration of action). In certain embodiments, methods of
specifically localizing a pharmaceutical agent, such as by bolus
injection, decreases median effective concentration (EC50) by a
factor of about 50 (e.g. 50 fold less concentration in tissue is
required to achieve the same or similar pharmacodynamic effect). In
certain embodiments, methods of specifically localizing a
pharmaceutical agent, such as by bolus injection, decreases median
effective concentration (EC50) by a factor of 20, 25, 30, 35, 40,
45 or 50. In certain embodiments the pharmaceutical agent in an
antisense compound as further described herein. In certain
embodiments, the targeted tissue is brain tissue. In certain
embodiments the targeted tissue is striatal tissue.
[0436] In certain embodiments, decreasing EC50 is desirable because
it reduces the dose required to achieve a pharmacological result in
a patient in need thereof.
[0437] The half-life of MOE gapmer oligonucleotides in CD1 mice
liver tissue is about 21 days (see Examples 12).
[0438] In certain embodiments, an antisense oligonucleotide is
delivered by injection or infusion once every month, every two
months, every 90 days, every 3 months, every 6 months, twice a year
or once a year.
Certain Combination Therapies
[0439] In certain embodiments, one or more pharmaceutical
compositions of the present invention are co-administered with one
or more other pharmaceutical agents. In certain embodiments, such
one or more other pharmaceutical agents are designed to treat the
same disease, disorder, or condition as the one or more
pharmaceutical compositions described herein. In certain
embodiments, such one or more other pharmaceutical agents are
designed to treat a different disease, disorder, or condition as
the one or more pharmaceutical compositions described herein. In
certain embodiments, such one or more other pharmaceutical agents
are designed to treat an undesired side effect of one or more
pharmaceutical compositions as described herein. In certain
embodiments, one or more pharmaceutical compositions are
co-administered with another pharmaceutical agent to treat an
undesired effect of that other pharmaceutical agent. In certain
embodiments, one or more pharmaceutical compositions are
co-administered with another pharmaceutical agent to produce a
combinational effect. In certain embodiments, one or more
pharmaceutical compositions are co-administered with another
pharmaceutical agent to produce a synergistic effect.
[0440] In certain embodiments, one or more pharmaceutical
compositions and one or more other pharmaceutical agents are
administered at the same time. In certain embodiments, one or more
pharmaceutical compositions and one or more other pharmaceutical
agents are administered at different times. In certain embodiments,
one or more pharmaceutical compositions and one or more other
pharmaceutical agents are prepared together in a single
formulation. In certain embodiments, one or more pharmaceutical
compositions and one or more other pharmaceutical agents are
prepared separately.
[0441] In certain embodiments, the second compound is administered
prior to administration of a pharmaceutical composition of the
present invention. In certain embodiments, the second compound is
administered following administration of a pharmaceutical
composition of the present invention. In certain embodiments, the
second compound is administered at the same time as a
pharmaceutical composition of the present invention. In certain
embodiments, the dose of a co-administered second compound is the
same as the dose that would be administered if the second compound
was administered alone. In certain embodiments, the dose of a
co-administered second compound is lower than the dose that would
be administered if the second compound was administered alone. In
certain embodiments, the dose of a co-administered second compound
is greater than the dose that would be administered if the second
compound was administered alone.
[0442] In certain embodiments, the co-administration of a second
compound enhances the effect of a first compound, such that
co-administration of the compounds results in an effect that is
greater than the effect of administering the first compound alone.
In certain embodiments, the co-administration results in effects
that are additive of the effects of the compounds when administered
alone. In certain embodiments, the co-administration results in
effects that are supra-additive of the effects of the compounds
when administered alone. In certain embodiments, the first compound
is an antisense compound. In certain embodiments, the second
compound is an antisense compound.
[0443] In certain embodiments, pharmaceutical agents that may be
co-administered with a pharmaceutical composition of the present
invention include antipsychotic agents, such as, e.g., haloperidol,
chlorpromazine, clozapine, quetapine, and olanzapine;
antidepressant agents, such as, e.g., fluoxetine, sertraline
hydrochloride, venlafaxine and nortriptyline; tranquilizing agents
such as, e.g., benzodiazepines, clonazepam, paroxetine, venlafaxin,
and beta-blockers; mood-stabilizing agents such as, e.g., lithium,
valproate, lamotrigine, and carbamazepine; paralytic agents such
as, e.g., Botulinum toxin; and/or other experimental agents
including, but not limited to, tetrabenazine (Xenazine), creatine,
conezyme Q10, trehalose, docosahexanoic acids, ACR16, ethyl-EPA,
atomoxetine, citalopram, dimebon, memantine, sodium phenylbutyrate,
ramelteon, ursodiol, zyprexa, xenasine, tiapride, riluzole,
amantadine, [123I]MNI-420, atomoxetine, tetrabenazine, digoxin,
detromethorphan, warfarin, alprozam, ketoconazole, omeprazole, and
minocycline.
[0444] In certain embodiments, pharmaceutical agents that may be
co-administered with a pharmaceutical composition of the present
invention include analgesics, such as, paracetamol (acetaminophen);
non-steroidal anti-inflammatory drugs (NSAIDs), such as,
salicylates; narcotic drugs, such as, morphine, and synthetic drugs
with narcotic properties such as tramadol.
In certain embodiments, pharmaceutical agents that may be
co-administered with a pharmaceutical composition of the present
invention include muscle relaxants, such as, benzodiapines and
methocarbamol.
Formulations
[0445] The compounds of the invention may also be admixed,
conjugated or otherwise associated with other molecules, molecule
structures or mixtures of compounds, as for example, liposomes,
receptor-targeted molecules, 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.
[0446] 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.
[0447] 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.
[0448] 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 parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intracerebral administration, intrathecal
administration, intraventricular administration, ventricular
administration, intracerebroventricular administration, cerebral
intraventricular administration or cerebral ventricular
administration.
[0449] Administration intraventricularly, is preferred to target
transthyretin expression in the choroid plexus. 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.
[0450] 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.
[0451] 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.
[0452] 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.
[0453] 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 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.
[0454] 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.
[0455] 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.
[0456] In another embodiment of the invention, formulations of the
present invention include saline formulations. In certain
embodiment of the invention, a formulation consists of the
compounds described herein and saline. In certain embodiments, a
formulation consists essentially of the compounds described herein
and saline. In certain embodiments, the saline is pharmaceutically
acceptable grade saline. In certain embodiments, the saline is
buffered saline. In certain embodiments, the saline is phosphate
buffered saline (PBS).
[0457] In certain embodiments, a formulation excludes liposomes. In
certain embodiments, the formulation excludes sterically stabilized
liposomes. In certain embodiments, a formulation excludes
phospholipids. In certain embodiments, the formulation consists
essentially of the compounds described herein and saline and
excludes liposomes.
[0458] 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.
[0459] In one embodiment, the present invention employs various
penetration enhancers to affect 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.
[0460] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0461] 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).
[0462] Compositions and formulations for parenteral administration,
including intravenous, intraarterial, subcutaneous,
intraperitoneal, intramuscular injection or infusion, or
intracranial 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.
[0463] 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.
[0464] 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.
Dosing
[0465] 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 EC5s found to be effective in in vitro and in vivo animal
models. In general, dosage is from 0.01 .mu.g 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 .mu.g to 100 g per kg of body weight, once or more daily.
[0466] 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
Non-Limiting Disclosure and Incorporation by Reference
[0467] While certain compounds, compositions and methods described
herein have been described with specificity in accordance with
certain embodiments, the following examples serve only to
illustrate the compounds described herein and are not intended to
limit the same. Each of the references recited in the present
application is incorporated herein by reference in its
entirety.
Example 1: Antisense Inhibition of Human Transthyretin in HepG2
Cells
[0468] Antisense oligonucleotides were designed targeting a
transthyretin nucleic acid and were tested for their effects on
transthyretin mRNA in vitro. Cultured HepG2 cells at a density of
10,000 cells per well were transfected using lipofectin reagent
with 50 nM antisense oligonucleotide. After a treatment period of
approximately 24 hours, RNA was isolated from the cells and
transthyretin mRNA levels were measured by quantitative real-time
PCR. Human primer probe set RTS1396 (forward sequence
CCCTGCTGAGCCCCTACTC, designated herein as SEQ ID NO: 5; reverse
sequence TCCCTCATTCCTTGGGATTG, designated herein as SEQ ID NO: 6;
probe sequence ATTCCACCACGGCTGTCGTCAX, designated herein as SEQ ID
NO: 7). Transthyretin mRNA levels were adjusted according to total
RNA content, as measured by RIBOGREEN.RTM.. Results are presented
as percent inhibition of transthyretin, relative to untreated
control cells.
[0469] The chimeric antisense oligonucleotides in Tables 1 and 2
were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleotides
in length, wherein the central gap segment is comprised of ten
2'-deoxynucleotides and is flanked on both sides (in the 5' and 3'
directions) by wings comprising five nucleotides each. Each
nucleotide in the 5' wing segment and each nucleotide in the 3'
wing segment has a 2'-MOE modification. The internucleoside
linkages throughout each gapmer are phosphorothioate (P.dbd.S)
linkages. All cytidine residues throughout each gapmer are
5-methylcytidines. "Human Target start site" indicates the 5'-most
nucleotide to which the gapmer is targeted in the human gene
sequence. "Human Target stop site" indicates the 3'-most nucleotide
to which the gapmer is targeted human gene sequence. Each gapmer
listed in Table 1 is targeted to human transthyretin mRNA,
designated herein as SEQ ID NO: 1 (GENBANK Accession No.
NM_000371.2). Certain gapmers were also designed which targeted
intronic sequences or intron-exon junctions of the human
transthyretin genomic sequence, designated herein as SEQ ID NO: 2
(GENBANK Accession No. NT_010966.10 truncated from nucleotides
2009236 to 2017289) and are listed in Table 2.
[0470] The human oligonucleotides of Tables 1 and 2 are also
cross-reactive with rhesus monkey gene sequences. `Mismatches`
indicate the number of nucleobases by which the human
oligonucleotide is mismatched with a rhesus monkey gene sequence.
The greater the complementarity between the human oligonucleotide
and the rhesus monkey sequence, the more likely the human
oligonucleotide can cross-react with the rhesus monkey sequence.
The human oligonucleotides in Table 1 were compared to exons 1-4
extracted from the rhesus monkey genomic sequence GENBANK Accession
No. NW_001105671.1, based on similarity to human exons. The human
oligonucleotides in Table 2 were compared to the rhesus monkey
genomic sequence, designated herein as SEQ ID NO: 4 (GENBANK
Accession No. NW_001105671.1 truncated from nucleotides 628000 to
638000). "Rhesus monkey Target start site" indicates the 5'-most
nucleotide to which the gapmer is targeted in the rhesus monkey
gene sequence. "Rhesus monkey Target stop site" indicates the
3'-most nucleotide to which the gapmer is targeted rhesus monkey
gene sequence.
TABLE-US-00001 TABLE 1 Inhibition of human transthyretin mRNA
levels by chimeric antisense oligonucleotides having 5-10-5 MOE
wings and deoxy gap targeted to SEQ ID NO: 1 and SEQ ID NO: 4 Human
Human % Rhesus Rhesus SEQ ISIS Start Stop inhi- monkey monkey Mis-
ID NO Site Site Region Sequence bition start site stop site matches
NO 304267 217 236 coding ACTGGTTTTC 53 217 236 0 8 CCAGAGGCAA
304268 222 241 coding GACTCACTGG 16 222 241 0 9 TTTTCCCAGA 304280
353 372 coding TGAATACCAC 51 353 372 0 10 CTCTGCATGC 304284 425 444
coding CCGTGGTGGA 82 425 444 0 11 ATAGGAGTAG 304285 427 446 coding
AGCCGTGGTG 89 427 446 0 12 GAATAGGAGT 304286 431 450 coding
CCACAGCCGT 63 431 450 0 13 GGTGGAATAG 304287 438 457 coding
TTGGTGACGA 88 438 457 0 14 CAGCCGTGGT 304288 440 459 coding
GATTGGTGAC 82 440 459 0 15 GACAGCCGTG 304289 442 461 coding
GGGATTGGTG 78 442 461 0 16 ACGACAGCCG 304290 443 462 coding
TGGGATTGGT 85 443 462 0 17 GACGACAGCC 304291 449 468 coding-
ATTCCTTGGG 52 449 468 0 18 stop codon ATTGGTGACG 304292 450 469
coding- CATTCCTTGG 34 450 469 0 19 stop codon GATTGGTGAC 304293 451
470 coding- TCATTCCTTG 29 451 470 0 20 stop codon GGATTGGTGA 304294
460 479 coding- stop AGAAGTCCCT 32 460 479 0 21 codon- 3' UTR
CATTCCTTGG 304296 481 500 3'-UTR GTCCTTCAGG 84 478 497 2 22
TCCACTGGAG 304297 489 508 3'-UTR CATCCCTCGT 0 486 505 1 23
CCTTCAGGTC 304298 501 520 3'-UTR TACATGAAAT 26 498 517 0 24
CCCATCCCTC 304299 507 526 3'-UTR CTTGGTTACA 85 504 523 0 25
TGAAATCCCA 304300 513 532 3'-UTR AATACTCTTG 49 510 529 0 26
GTTACATGAA 304301 526 545 3'UTR TTAGTAAAAA 0 523 542 0 27
TGGAATACTC 304302 532 551 3'UTR ACTGCTTTAG 42 529 548 0 28
TAAAAATGGA 304303 539 558 3'UTR TGAAAACACT 41 536 555 0 29
GCTTTAGTAA 304304 546 565 3'UTR TATGAGGTGA 49 543 562 0 30
AAACACTGCT 304307 564 583 3'UTR TGGACTTCTA 73 561 580 2 31
ACATAGCATA 304308 572 591 3'UTR TCTCTGCCTG 55 569 588 1 32
GACTTCTAAC 304309 578 597 3'-UTR TTATTGTCTC 77 575 594 0 33
TGCCTGGACT 304311 597 616 3'-UTR TGCCTTTCAC 80 594 613 0 34
AGGAATGTTT 304312 598 617 3'-UTR GTGCCTTTCA 71 595 614 0 35
CAGGAATGTT 420871 36 55 coding CAGAGGAGGA 48 36 55 0 36 GCAGACGATG
420872 120 139 coding TCTAGAACTT 55 120 139 0 37 TGACCATCAG 420873
212 231 coding TTTTCCCAGA 54 212 231 0 38 GGCAAATGGC 420874 226 245
coding TCCAGACTCA 63 226 245 0 39 CTGGTTTTCC 420875 271 290 coding
TATCCCTTCT 40 271 290 0 40 ACAAATTCCT 420876 285 304 coding
ATTTCCACTT 42 285 304 0 41 TGTATATCCC 420877 293 312 coding
TGGTGTCTAT 76 293 312 0 42 TTCCACTTTG 420878 303 322 coding
CAGTAAGATT 80 303 322 0 43 TGGTGTCTAT 420879 307 326 coding
CTTCCAGTAA 73 307 326 0 44 GATTTGGTGT 420880 347 366 coding
CCACCTCTGC 76 347 366 0 45 ATGCTCATGG 420881 355 374 coding
TGTGAATACC 58 355 374 0 46 ACCTCTGCAT 420882 357 376 coding
GCTGTGAATA 69 357 376 0 47 CCACCTCTGC 420883 362 381 coding
CGTTGGCTGT 64 362 381 0 48 GAATACCACC 420884 428 447 coding
CAGCCGTGGT 93 428 447 0 49 GGAATAGGAG 420885 430 449 coding
GACAGCCGTG 93 430 449 0 50 GTGGAATAGG 420886 432 451 coding
ACGACAGCCG 92 432 451 0 51 TGGTGGAATA 420887 433 452 coding
GACGACAGCC 93 433 452 0 52 GTGGTGGAAT 420888 434 453 coding
TGACGACAGC 95 434 453 0 53 CGTGGTGGAA 420889 435 454 coding
GTGACGACAG 93 435 454 0 54 CCGTGGTGGA 420890 436 455 coding
GGTGACGACA 97 436 455 0 55 GCCGTGGTGG 420891 437 456 coding
TGGTGACGAC 97 437 456 0 56 AGCCGTGGTG 420892 439 458 coding
ATTGGTGACG 93 439 458 0 57 ACAGCCGTGG 420893 441 460 coding
GGATTGGTGA 96 441 460 0 58 CGACAGCCGT 420894 444 463 coding
TTGGGATTGG 88 444 463 0 59 TGACGACAGC 420895 445 464 coding
CTTGGGATTG 95 445 464 0 60 GTGACGACAG 420896 446 465 coding
CCTTGGGATT 95 446 465 0 61 GGTGACGACA 420897 447 466 coding
TCCTTGGGAT 94 447 466 0 62 TGGTGACGAC 420898 448 467 coding-
TTCCTTGGGA 86 448 467 0 63 stop codon TTGGTGACGA 420899 452 471
coding- stop CTCATTCCTT 94 452 471 0 64 codon- 3'UTR GGGATTGGTG
420900 453 472 coding- stop CCTCATTCCT 92 453 472 0 65 codon- 3'UTR
TGGGATTGGT 420901 454 473 coding- stop CCCTCATTCC 93 454 473 0 66
codon- 3'UTR TTGGGATTGG 420902 455 474 coding- stop TCCCTCATTC 75
455 474 0 67 codon- 3'UTR CTTGGGATTG 420903 456 475 coding- stop
GTCCCTCATT 57 456 475 0 68 codon- 3'UTR CCTTGGGATT 420904 457 476
coding- stop AGTCCCTCAT 62 457 476 0 69 codon- 3'UTR TCCTTGGGAT
420905 458 477 coding- stop AAGTCCCTCA 58 458 477 0 70 codon- 3'UTR
TTCCTTGGGA 420906 459 478 coding- stop GAAGTCCCTC 79 459 478 0 71
codon- 3'UTR ATTCCTTGGG 420907 461 480 coding- stop GAGAAGTCCC 59
461 480 0 72 codon- 3'UTR TCATTCCTTG 420908 462 481 coding- stop
GGAGAAGTCC 75 462 481 0 73 codon- 3'UTR CTCATTCCTT 420909 500 519
3'UTR ACATGAAATC 82 497 516 0 74 CCATCCCTCG 420910 502 521 3'UTR
TTACATGAAA 74 499 518 0 75 TCCCATCCCT 420911 503 522 3'UTR
GTTACATGAA 81 500 519 0 76 ATCCCATCCC 420912 504 523 3'UTR
GGTTACATGA 92 501 520 0 77 AATCCCATCC 420913 505 524 3'UTR
TGGTTACATG 95 502 521 0 78 AAATCCCATC 420914 506 525 3'UTR
TTGGTTACAT 93 503 522 0 79 GAAATCCCAT 420915 508 527 3'UTR
TCTTGGTTAC 92 505 524 0 80 ATGAAATCCC 420916 509 528 3'UTR
CTCTTGGTTA 88 506 525 0 81 CATGAAATCC 420917 510 529 3'UTR
ACTCTTGGTT 92 507 526 0 82 ACATGAAATC 420918 511 530 3'UTR
TACTCTTGGT 88 508 527 0 83 TACATGAAAT 420919 512 531 3'UTR
ATACTCTTGG 89 509 528 0 84 TTACATGAAA 420920 514 533 3'UTR
GAATACTCTT 87 511 530 0 85 GGTTACATGA 420921 515 534 3'UTR
GGAATACTCT 92 512 531 0 86 TGGTTACATG 420922 516 535 3'UTR
TGGAATACTC 95 513 532 0 87 TTGGTTACAT
420923 517 536 3'UTR ATGGAATACT 90 514 533 0 88 CTTGGTTACA 420924
518 537 3'UTR AATGGAATAC 75 515 534 0 89 TCTTGGTTAC 420925 519 538
3'UTR AAATGGAATA 87 516 535 0 90 CTCTTGGTTA 420926 520 539 3'UTR
AAAATGGAAT 88 517 536 0 91 ACTCTTGGTT 420927 521 540 3'UTR
AAAAATGGAA 50 518 537 0 92 TACTCTTGGT 420928 522 541 3'UTR
TAAAAATGGA 26 519 538 0 93 ATACTCTTGG 420929 523 542 3'UTR
GTAAAAATGG 56 520 539 0 94 AATACTCTTG 420930 524 543 3'UTR
AGTAAAAATG 18 521 540 0 95 GAATACTCTT 420931 525 544 3'UTR
TAGTAAAAAT 12 522 541 0 96 GGAATACTCT 420932 527 546 3'UTR
TTTAGTAAAA 1 524 543 0 97 ATGGAATACT 420933 528 547 3'UTR
CTTTAGTAAA 0 525 544 0 98 AATGGAATAC 420934 529 548 3'UTR
GCTTTAGTAA 6 526 545 0 99 AAATGGAATA 420935 530 549 3'UTR
TGCTTTAGTA 0 527 546 0 100 AAAATGGAAT 420936 531 550 3'UTR
CTGCTTTAGT 40 528 547 0 101 AAAAATGGAA 420937 533 552 3'UTR
CACTGCTTTA 47 530 549 0 102 GTAAAAATGG 420938 534 553 3'UTR
ACACTGCTTT 30 531 550 0 103 AGTAAAAATG 420939 535 554 3'UTR
AACACTGCTT 0 532 551 0 104 TAGTAAAAAT 420940 536 555 3'UTR
AAACACTGCT 0 533 552 0 105 TTAGTAAAAA 420941 537 556 3'UTR
AAAACACTGC 0 534 553 0 106 TTTAGTAAAA 420942 538 557 3'UTR
GAAAACACTG 0 535 554 0 107 CTTTAGTAAA 420943 540 559 3'UTR
GTGAAAACAC 14 537 556 0 108 TGCTTTAGTA 420944 541 560 3'UTR
GGTGAAAACA 43 538 557 0 109 CTGCTTTAGT 420945 542 561 3'UTR
AGGTGAAAAC 41 539 558 0 110 ACTGCTTTAG 420946 543 562 3'UTR
GAGGTGAAAA 20 540 559 0 111 CACTGCTTTA 420947 544 563 3'UTR
TGAGGTGAAA 69 541 560 0 112 ACACTGCTTT 420948 545 564 3'UTR
ATGAGGTGAA 63 542 561 0 113 AACACTGCTT 420949 579 598 3'UTR
TTTATTGTCT 84 576 595 0 114 CTGCCTGGAC 420950 580 599 3'UTR
TTTTATTGTC 69 577 596 0 115 TCTGCCTGGA 420951 581 600 3'UTR
GTTTTATTGT 87 578 597 0 116 CTCTGCCTGG 420952 582 601 3'UTR
TGTTTTATTG 67 579 598 0 117 TCTCTGCCTG 420953 583 602 3'UTR
ATGTTTTATT 51 580 599 0 118 GTCTCTGCCT 420954 584 603 3'UTR
AATGTTTTAT 60 581 600 0 119 TGTCTCTGCC 420955 585 604 3'UTR
GAATGTTTTA 65 582 601 0 120 TTGTCTCTGC 420956 586 605 3'UTR
GGAATGTTTT 67 583 602 0 121 ATTGTCTCTG 420957 587 606 3'UTR
AGGAATGTTT 68 584 603 0 122 TATTGTCTCT 420958 588 607 3'UTR
CAGGAATGTT 45 585 604 0 123 TTATTGTCTC 420959 589 608 3'UTR
ACAGGAATGT 28 586 605 0 124 TTTATTGTCT
TABLE-US-00002 TABLE 2 Inhibition of human transthyretin mRNA
levels by chimeric antisense oligonucleotides having 5-10-5 MOE
wings and deoxy gap targeted to SEQ ID NO: 2 and SEQ ID NO: 4
Rhesus Rhesus Human Human % monkey monkey SEQ ISIS Start Stop inhi-
start stop Mis- ID NO Site Site Region Sequence bition site site
matches NO 420960 606 625 exon1- GATGTCACAG 13 1755 1774 0 125
intron1 AAACACTCAC 420961 665 684 intron GCAAAGCTGG 7 1814 1833 0
126 1 AAGGAGTCAC 420962 748 767 intron GAACTTCATT 0 1897 1916 0 127
1 CTTTTTGAAG 420963 882 901 intron AGCTTCCTTA 0 2031 2050 0 128 1
ATATCATATC 420964 966 985 intron TATAGGGCCA 10 2115 2134 0 129 1
GAATATAATC 420965 1010 1029 intron ACTAAGCCTT 17 2159 2178 0 130 1
TTAAAGATTA 420966 1208 1227 intron TGGAATTACT 35 2356 2375 0 131 1
GAAAAGATGT 420967 1289 1308 intron ACCAGGGATG 43 2437 2456 0 132 1
TGTATAATGA 420968 1364 1383 intron TCCCTACTCA 0 2512 2531 0 133 1
GTATAACACA 420969 1472 1491 intron GATCAGAGTG 0 2620 2639 0 134 1
AAAGGATTTA 420970 1687 1706 intron GGGAAGATAA 46 2826 2845 0 135 2
AACCAAGTCC 420971 1739 1758 intron TAAATTCTTT 0 2878 2897 0 136 2
AGCAGATGAT 420972 1842 1861 intron AATGATGCTC 23 2980 2999 0 137 2
AGGTTCCTGG 420973 2051 2070 intron TTGGTGTTAC 0 3187 3206 0 138 2
CCAGGGACAC 420974 2207 2226 intron AAAGTGTTCA 29 3344 3363 0 139 2
TTAGGCAAAA 420975 2655 2674 intron GGCATTTTAT 0 3798 3817 0 140 2
ATAAACATAA 420976 2733 2752 intron AAGAACATTG 0 3876 3895 0 141 2
GAATATTTTT 420977 2874 2893 intron GTTGGAAATT 9 4017 4036 0 142 2
GCTTCCCATT 420978 3015 3034 intron AGTGGAAAAC 0 4156 4175 0 143 2
CTAAAGTAGG 420979 3618 3637 intron TTCCCCTCAA 0 4795 4814 0 144 2
CTAAGTCAGA 420980 3735 3754 intron2- CCTATAAGGT 0 4930 4949 0 145
exon 3 GTGAAAGTCT 420981 4096 4115 intron TGTAAGTTCA 0 5291 5310 0
146 3 AGTCATGTTA 420982 4306 4325 intron GTGTTGCCAA 0 5502 5521 0
147 3 GAATCACTTG 420983 4404 4423 intron AAAACACTTA 0 5600 5619 0
148 3 TAATTGTGTC 420984 4518 4537 intron CTTTGACAAG 0 5714 5733 0
149 3 TTATTTGACT 420985 4880 4899 intron ATCCATGACT 0 6073 6092 0
150 3 AAGCCAGAGA 420986 5185 5204 intron ATGGTTCCCA 0 6379 6398 0
151 3 TCAGGCTGAG 420987 5542 5561 intron GCATTTATCA 0 6732 6751 0
152 3 GAAGAAGCTG 420988 6030 6049 intron TTGACCTTCA 0 7226 7245 0
153 3 GCCCACTTGA 420989 6133 6152 intron AGGAAGTGAG 0 7641 7660 0
154 3 AATCACCTAA 420990 6320 6339 intron AGAAGACAGT 0 7828 7847 0
155 3 AAAGATGTGT 420991 6457 6476 intron AAATTGTGGA 0 7966 7985 0
156 3 TCAAAATGCT 420992 6736 6755 intron AACCAGACTT 0 8246 8265 0
157 3 GAATTATTGT 420993 6811 6830 intron AGTGGCTGCC 0 8321 8340 0
158 3 AACCACAGAC 420994 7106 7125 intron GGAAGTCCAG 0 8615 8634 0
159 3 TGCCAACTTA 420995 7162 7181 intron ATCCATTTCC 0 8670 8689 0
160 3 ACCAGAGCCC
[0471] Due to the short length of the human transthyretin mRNA, a
second primer probe set was designed away from the first primer
probe set, RTS1396, to avoid amplicon oligonucleotides. The
antisense oligonucleotides were also tested for their effects on
transthyretin mRNA in vitro using new human primer probe set
RTS3029 (forward sequence CTTGCTGGACTGGTATTTGTGTCT, designated
herein as SEQ ID NO: 161, reverse sequence
AGAACTTTGACCATCAGAGGACACT, designated herein as SEQ ID NO: 162;
probe sequence CCCTACGGGCACCGGTGAATCCX, designated herein as SEQ ID
NO: 163). Cultured HepG2 cells at a density of 10,000 cells per
well were transfected using lipofectin reagent with 50 nM antisense
oligonucleotide. After a treatment period of approximately 24
hours, RNA was isolated from the cells and transthyretin mRNA
levels were measured by quantitative real-time PCR. Transthyretin
mRNA levels were adjusted according to total RNA content, as
measured by RIBOGREEN.RTM.. Results are presented as percent
inhibition of transthyretin, relative to untreated control cells.
The results are presented in Table 3 as percent inhibition of the
PBS control cell set.
TABLE-US-00003 TABLE 3 Inhibition of human transthyretin mRNA
levels by chimeric antisense oligonucleotides having 5-10-5 MOE
wings and deoxy gap with primer probe set RTS3029 ISIS NO Region %
inhibition 304267 coding 13 304268 coding 10 304280 coding 23
304284 coding 10 304285 coding 34 304286 coding 0 304287 coding 34
304288 coding 45 304289 coding 3 304290 coding 16 304291
coding-stop codon 4 304292 coding-stop codon 10 304293 coding-stop
codon 14 304294 stop codon-3' UTR 30 304296 exon 4 78 304297 exon 4
29 304298 exon 4 19 304299 exon 4 85 304300 exon 4 52 304301 exon 4
15 304302 exon 4 45 304303 exon 4 51 304304 exon 4 62 304307 exon 4
76 304308 exon 4 63 304309 exon 4 75 304311 exon 4 81 304312 exon 4
68 420871 coding 0 420872 coding 5 420873 coding 19 420874 coding 0
420875 coding 6 420876 coding 20 420877 coding 28 420878 coding 37
420879 coding 34 420880 coding 36 420881 coding 10 420882 coding 27
420883 coding 13 420884 coding 28 420885 coding 4 420886 coding 21
420887 coding 39 420888 coding 37 420889 coding 9 420890 coding 27
420891 coding 39 420892 coding 43 420893 coding 39 420894 coding 0
420895 coding 0 420896 coding 24 420897 coding 31 420898 coding- 0
420899 stop codon-3'UTR 41 420900 stop codon-3'UTR 26 420901 stop
codon-3'UTR 28 420902 stop codon-3'UTR 20 420903 stop codon-3'UTR
20 420904 stop codon-3'UTR 22 420905 stop codon-3'UTR 32 420906
stop codon-3'UTR 13 420907 -stop codon-3'UTR 0 420908 stop
codon-3'UTR 45 420909 3'UTR 41 420910 3'UTR 14 420911 3'UTR 45
420912 3'UTR 62 420913 3'UTR 81 420914 3'UTR 68 420915 3'UTR 71
420916 3'UTR 54 420917 3'UTR 50 420918 3'UTR 43 420919 3'UTR 65
420920 3'UTR 61 420921 3'UTR 65 420922 3'UTR 68 420923 3'UTR 62
420924 3'UTR 9 420925 3'UTR 17 420926 3'UTR 47 420927 3'UTR 57
420928 3'UTR 51 420929 3'UTR 46 420930 3'UTR 39 420931 3'UTR 14
420932 3'UTR 6 420933 3'UTR 1 420934 3'UTR 48 420935 3'UTR 13
420936 3'UTR 62 420937 3'UTR 65 420938 3'UTR 48 420939 3'UTR 7
420940 3'UTR 3 420941 3'UTR 31 420942 3'UTR 0 420943 3'UTR 40
420944 3'UTR 78 420945 3'UTR 58 420946 3'UTR 52 420947 3'UTR 71
420948 3'UTR 73 420949 3'UTR 88 420950 3'UTR 82 420951 3'UTR 90
420952 3'UTR 82 420953 3'UTR 71 420954 3'UTR 67 420955 3'UTR 73
420956 3'UTR 65 420957 3'UTR 74 420958 3'UTR 69 420959 3'UTR 63
420960 exon1-intron1 14 420961 intron 1 16 420962 intron 1 0 420963
intron 1 0 420964 intron 1 14 420965 intron 1 23 420966 intron 1 25
420967 intron 1 12 420968 intron 1 0 420969 intron 1 0 420970
intron 2 25 420971 intron 2 0 420972 intron 2 25 420973 intron 2 7
420974 intron 2 28 420975 intron 2 9 420976 intron 2 21 420977
intron 2 14 420978 intron 2 37 420979 intron 2 37 420980
intron2-exon 3 16 420981 intron 3 0 420982 intron 3 28 420983
intron 3 0 420984 intron 3 0 420985 intron 3 0 420986 intron 3 7
420987 intron 3 0 420988 intron 3 0 420989 intron 3 0 420990 intron
3 6 420991 intron 3 15 420992 intron 3 0 420993 intron 3 0 420994
intron 3 0 420995 intron 3 10
[0472] Based on the inhibition results using the new primer probe
set RTS3029, antisense oligonucleotides exhibiting 50% or more
inhibition of transthyretin mRNA were selected for further
studies.
Example 2: Antisense Inhibition of Human Transthyretin in HepG2
Cells by Oligonucleotides Designed by Microwalk
[0473] Additional gapmers were designed based on the gapmers
presented in Table 3 that demonstrated an inhibition of at least
50%. These gapmers were designed by creating gapmers shifted
slightly upstream and downstream (i.e. "microwalk") of the original
gapmers from Table 3. Gapmers were also created with various
motifs, e.g. 5-10-5 MOE, 3-14-3 MOE, 2-13-5 MOE, and 4-11-5 MOE
motifs. These gapmers were tested in vitro. Cultured HepG2 cells at
a density of 10,000 cells per well were transfected using
lipofectin reagent with 50 nM antisense oligonucleotide. After a
treatment period of approximately 24 hours, RNA was isolated from
the cells and transthyretin mRNA levels were measured by
quantitative real-time PCR. The human primer probe set RTS3029 was
used to measure transthyretin mRNA levels. Transthyretin mRNA
levels were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
transthyretin, relative to untreated control cells. The results are
presented in Table 4.
[0474] The chimeric antisense oligonucleotides in Table 4 were
designed as 5-10-5 MOE, 3-14-3 MOE, 2-13-5 MOE or 4-11-5 MOE
gapmers. The gapmers designated with an asterisk (*) in Table 4 are
the original gapmers from which gapmers, ISIS 425650-425763, were
designed via microwalk. The 5-10-5 gapmers are 20 nucleotides in
length, wherein the central gap segment is comprised of ten
2'-deoxynucleotides and is flanked on both sides (in the 5' and 3'
directions) by wings comprising five nucleotides each. The 3-14-3
gapmers are 20 nucleotides in length, wherein the central gap
segment is comprised of fourteen 2'-deoxynucleotides and is flanked
on both sides (in the 5' and 3' directions) by wings comprising
three nucleotides each. The 2-13-5 gapmers are 20 nucleotides in
length, wherein the central gap segment is comprised of thirteen
2'-deoxynucleotides and is flanked on the 5' and the 3' directions
with wings comprising two and five nucleotides respectively. The
4-11-5 gapmers are 20 nucleotides in length, wherein the central
gap segment is comprised of eleven 2'-deoxynucleotides and is
flanked on the 5' and the 3' directions with wings comprising four
and five nucleotides respectively. For each of the motifs (5-10-5,
3-14-3, 2-113-5, and 4-11-5), each nucleotide in the 5' wing
segment and each nucleotide in the 3' wing segment has a 2'-MOE
modification. The internucleoside linkages throughout each gapmer
are phosphorothioate (P.dbd.S) linkages. All cytidine residues
throughout each gapmer are 5-methylcytidines. "Target start site"
indicates the 5'-most nucleotide to which the gapmer is targeted.
"Target stop site" indicates the 3'-most nucleotide to which the
gapmer is targeted. Each gapmer listed in Table 4 is targeted to
the target region spanning nucleobases 481-619 of SEQ ID NO: 1
(GENBANK Accession No. NM_000371.2).
[0475] As shown in Table 4, several of the gapmers exhibited at
least 50% inhibition, including ISIS numbers: 304296, 425655,
425695, 425735, 425649, 425656, 425696, 425736, 420912, 425657,
425697, 425737, 420913, 425658, 425698, 425738, 420914, 425659,
425699, 425739, 304299, 425660, 425700, 425740, 420915, 420916,
425662, 425702, 420919, 425703, 420920, 425664, 425704, 425742,
420921, 425665, 425705, 425743, 420922, 425666, 425706, 420923,
420937, 420944, 425669, 425709, 425746, 425710, 425711, 425747,
420948, 425712, 425748, 425673, 425713, 425749, 425651, 425675,
425715, 425751, 304309, 425676, 425716, 425752, 420949, 425677,
425717, 425753, 420950, 425678, 425718, 425754, 420951, 425679,
425719, 425755, 420952, 425680, 425720, 425756, 420953, 425681,
425721, 425757, 420954, 425722, 425758, 420955, 425759, 425724,
425760, 425762, 304310, 425729, 425764, 425653, 425690, 425730,
425765, 304311, 425691, 425731, 425766, 304312, 425692, 425732,
425767, 425654, 425693, 425733, 425768, 304313, 425734, and
425769.
[0476] Several of the gapmers exhibited at least 60% inhibition,
including ISIS numbers: 304296, 425655, 425695, 425735, 425649,
425656, 425696, 425736, 420912, 425657, 425697, 425737, 420913,
425658, 425698, 425738, 420914, 425659, 425739, 304299, 425740,
420915, 425702, 420919, 420920, 425742, 420921, 425665, 425705,
425706, 420923, 425746, 425711, 425747, 420948, 425712, 425748,
425651, 425715, 425751, 304309, 425716, 425752, 425677, 425717,
425753, 420950, 425718, 425754, 420951, 425679, 425719, 425755,
420952, 425680, 425720, 420953, 425681, 425721, 425757, 420954,
425722, 425758, 420955, 425724, 425760, 425764, 425653, 425690,
425730, 425765, 304311, 425691, 425731, 425766, 304312, 425692,
425732, 425767, 425654, 425693, 425733, 304313, and 425769.
[0477] Several of the gapmers exhibited at least 70% inhibition,
including ISIS numbers: 304296, 425655, 425695, 425735, 425649,
425656, 425696, 425736, 420912, 425657, 425737, 420913, 425738,
420914, 425659, 304299, 420915, 420920, 425742, 425712, 425748,
425716, 425754, 420951, 425679, 425719, 425755, 425680, 425721,
425757, 425760, 425653, 425690, 425730, 425765, 304311, 425691,
425731, 425766, 304312, 425767, 425693, and 304313.
[0478] Several of the gapmers exhibited at least 80% inhibition,
including ISIS numbers: 304296, 425655, 425695, 425736, 420913,
425659, 304299, 420915, 425716, 425754, 425719, 425757, 425765, and
425767.
[0479] Several of the gapmers exhibited at least 85% inhibition,
including ISIS numbers: 420913, 425716, 425754, and 425719.
[0480] One gapmer, ISIS 425719, exhibited 90% inhibition.
TABLE-US-00004 TABLE 4 Inhibition of human transthyretin mRNA
levels by chimeric antisense oligonucleotides targeted to SEQ ID
NO: 1 (GENBANK Accession No. NM_000371.2) % SEQ Start Stop inhi- ID
OligoID Site Site Sequence Motif bition NO *304296 481 500
GTCCTTCAGGTCCACTGGAG 5-10-5 83 22 425655 481 500
GTCCTTCAGGTCCACTGGAG 3-14-3 80 22 425695 481 500
GTCCTTCAGGTCCACTGGAG 2-13-5 80 22 425735 481 500
GTCCTTCAGGTCCACTGGAG 4-11-5 72 22 425649 482 501
CGTCCTTCAGGTCCACTGGA 5-10-5 75 170 425656 482 501
CGTCCTTCAGGTCCACTGGA 3-14-3 78 170 425696 482 501
CGTCCTTCAGGTCCACTGGA 2-13-5 74 170 425736 482 501
CGTCCTTCAGGTCCACTGGA 4-11-5 83 170 *420912 504 523
GGTTACATGAAATCCCATCC 5-10-5 73 77 425657 504 523
GGTTACATGAAATCCCATCC 3-14-3 76 77 425697 504 523
GGTTACATGAAATCCCATCC 2-13-5 69 77 425737 504 523
GGTTACATGAAATCCCATCC 4-11-5 78 77 *420913 505 524
TGGTTACATGAAATCCCATC 5-10-5 89 78 425658 505 524
TGGTTACATGAAATCCCATC 3-14-3 69 78 425698 505 524
TGGTTACATGAAATCCCATC 2-13-5 61 78 425738 505 524
TGGTTACATGAAATCCCATC 4-11-5 78 78 *420914 506 525
TTGGTTACATGAAATCCCAT 5-10-5 70 79 425659 506 525
TTGGTTACATGAAATCCCAT 3-14-3 83 79 425699 506 525
TTGGTTACATGAAATCCCAT 2-13-5 56 79 425739 506 525
TTGGTTACATGAAATCCCAT 4-11-5 69 79 *304299 507 526
CTTGGTTACATGAAATCCCA 5-10-5 83 25 425660 507 526
CTTGGTTACATGAAATCCCA 3-14-3 59 25 425700 507 526
CTTGGTTACATGAAATCCCA 2-13-5 52 25 425740 507 526
CTTGGTTACATGAAATCCCA 4-11-5 69 25 *420915 508 527
TCTTGGTTACATGAAATCCC 5-10-5 81 80 425661 508 527
TCTTGGTTACATGAAATCCC 3-14-3 48 80 425701 508 527
TCTTGGTTACATGAAATCCC 2-13-5 41 80 425741 508 527
TCTTGGTTACATGAAATCCC 4-11-5 37 80 *420916 509 528
CTCTTGGTTACATGAAATCC 5-10-5 52 81 425662 509 528
CTCTTGGTTACATGAAATCC 3-14-3 57 81 425702 509 528
CTCTTGGTTACATGAAATCC 2-13-5 63 81 *420919 512 531
ATACTCTTGGTTACATGAAA 5-10-5 69 84 425663 512 531
ATACTCTTGGTTACATGAAA 3-14-3 46 84 425703 512 531
ATACTCTTGGTTACATGAAA 2-13-5 52 84 *420920 514 533
GAATACTCTTGGTTACATGA 5-10-5 71 85 425664 514 533
GAATACTCTTGGTTACATGA 3-14-3 57 85 425704 514 533
GAATACTCTTGGTTACATGA 2-13-5 58 85 425742 514 533
GAATACTCTTGGTTACATGA 4-11-5 71 85 *420921 515 534
GGAATACTCTTGGTTACATG 5-10-5 68 86 425665 515 534
GGAATACTCTTGGTTACATG 3-14-3 65 86 425705 515 534
GGAATACTCTTGGTTACATG 2-13-5 60 86 425743 515 534
GGAATACTCTTGGTTACATG 4-11-5 56 86 *420922 516 535
TGGAATACTCTTGGTTACAT 5-10-5 54 87 425666 516 535
TGGAATACTCTTGGTTACAT 3-14-3 56 87 425706 516 535
TGGAATACTCTTGGTTACAT 2-13-5 64 87 425744 516 535
TGGAATACTCTTGGTTACAT 4-11-5 39 87 *420923 517 536
ATGGAATACTCTTGGTTACA 5-10-5 62 88 425667 517 536
ATGGAATACTCTTGGTTACA 3-14-3 44 88 425707 517 536
ATGGAATACTCTTGGTTACA 2-13-5 30 88 *420937 533 552
CACTGCTTTAGTAAAAATGG 5-10-5 59 102 425668 533 552
CACTGCTTTAGTAAAAATGG 3-14-3 37 102 425708 533 552
CACTGCTTTAGTAAAAATGG 2-13-5 32 102 425745 533 552
CACTGCTTTAGTAAAAATGG 4-11-5 43 102 *420944 541 560
GGTGAAAACACTGCTTTAGT 5-10-5 52 109 425669 541 560
GGTGAAAACACTGCTTTAGT 3-14-3 54 109 425709 541 560
GGTGAAAACACTGCTTTAGT 2-13-5 54 109 425746 541 560
GGTGAAAACACTGCTTTAGT 4-11-5 60 109 *420945 542 561
AGGTGAAAACACTGCTTTAG 5-10-5 38 110 425670 542 561
AGGTGAAAACACTGCTTTAG 3-14-3 38 110 425710 542 561
AGGTGAAAACACTGCTTTAG 2-13-5 52 110 *420947 544 563
TGAGGTGAAAACACTGCTTT 5-10-5 34 112 425671 544 563
TGAGGTGAAAACACTGCTTT 3-14-3 27 112 425711 544 563
TGAGGTGAAAACACTGCTTT 2-13-5 68 112 425747 544 563
TGAGGTGAAAACACTGCTTT 4-11-5 61 112 *420948 545 564
ATGAGGTGAAAACACTGCTT 5-10-5 66 113 425672 545 564
ATGAGGTGAAAACACTGCTT 3-14-3 47 113 425712 545 564
ATGAGGTGAAAACACTGCTT 2-13-5 70 113 425748 545 564
ATGAGGTGAAAACACTGCTT 4-11-5 71 113 *304304 546 565
TATGAGGTGAAAACACTGCT 5-10-5 46 30 425673 546 565
TATGAGGTGAAAACACTGCT 3-14-3 51 30 425713 546 565
TATGAGGTGAAAACACTGCT 2-13-5 50 30 425749 546 565
TATGAGGTGAAAACACTGCT 4-11-5 58 30 425650 547 566
ATATGAGGTGAAAACACTGC 5-10-5 28 171 425674 547 566
ATATGAGGTGAAAACACTGC 3-14-3 40 171 425714 547 566
ATATGAGGTGAAAACACTGC 2-13-5 44 171 425750 547 566
ATATGAGGTGAAAACACTGC 4-11-5 47 171 425651 577 596
TATTGTCTCTGCCTGGACTT 5-10-5 65 172 425675 577 596
TATTGTCTCTGCCTGGACTT 3-14-3 55 172 425715 577 596
TATTGTCTCTGCCTGGACTT 2-13-5 65 172 425751 577 596
TATTGTCTCTGCCTGGACTT 4-11-5 62 172 *304309 578 597
TTATTGTCTCTGCCTGGACT 5-10-5 66 33 425676 578 597
TTATTGTCTCTGCCTGGACT 3-14-3 59 33 425716 578 597
TTATTGTCTCTGCCTGGACT 2-13-5 87 33 425752 578 597
TTATTGTCTCTGCCTGGACT 4-11-5 67 33 *420949 579 598
TTTATTGTCTCTGCCTGGAC 5-10-5 57 114 425677 579 598
TTTATTGTCTCTGCCTGGAC 3-14-3 67 114 425717 579 598
TTTATTGTCTCTGCCTGGAC 2-13-5 68 114 425753 579 598
TTTATTGTCTCTGCCTGGAC 4-11-5 69 114 *420950 580 599
TTTTATTGTCTCTGCCTGGA 5-10-5 61 115 425678 580 599
TTTTATTGTCTCTGCCTGGA 3-14-3 59 115 425718 580 599
TTTTATTGTCTCTGCCTGGA 2-13-5 69 115 425754 580 599
TTTTATTGTCTCTGCCTGGA 4-11-5 86 115 *420951 581 600
GTTTTATTGTCTCTGCCTGG 5-10-5 78 116 425679 581 600
GTTTTATTGTCTCTGCCTGG 3-14-3 73 116 425719 581 600
GTTTTATTGTCTCTGCCTGG 2-13-5 90 116 425755 581 600
GTTTTATTGTCTCTGCCTGG 4-11-5 73 116 *420952 582 601
TGTTTTATTGTCTCTGCCTG 5-10-5 61 117 425680 582 601
TGTTTTATTGTCTCTGCCTG 3-14-3 77 117 425720 582 601
TGTTTTATTGTCTCTGCCTG 2-13-5 67 117 425756 582 601
TGTTTTATTGTCTCTGCCTG 4-11-5 57 117 *420953 583 602
ATGTTTTATTGTCTCTGCCT 5-10-5 65 118 425681 583 602
ATGTTTTATTGTCTCTGCCT 3-14-3 61 118 425721 583 602
ATGTTTTATTGTCTCTGCCT 2-13-5 77 118 425757 583 602
ATGTTTTATTGTCTCTGCCT 4-11-5 83 118 *420954 584 603
AATGTTTTATTGTCTCTGCC 5-10-5 63 119 425682 584 603
AATGTTTTATTGTCTCTGCC 3-14-3 42 119 425722 584 603
AATGTTTTATTGTCTCTGCC 2-13-5 69 119 425758 584 603
AATGTTTTATTGTCTCTGCC 4-11-5 61 119 *420955 585 604
GAATGTTTTATTGTCTCTGC 5-10-5 65 120 425683 585 604
GAATGTTTTATTGTCTCTGC 3-14-3 30 120 425723 585 604
GAATGTTTTATTGTCTCTGC 2-13-5 44 120 425759 585 604
GAATGTTTTATTGTCTCTGC 4-11-5 50 120 *420956 586 605
GGAATGTTTTATTGTCTCTG 5-10-5 47 121 425684 586 605
GGAATGTTTTATTGTCTCTG 3-14-3 44 121 425724 586 605
GGAATGTTTTATTGTCTCTG 2-13-5 65 121 *420957 587 606
AGGAATGTTTTATTGTCTCT 5-10-5 37 122 425685 587 606
AGGAATGTTTTATTGTCTCT 3-14-3 46 122 425725 587 606
AGGAATGTTTTATTGTCTCT 2-13-5 43 122 425760 587 606
AGGAATGTTTTATTGTCTCT 4-11-5 78 122 *420958 588 607
CAGGAATGTTTTATTGTCTC 5-10-5 41 123 425686 588 607
CAGGAATGTTTTATTGTCTC 3-14-3 6 123
425726 588 607 CAGGAATGTTTTATTGTCTC 2-13-5 41 123 425761 588 607
CAGGAATGTTTTATTGTCTC 4-11-5 39 123 *420959 589 608
ACAGGAATGTTTTATTGTCT 5-10-5 43 124 425687 589 608
ACAGGAATGTTTTATTGTCT 3-14-3 22 124 425727 589 608
ACAGGAATGTTTTATTGTCT 2-13-5 25 124 425762 589 608
ACAGGAATGTTTTATTGTCT 4-11-5 57 124 425652 590 609
CACAGGAATGTTTTATTGTC 5-10-5 23 173 425688 590 609
CACAGGAATGTTTTATTGTC 3-14-3 11 173 425728 590 609
CACAGGAATGTTTTATTGTC 2-13-5 37 173 425763 590 609
CACAGGAATGTTTTATTGTC 4-11-5 38 173 304310 595 614
CCTTTCACAGGAATGTTTTA 5-10-5 57 174 425689 595 614
CCTTTCACAGGAATGTTTTA 3-14-3 38 174 425729 595 614
CCTTTCACAGGAATGTTTTA 2-13-5 58 174 425764 595 614
CCTTTCACAGGAATGTTTTA 4-11-5 60 174 425653 596 615
GCCTTTCACAGGAATGTTTT 5-10-5 79 175 425690 596 615
GCCTTTCACAGGAATGTTTT 3-14-3 73 175 425730 596 615
GCCTTTCACAGGAATGTTTT 2-13-5 76 175 425765 596 615
GCCTTTCACAGGAATGTTTT 4-11-5 83 175 *304311 597 616
TGCCTTTCACAGGAATGTTT 5-10-5 71 34 425691 597 616
TGCCTTTCACAGGAATGTTT 3-14-3 74 34 425731 597 616
TGCCTTTCACAGGAATGTTT 2-13-5 73 34 425766 597 616
TGCCTTTCACAGGAATGTTT 4-11-5 79 34 *304312 598 617
GTGCCTTTCACAGGAATGTT 5-10-5 71 35 425692 598 617
GTGCCTTTCACAGGAATGTT 3-14-3 69 35 425732 598 617
GTGCCTTTCACAGGAATGTT 2-13-5 67 35 425767 598 617
GTGCCTTTCACAGGAATGTT 4-11-5 83 35 425654 599 618
AGTGCCTTTCACAGGAATGT 5-10-5 64 176 425693 599 618
AGTGCCTTTCACAGGAATGT 3-14-3 79 176 425733 599 618
AGTGCCTTTCACAGGAATGT 2-13-5 68 176 425768 599 618
AGTGCCTTTCACAGGAATGT 4-11-5 50 176 304313 600 619
AAGTGCCTTTCACAGGAATG 5-10-5 73 177 425694 600 619
AAGTGCCTTTCACAGGAATG 3-14-3 45 177 425734 600 619
AAGTGCCTTTCACAGGAATG 2-13-5 55 177 425769 600 619
AAGTGCCTTTCACAGGAATG 4-11-5 62 177
Example 3: Dose-Dependent Antisense Inhibition of Human
Transthyretin in HepG2 Cells
[0481] Gapmers from Example 2 exhibiting significant in vitro
inhibition of human transthyretin were tested at various doses in
HepG2 cells. Cells were plated at a density of 20,000 cells per
well and transfected using electroporation with 625 nM, 1250 nM,
2500 nM, 5000 nM and 10000 nM concentrations of antisense
oligonucleotide, as specified in Table 5. After a treatment period
of approximately 16 hours, RNA was isolated from the cells and
transthyretin mRNA levels were measured by quantitative real-time
PCR. Human transthyretin primer probe set RTS3029 was used to
measure mRNA levels. Transthyretin mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN.RTM..
Results are presented as percent inhibition of transthyretin,
relative to untreated control cells.
[0482] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is also presented in Table 5 and was
calculated by plotting the concentrations of oligonucleotides used
versus the percent inhibition of transthyretin mRNA expression
achieved at each concentration, and noting the concentration of
oligonucleotide at which 50% inhibition of transthyretin mRNA
expression was achieved compared to the control. As illustrated in
Table 5, transthyretin mRNA levels were significantly reduced in a
dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00005 TABLE 5 Dose-dependent antisense inhibition of human
transthyretin in HepG2 cells using electroporation ISIS 10000
IC.sub.50 NO 625 nM 1250 nM 2500 nM 5000 nM nM (.mu.M) 304296 57 74
83 91 96 <0.625 304299 43 76 82 95 94 0.627 420913 59 75 90 88
98 <0.625 420915 60 85 91 95 99 <0.625 420951 64 77 90 97 99
<0.625 425653 70 86 86 88 82 <0.625 425655 48 80 85 97 96
<0.625 425656 70 89 92 92 96 <0.625 425659 46 56 68 82 93 0.8
425679 63 77 72 94 97 <0.625 425680 28 79 85 93 98 0.8 425693 2
64 74 76 81 1.7 425695 74 87 91 97 98 <0.625 425716 69 84 95 97
98 <0.625 425719 58 84 92 96 98 <0.625 425721 40 75 89 95 98
0.7 425736 64 71 86 93 93 <0.625 425737 78 93 95 97 98 <0.625
425738 40 77 88 94 95 0.7 425754 56 75 87 96 99 <0.625 425755 58
84 88 94 97 <0.625 425757 62 82 94 97 99 <0.625 425760 58 42
74 85 93 <0.625 425765 81 86 87 83 88 <0.625 425766 83 89 81
75 74 <0.625 425767 56 75 83 81 80 <0.625
[0483] Gapmers from Example 2 were also tested at various doses in
HepG2 cells using the transfection reagent, lipofectin. Cells were
plated at a density of 10,000 cells per well and transfected using
electroporation with 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM
concentrations of antisense oligonucleotide, as specified in Table
6. After a treatment period of approximately 16 hours, RNA was
isolated from the cells and transthyretin mRNA levels were measured
by quantitative real-time PCR. Human transthyretin primer probe set
RTS3029 was used to measure mRNA levels. Transthyretin mRNA levels
were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
transthyretin, relative to untreated control cells. As illustrated
in Table 6, transthyretin mRNA levels were significantly reduced in
a dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00006 TABLE 6 Dose-dependent antisense inhibition of human
transthyretin in HepG2 cells using lipofectin reagent ISIS
IC.sub.50 NO 6.25 nM 12.5 nM 25 nM 50 nM 100 nM (nM) 304296 26 41
43 52 65 39 304299 22 70 43 74 83 20 420913 4 60 60 68 82 30 420915
36 31 46 64 67 28 420951 10 37 56 85 84 19 425653 25 38 60 74 77 18
425655 27 15 62 79 81 16 425656 37 62 47 69 82 15 425659 17 35 33
79 73 30 425679 32 6 63 79 77 14 425680 16 48 41 84 84 28 425693 10
19 51 66 61 26 425695 36 23 54 76 84 28 425716 57 52 36 85 81 38
425719 25 39 28 60 76 45 425721 0 22 38 73 75 32 425736 25 60 30 77
80 22 425737 36 52 50 60 76 14 425738 13 15 19 65 70 27 425754 8 18
38 75 71 42 425755 26 46 54 77 86 20 425757 0 37 81 83 71 19 425760
28 46 72 70 80 18 425765 0 52 48 66 69 29 425766 24 19 48 69 71 29
425767 41 49 48 65 75 14
Example 4: Dose-Dependent Antisense Inhibition of Human
Transthyretin in HepG2 Cells
[0484] Gapmers selected from Example 3 were tested at various doses
in HepG2 cells. Cells were plated at a density of 20,000 cells per
well and transfected using electroporation with 0.0617 .mu.M,
0.1852 .mu.M, 0.5556 .mu.M, 1.6667 .mu.M and 5 .mu.M concentrations
of antisense oligonucleotide, as specified in Table 7. After a
treatment period of approximately 16 hours, RNA was isolated from
the cells and transthyretin mRNA levels were measured by
quantitative real-time PCR. Human transthyretin primer probe set
RTS3029 was used to measure mRNA levels. Transthyretin mRNA levels
were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
transthyretin, relative to untreated control cells. As illustrated
in Table 7, transthyretin mRNA levels were reduced in a
dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00007 TABLE 7 Dose-dependent antisense inhibition of human
transthyretin in HepG2 cells using electroporation ISIS 0.0617
0.1852 IC.sub.50 NO .mu.M .mu.M 0.5556 .mu.M 1.6667 .mu.M 5 .mu.M
(.mu.M) 304296 0 6 44 58 83 1.2 304299 38 10 57 83 92 0.6 420913 51
51 54 73 93 0.2 420915 33 35 62 65 93 0.2 420951 40 33 36 82 96 0.4
425653 55 58 74 72 84 <0.06 425655 8 35 54 57 90 0.5 425656 12
43 43 78 94 0.4 425659 14 35 19 46 82 0.6 425679 30 13 23 69 91 0.8
425680 0 35 45 74 84 0.7 425693 0 6 14 32 59 3.4 425695 15 47 61 81
91 0.3 425716 20 17 53 77 91 0.6 425719 0 14 45 78 94 0.8 425721 0
0 22 74 84 0.9 425736 42 43 56 76 91 0.3 425737 21 29 61 81 97 0.3
425738 14 39 57 74 93 0.4 425754 29 34 45 78 94 0.4 425755 8 21 57
78 95 0.5 425757 29 28 62 83 95 0.4 425760 3 6 9 56 77 1.4 425765
24 51 75 77 88 0.3 425766 7 41 59 73 77 0.3 425767 1 18 49 66 79
1.0
Example 5: Dose Response Confirmation of Antisense Oligonucleotides
Targeting Human Transthyretin in Hep3B Cells
[0485] Gapmers from Example 4 exhibiting significant in vitro
inhibition of human transthyretin were tested at various doses in
Hep3B cells. Cells were plated at a density of 20,000 cells per
well and transfected using electroporation with 0.0206 .mu.M, 0.062
.mu.M, 0.185 .mu.M, 0.556 .mu.M, 1.667 .mu.M and 5 .mu.M
concentrations of antisense oligonucleotide, as specified in Table
8. After a treatment period of approximately 16 hours, RNA was
isolated from the cells and transthyretin mRNA levels were measured
by quantitative real-time PCR. Human transthyretin primer probe set
RTS1396 was used to measure mRNA levels. Transthyretin mRNA levels
were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
transthyretin, relative to untreated control cells. As illustrated
in Table 8, transthyretin mRNA levels were reduced in a
dose-dependent manner in antisense oligonucleotide treated cells.
The IC.sub.50 of each oligonucleotide is also presented in Table
8.
TABLE-US-00008 TABLE 8 Dose-dependent antisense inhibition of human
transthyretin in Hep3B cells using electroporation ISIS 0.0206
0.185 1.667 IC.sub.50 NO .mu.M 0.062 .mu.M .mu.M 0.556 .mu.M .mu.M
5 .mu.M (.mu.M) 304299 27 2 25 52 76 96 0.5 420915 0 12 27 30 69 93
0.8 425653 23 13 55 86 88 91 0.1 425655 3 30 32 62 84 94 0.3 425656
0 0 29 66 82 95 0.5 425679 0 21 36 71 92 97 0.3 425695 37 23 63 79
94 98 0.1 425736 31 43 40 64 82 95 0.1 425737 0 13 62 82 95 98 0.2
425755 17 8 18 69 86 98 0.4 425757 22 47 53 79 96 98 0.2
Example 6: Dose Response Confirmation of Antisense Oligonucleotides
Targeting Human Transthyretin in Human Transthyretin-Transgenic
Mouse Primary Hepatocytes
[0486] Gapmers from Example 5 were also tested at various doses in
primary hepatocytes of human transthyretin-transgenic mice. ISIS
304309, ISIS 304311, ISIS 304312 and ISIS 420951 (see Example 2)
were also retested along with these gapmers under the same culture
conditions. Cells were plated at a density of 10,000 cells per well
and transfected using cytofectin with 18.75 nM, 37.5 nM, 75 nM, 150
nM and 300 nM concentrations of antisense oligonucleotide, as
specified in Table 9. After a treatment period of approximately 16
hours, RNA was isolated from the cells and transthyretin mRNA
levels were measured by quantitative real-time PCR. Human
transthyretin primer probe set RTS1396 was used to measure mRNA
levels. Transthyretin mRNA levels were adjusted according to total
RNA content, as measured by RIBOGREEN.RTM.. Results are presented
as percent inhibition of transthyretin, relative to untreated
control cells. As illustrated in Table 9, transthyretin mRNA levels
were reduced in a dose-dependent manner in antisense
oligonucleotide treated cells.
TABLE-US-00009 TABLE 9 Dose-dependent antisense inhibition of human
transthyretin in mouse primary hepatocytes using cytofectin ISIS NO
18.75 nM 37.5 nM 75 nM 150 nM 300 nM Motif 304299 54 79 97 98 99
5-10-5 304309 48 77 94 99 99 5-10-5 304311 45 79 92 96 98 5-10-5
304312 33 71 89 96 98 5-10-5 420915 40 70 92 98 99 5-10-5 420951 41
86 96 98 99 5-10-5 425653 44 81 93 96 99 5-10-5 425655 61 88 96 99
99 3-14-3 425656 61 84 94 98 99 3-14-3 425679 74 78 97 98 99 3-14-3
425695 66 84 96 98 99 2-13-5 425736 58 84 95 98 99 4-11-5 425737 57
77 95 98 99 4-11-5 425755 61 82 96 99 99 4-11-5 425757 37 77 93 98
98 4-11-5
Example 7: Dose Response Confirmation of Antisense Oligonucleotides
Targeting Human Transthyretin in HepG2 Cells
[0487] Gapmers from Example 6 were tested at various doses in HepG2
cells. Cells were plated at a density of 10,000 cells per well and
transfected using electroporation with 0.062 .mu.M, 0.185 .mu.M,
0.556 .mu.M, 1.66 .mu.M and 5000 .mu.M concentrations of antisense
oligonucleotide, as specified in Table 10. After a treatment period
of approximately 16 hours, RNA was isolated from the cells and
transthyretin mRNA levels were measured by quantitative real-time
PCR. Human transthyretin primer probe set RTS1396 was used to
measure mRNA levels. Transthyretin mRNA levels were adjusted
according to total RNA content, as measured by RIBOGREEN.RTM..
Results are presented as percent inhibition of transthyretin,
relative to untreated control cells. As illustrated in Table 10,
transthyretin mRNA levels were reduced in a dose-dependent manner
in antisense oligonucleotide treated cells.
TABLE-US-00010 TABLE 10 Dose-dependent antisense inhibition of
human transthyretin in HepG2 cells using electroporation ISIS 0.062
0.556 5.000 IC.sub.50 NO .mu.M 0.185 .mu.M .mu.M 1.667 .mu.M .mu.M
(.mu.M) Motif 304299 55 66 72 87 96 0.037 5-10-5 304309 41 65 72 91
96 0.087 5-10-5 304311 57 83 88 89 83 0.001 5-10-5 304312 46 69 74
84 81 0.038 5-10-5 420915 38 62 80 90 98 0.096 5-10-5 420951 45 71
84 93 97 0.049 5-10-5 425653 48 73 87 88 82 0.017 5-10-5 425655 40
57 77 85 95 0.105 3-14-3 425656 28 54 70 94 97 0.177 3-14-3 425679
43 51 81 95 99 0.106 3-14-3 425695 49 67 90 96 99 0.043 2-13-5
425736 32 63 85 95 98 0.108 4-11-5 425737 42 71 90 98 99 0.053
4-11-5 425755 24 63 85 95 99 0.137 4-11-5 425757 21 62 86 96 99
0.148 4-11-5
Example 8: Dose Response Confirmation of Antisense Oligonucleotides
Targeting Human Transthyretin in Human Transthyretin-Transgenic
Mouse Primary Hepatocytes
[0488] Gapmers from Example 6 were also tested at various doses in
primary hepatocytes of human transthyretin-transgenic mice. Cells
were plated at a density of 10,000 cells per well and transfected
using cytofectin with 5 nM, 10 nM, 20 nM, 40 nM and 80 nM
concentrations of antisense oligonucleotide, as specified in Table
11. After a treatment period of approximately 16 hours, RNA was
isolated from the cells and transthyretin mRNA levels were measured
by quantitative real-time PCR. Human transthyretin primer probe set
RTS3029 was used to measure mRNA levels. Transthyretin mRNA levels
were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
transthyretin, relative to untreated control cells. As illustrated
in Table 11, transthyretin mRNA levels were reduced in a
dose-dependent manner in antisense oligonucleotide treated
cells.
TABLE-US-00011 TABLE 11 Dose-dependent antisense inhibition of
human transthyretin in mouse primary hepatocytes using cytofectin
ISIS NO 5 nM 10 nM 20 nM 40 nM 80 nM Motif 304299 0 8 37 69 90
5-10-5 304309 0 9 39 75 93 5-10-5 304311 1 13 43 70 81 5-10-5
304312 0 3 32 64 76 5-10-5 420915 0 0 34 59 87 5-10-5 420951 0 12
57 84 92 5-10-5 425653 0 9 44 72 84 5-10-5 425655 0 19 45 80 91
3-14-3 425656 0 2 33 70 93 3-14-3 425679 0 13 42 72 90 3-14-3
425695 3 12 33 70 90 2-13-5 425736 2 7 37 70 89 4-11-5 425737 0 4
36 65 89 4-11-5 425755 0 25 50 75 94 4-11-5 425757 0 5 43 72 92
4-11-5
[0489] Gapmers were also tested using electroporation as the
transfection agent. Cells were plated at a density of 35,000 cells
per well and transfected using electroporation with 148.148 nM,
444.444 nM, 1,333.333 nM, 4,000 nM and 12,000 nM concentrations of
antisense oligonucleotide, as specified in Table 12. After a
treatment period of approximately 16 hours, RNA was isolated from
the cells and transthyretin mRNA levels were measured by
quantitative real-time PCR. Human transthyretin primer probe set
RTS3029 was used to measure mRNA levels. Transthyretin mRNA levels
were adjusted according to total RNA content, as measured by
RIBOGREEN.RTM.. Results are presented as percent inhibition of
transthyretin, relative to untreated control cells.
TABLE-US-00012 TABLE 12 Dose-dependent antisense inhibition of
human transthyretin in mouse primary hepatocytes using
electroporation ISIS 148.148 444.444 1333.333 NO nM nM nM 4000 nM
12000 nM Motif 304299 75 96 98 98 99 5-10-5 304309 72 96 98 98 98
5-10-5 304311 68 92 93 94 97 5-10-5 304312 50 84 92 93 97 5-10-5
420915 55 89 96 96 97 5-10-5 420951 65 92 95 96 98 5-10-5 425653 68
89 91 93 95 5-10-5 425655 63 94 96 96 96 3-14-3 425656 69 93 98 98
98 3-14-3 425679 63 92 97 98 98 3-14-3 425695 69 92 96 96 95 2-13-5
425736 75 93 96 96 96 4-11-5 425737 71 94 96 96 95 4-11-5 425755 70
93 95 95 95 4-11-5 425757 61 91 95 95 95 4-11-5
Example 9: Dose Response Confirmation of Antisense Oligonucleotides
Targeting Human Transthyretin in Cynomolgus Monkey Primary
Hepatocytes
[0490] Gapmers from Example 6 were also tested at various doses in
primary hepatocytes of cynomolgus monkeys. Cells were plated at a
density of 35,000 cells per well and transfected using
electroporation with 1,250 nM, 2,500 nM, 5,000 nM, 10,000 nM and
20,000 nM concentrations of antisense oligonucleotide, as specified
in Table 13. After a treatment period of approximately 16 hours,
RNA was isolated from the cells and transthyretin mRNA levels were
measured by quantitative real-time PCR. Human transthyretin primer
probe set RTS1396 was used to measure mRNA levels. Transthyretin
mRNA levels were adjusted according to total RNA content, as
measured by RIBOGREEN.RTM.. Results are presented as percent
inhibition of transthyretin, relative to untreated control cells.
As illustrated in Table 13, transthyretin mRNA levels were reduced
in a dose-dependent manner in hepatocytes treated with ISIS
oligonucleotides.
[0491] In absence of a complete cynomolgus monkey gene sequence in
the NCBI database, the oligonucleotides were tested for
cross-reactivity against the rhesus monkey gene sequence, since the
two species are from the same genus, `Macaca`. The human
oligonucleotides are cross-reactive with rhesus monkey
transthyretin gene, designated herein as SEQ ID NO: 4 (exons 1-4
extracted from GENBANK Accession No. NW_001105671.1). `Mismatches`
indicates the number of mismatches between the human
oligonucleotide and the rhesus monkey transthyretin gene. `n/a`
indicates that the human oligonucleotide has more than 3 mismatches
with the rhesus monkey transthyretin gene and therefore does not
cross-react with it.
TABLE-US-00013 TABLE 13 Dose-dependent antisense inhibition of
human transthyretin in Rhesus monkey primary hepatocytes using
electroporation Rhesus monkey Rhesus monkey 1,250 2,500 5,000
10,000 2,0000 IC.sub.50 Target start Target stop ISIS NO nM nM nM
nM nM (.mu.M) site site Mismatches 304299 21 45 69 80 95 3.1 504
523 0 304309 53 66 79 85 93 <1.25 575 594 0 304311 75 78 82 86
90 <1.25 594 613 0 304312 37 53 65 75 80 2.3 595 614 0 420915 59
54 77 87 94 <1.25 505 524 0 420951 67 77 91 93 96 <1.25 578
597 0 425653 56 72 84 83 85 <1.25 593 612 0 425655 0 7 0 21 45
>20 478 497 2 425656 41 20 38 53 51 8.7 479 498 2 425679 68 74
88 94 98 <1.25 578 597 0 425695 42 29 41 49 65 25.8 478 497 2
425736 36 27 37 49 74 8.2 479 498 2 425737 76 78 89 95 97 <1.25
501 520 0 425755 79 80 92 94 97 <1.25 578 597 0 425757 68 74 88
95 96 <1.25 580 599 0
Example 10: In Vivo Inhibition of Human Transthyretin in Human
Transthyretin-Transgenic Mice
[0492] Gapmers from Example 6, demonstrating significant inhibition
of transthyretin mRNA, were tested in transgenic mice containing
the human transthyretin gene and the efficacy of the gapmers was
evaluated.
Treatment
[0493] Fifteen groups of four hTTR transgenic female mice each were
administered subcutaneously twice a week for four weeks with 25
mg/kg of ISIS 304299, ISIS 304309, ISIS 304311, ISIS 304312, ISIS
420915, ISIS 420951, ISIS 425653, ISIS 425655, ISIS 425656, ISIS
425679, ISIS 425695, ISIS 425736, ISIS 425737, ISIS 425755, or ISIS
425757. Another group of four female hTTR transgenic mice was
injected with 25 mg/kg of control oligonucleotide ISIS 141923
(CCTTCCCTGAAGGTTCCTCC, designated herein as SEQ ID NO: 165) twice a
week for four weeks. Another group of four hTTR transgenic female
mice were injected subcutaneously with PBS twice a week for four
weeks. The mice injected with PBS served as a control group. Blood
samples were collected from all groups on weeks 0, 1, 2, 3, and 4
for plasma transthyretin level analysis. The mice were sacrificed
two days after the last dose and livers were harvested for target
mRNA analysis.
RNA Analysis
[0494] RNA was extracted from liver tissue for real-time PCR
analysis of transthyretin using primer probe set RTS3029. Results
are presented as percent inhibition of human transthyretin,
relative to PBS control. As shown in Table 14, treatment with ISIS
antisense oligonucleotides resulted in significant reduction of
human transthyretin mRNA in comparison to the PBS control.
Treatment with the control oligonucleotide, ISIS 141923 did not
result in significant reduction of transthyretin, as expected.
TABLE-US-00014 TABLE 14 Inhibition of human transthyretin mRNA in
the hTTR transgenic mice liver relative to the PBS control ISIS NO
% inhibition 304299 79 304309 83 304311 63 304312 64 420915 82
420951 92 425653 66 425655 76 425656 76 425679 93 425695 82 425736
63 425737 76 425755 91 425757 91 141923 28
Protein Analysis
[0495] Human transthyretin protein levels were measured in
transgenic mice plasma by ELISA using an anti-transthyretin
polyclonal antibody (Abcam Ab37774) and a sheep anti-TTR horse
radish peroxidase detection antibody (Abcam cat. no. 35217). The
color reaction was developed by the ImmunoPure.RTM. TMB Substrate
Kit and absorbance measured at 450 nm using a microtiter plate
spectrophotometer. Plasma samples were taken predose and on days 7,
14 and 28. The results are presented in Table 15 expressed as
percentage inhibition compared to the predose levels and
demonstrate a time-dependent reduction in protein levels with
treatment with ISIS oligonucleotides.
TABLE-US-00015 TABLE 15 Inhibition of human transthyretin protein
in the hTTR transgenic mice plasma relative to predose levels ISIS
ISIS ISIS ISIS ISIS ISIS ISIS ISIS PBS 304299 304309 420915 420951
425679 425695 425755 141923 Day 7 0 50 63 71 92 99 69 57 3 Day 14 3
76 78 90 98 100 80 72 3 Day 21 20 88 81 95 100 99 88 78 13 Day 28
13 89 83 98 100 100 91 79 8
Body Weight and Organ Weight
[0496] The body weights of the mice were measured predose and at
the end of the treatment period. The body weights are presented in
Table 16 and are expressed as percent increase over the PBS control
weight taken before the start of treatment. Liver, spleen and
kidney weights were measured at the end of the study, and are also
presented in Table 16 as a percent change over the respective organ
weights of the PBS control. As shown in Table 16, there was no
significant change in body or organ weights as a result of
antisense oligonucleotide treatment.
TABLE-US-00016 TABLE 16 Percent change in body and organ weights of
transgenic mice after antisense oligonucleotide treatment Body
weight Liver Spleen Kidney PBS 1.1 1.0 1.0 1.0 ISIS 304299 1.1 1.1
1.0 0.8 ISIS 304309 1.1 1.1 1.0 1.0 ISIS 304311 1.1 1.2 1.0 1.2
ISIS 304312 1.1 1.3 1.0 0.8 ISIS 420915 1.1 1.1 1.0 1.1 ISIS 420951
1.1 1.2 1.0 1.5 ISIS 425653 1.1 1.1 0.9 1.0 ISIS 425655 1.1 1.3 1.0
1.2 ISIS 425656 1.2 1.3 1.0 1.3 ISIS 425679 1.2 1.2 1.0 1.6 ISIS
425695 1.1 1.3 1.0 1.0 ISIS 425736 1.2 1.2 1.0 1.0 ISIS 425737 1.1
1.2 1.1 1.2 ISIS 425755 1.2 1.3 1.1 1.3 ISIS 425757 1.1 1.9 1.0 1.5
ISIS 141923 1.1 1.1 1.0 0.8
Liver Function
[0497] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Table 17, expressed in IU/L. Plasma
levels of bilirubin were also measured using the same clinical
chemistry analyzer; results are also presented in Table 17 and
expressed in mg/dL.
TABLE-US-00017 TABLE 17 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of transgenic mice ALT
AST Bilirubin (IU/L) (IU/L) (mg/dL) PBS 31 78 0.23 ISIS 304299 40
121 0.19 ISIS 304309 38 119 0.20 ISIS 304311 34 60 0.16 ISIS 304312
43 67 0.17 ISIS 420915 34 75 0.26 ISIS 420951 75 124 0.17 ISIS
425653 35 78 0.20 ISIS 425655 131 109 0.16 ISIS 425656 68 110 0.19
ISIS 425679 119 180 0.20 ISIS 425695 43 69 0.15 ISIS 425736 23 58
0.16 ISIS 425737 35 64 0.19 ISIS 425755 109 162 0.16 ISIS 425757
1904 937 0.24 ISIS 141923 31 76 0.19
Kidney Function
[0498] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) were
measured using an automated clinical chemistry analyzer (Hitachi
Olympus AU400e, Melville, N.Y.). Results are presented in Table 18,
expressed in mg/dL. The data indicates that antisense inhibition of
transthyretin has no effect on BUN levels in these transgenic
mice.
TABLE-US-00018 TABLE 18 Effect of antisense oligonucleotide
treatment on BUN (mg/dL) in the kidney of transgenic mice BUN
(mg/dL) PBS 26 ISIS 304299 24 ISIS 304309 29 ISIS 304311 28 ISIS
304312 26 ISIS 420915 25 ISIS 420951 25 ISIS 425653 24 ISIS 425655
28 ISIS 425656 25 ISIS 425679 26 ISIS 425695 28 ISIS 425736 25 ISIS
425737 23 ISIS 425755 24 ISIS 425757 25 ISIS 141923 23
Example 11: Tolerability of Antisense Oligonucleotides Targeting
Human Transthyretin in CD1 Mice
[0499] CD1.RTM. mice (Charles River, Mass.) are a multipurpose
model of mice, frequently utilized for safety and efficacy testing.
The mice were treated with ISIS antisense oligonucleotides selected
from studies described in Example 10 and evaluated for changes in
the levels of various metabolic markers.
Treatment
[0500] Groups of eight CD1 mice each were injected subcutaneously
twice a week with 50 mg/kg of ISIS 304299, ISIS 304309, ISIS
420915, ISIS 420951, ISIS 425655, ISIS 425656, ISIS 425679, ISIS
425695, ISIS 425736, ISIS 425737, and ISIS 425755. Four mice from
each group were evaluated at week 2 and week 6 of the treatment
period. Three days after the last dose at each time point, body
weights were taken, mice were euthanized and organs and plasma were
harvested for further analysis.
Body and Organ Weights
[0501] The body weights of the mice were measured pre-dose and at
the end of each treatment period (two weeks and six weeks). The
body weights are presented in Tables 19 and 20, and are expressed
as percent increase over the PBS control weight taken before the
start of treatment. Liver, spleen and kidney weights were measured
at the end of the study, and are also presented in Tables 19 and 20
as a percentage change over the respective organ weights of the PBS
control.
TABLE-US-00019 TABLE 19 Change in body and organ weights of CD1
mice after antisense oligonucleotide treatment (%) at week 2 Body
weight Liver Spleen Kidney PBS 1.1 1.0 1.0 1.0 ISIS 304299 1.1 1.1
1.1 1.1 ISIS 304309 1.1 1.1 1.1 1.0 ISIS 420915 1.1 1.1 1.1 1.0
ISIS 420951 1.1 1.3 1.7 1.2 ISIS 425655 1.1 1.2 1.2 0.9 ISIS 425656
1.1 1.1 1.1 1.0 ISIS 425679 1.1 1.1 1.4 1.1 ISIS 425695 1.1 1.1 0.9
1.1 ISIS 425736 1.1 1.1 1.0 1.1 ISIS 425737 1.2 1.1 1.1 1.1 ISIS
425755 1.2 1.2 1.3 1.2
TABLE-US-00020 TABLE 20 Change in body and organ weights of CD1
mice after antisense oligonucleotide treatment (%) at week 6 Body
weight Liver Spleen Kidney PBS 1.2 1.0 1.0 1.0 ISIS 304299 1.3 1.2
1.4 1.0 ISIS 304309 1.3 1.3 2.0 1.0 ISIS 420915 1.3 1.1 1.5 0.9
ISIS 420951 1.3 1.3 2.0 1.1 ISIS 425655 1.4 1.3 1.7 0.9 ISIS 425656
1.3 1.3 1.1 1.0 ISIS 425679 1.3 1.4 2.3 1.2 ISIS 425695 1.3 1.4 1.5
1.0 ISIS 425736 1.3 1.1 1.2 0.9 ISIS 425737 1.2 1.1 1.3 1.0 ISIS
425755 1.3 1.3 2.1 1.0
Liver Function
[0502] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Tables 21 and 22 expressed in IU/L.
Plasma levels of bilirubin and albumin were also measured using the
same clinical chemistry analyzer and the results are also presented
in Tables 21 and 22.
TABLE-US-00021 TABLE 21 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of CD1 mice at week 2
ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (g/dL) PBS 38 66
0.19 5.0 ISIS 304299 42 79 0.33 3.8 ISIS 304309 52 77 0.22 3.2 ISIS
420915 32 61 0.28 3.5 ISIS 420951 1184 804 0.17 3.7 ISIS 425655 60
70 0.20 3.9 ISIS 425656 37 53 0.31 3.5 ISIS 425679 88 147 0.23 3.7
ISIS 425695 25 50 0.23 3.6 ISIS 425736 31 79 0.23 3.2 ISIS 425737
39 43 0.23 3.1 ISIS 425755 104 85 0.29 3.6
TABLE-US-00022 TABLE 22 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of CD1 mice at week 6
ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (g/dL) PBS 31 67
0.20 5.6 ISIS 304299 54 71 0.20 5.2 ISIS 304309 1211 504 0.30 5.2
ISIS 420915 89 91 0.17 5.0 ISIS 420951 872 319 0.20 3.6 ISIS 425655
730 247 0.13 4.3 ISIS 425656 502 261 0.17 4.3 ISIS 425679 935 475
0.29 4.5 ISIS 425695 1627 563 0.16 4.0 ISIS 425736 41 47 0.15 4.1
ISIS 425737 32 55 0.16 4.1 ISIS 425755 233 176 0.16 4.3
Kidney Function
[0503] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) and
creatinine were measured using an automated clinical chemistry
analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are
presented in Tables 23 and 24, expressed in mg/dL.
TABLE-US-00023 TABLE 23 Effect of antisense oligonucleotide
treatment on metabolic markers (mg/dL) in the kidney of CD1 mice at
week 2 BUN Creatinine PBS 32 0.23 ISIS 304299 26 0.21 ISIS 304309
30 0.19 ISIS 420915 30 0.22 ISIS 420951 24 0.17 ISIS 425655 29 0.22
ISIS 425656 25 0.19 ISIS 425679 28 0.19 ISIS 425695 29 0.19 ISIS
425736 24 0.19 ISIS 425737 24 0.16 ISIS 425755 27 0.17
TABLE-US-00024 TABLE 24 Effect of antisense oligonucleotide
treatment on metabolic markers (mg/dL) in the kidney of CD1 mice at
week 6 BUN Creatinine PBS 24 0.15 ISIS 304299 19 0.11 ISIS 304309
20 0.14 ISIS 420915 24 0.18 ISIS 420951 19 0.08 ISIS 425655 22 0.11
ISIS 425656 21 0.10 ISIS 425679 20 0.06 ISIS 425695 21 0.08 ISIS
425736 22 0.07 ISIS 425737 18 0.07 ISIS 425755 22 0.09
Hematology Assays
[0504] Blood obtained from all mice groups were sent to Antech
Diagnostics for hematocrit (HCT), mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin
concentration (MCHC) measurements and analyses, as well as
measurements of the differential blood cell counts, such as that of
WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets,
and total hemoglobin content. The results are presented in Tables
25-28. Percentages given in the tables indicate the percent change
in total blood cell count compared to the PBS control. Those
antisense oligonucleotides which did not affect a decrease in
platelet count less than 70% of the PBS control or an increase in
monocyte count more than two-fold were selected for further
studies.
TABLE-US-00025 TABLE 25 Effect of antisense oligonucleotide
treatment on complete blood cell count (%) compared to the PBS
control in CD1 mice at week 2 ISIS NO. WBC RBC Hemoglobin HCT MCV
MCH MCHC 304299 -15 -3 -2 0 +3 +1 -1 304309 -13 -4 -7 -6 -2 -4 -2
420915 +7 -7 -7 -5 +2 +1 -2 420951 +79 -6 -5 -5 +1 +1 0 425655 +56
-3 -5 -4 -1 -2 -1 425656 +69 -5 -6 -5 0 -1 -2 425679 +30 -6 -7 -7
-1 -1 0 425695 +49 -3 -4 -4 0 0 +1 425736 +15 -6 -6 -4 +1 0 -2
425737 +19 -5 -7 -5 -1 -3 -2 425755 +85 -3 -6 -6 -4 -3 0
TABLE-US-00026 TABLE 26 Effect of antisense oligonucleotide
treatment on complete blood cell count (%) compared to the PBS
control in CD1 mice at week 6 ISIS NO. WBC RBC Hemoglobin HCT MCV
MCH MCHC 304299 -7 -9 -10 -13 -5 0 +4 304309 +10 -12 -11 -15 -5 +1
+6 420915 +11 -7 -8 -10 -4 -2 +2 420951 +81 -12 -20 -19 -9 -9 -1
425655 +29 -3 -11 -10 -8 -9 -2 425656 +72 -1 -5 -6 -4 -5 -1 425679
+154 -11 -20 -21 -10 -9 +2 425695 +118 +3 -9 -9 -2 -12 +3 425736
+51 +4 -5 -7 0 -10 +1 425737 +30 +8 -1 -2 0 -8 +1 425755 +54 -1 -11
-12 -8 -10 0
TABLE-US-00027 TABLE 27 Effect of antisense oligonucleotide
treatment on differential blood cell count (%) compared to the PBS
control in CD1 mice at week 2 ISIS NO. Neutrophils Monocytes
Lymphocytes Platelets 304299 11 -3 20 17 304309 -11 5 8 14 420915 1
4 -24 41 420951 18 -7 32 -9 425655 18 -5 20 18 425656 31 -7 -4 24
425679 2 -1 24 -19 425695 -50 15 20 29 425736 8 -1 0 10 425737 -29
10 -8 24 425755 -13 7 -4 9
TABLE-US-00028 TABLE 28 Effect of antisense oligonucleotide
treatment on differential blood cell count (%) compared to the PBS
control in CD1 mice at week 6 ISIS NO. Neutrophils Lymphocytes
Monocytes Platelets 304299 -60 +26 +10 -16 304309 -28 +12 +30 +2
420915 -29 +6 +50 -30 420951 -26 +11 0 -40 425655 -16 +8 -10 -19
425656 -22 +16 -50 -25 425679 -36 +19 -20 -27 425695 -25 +9 -15 -49
425736 -41 +16 -5 -46 425737 -53 +23 -20 -65 425755 -20 +4 +25
-41
Example 12: Measurement of Half-Life of Antisense Oligonucleotide
in CD1 Mouse Liver
[0505] CD1 mice were treated with ISIS antisense oligonucleotides
from studies described in Example 11 and the oligonucleotide
half-life as well as the elapsed time for oligonucleotide
degradation and elimination from the liver was evaluated.
Treatment
[0506] Groups of twelve CD1 mice each were injected subcutaneously
twice per week for 2 weeks with 50 mg/kg of ISIS 304299, ISIS
304309, ISIS 420915, ISIS 420951, ISIS 425655, ISIS 425656, ISIS
425679, ISIS 425695, ISIS 425736, ISIS 425737, and ISIS 425755.
Four mice from each group were sacrificed 3 days, 28 days and 56
days following the final dose. Livers were harvested for
analysis.
Measurement of Oligonucleotide Concentration
[0507] The concentration of the full-length oligonucleotide as well
as the total oligonucleotide concentration (including the degraded
form) was measured. The method used is a modification of previously
published methods (Leeds et al., 1996; Geary et al., 1999) which
consist of a phenol-chloroform (liquid-liquid) extraction followed
by a solid phase extraction. An internal standard (ISIS 355868, a
27-mer 2'-O-methoxyethyl modified phosphorothioate oligonucleotide,
GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166)
was added prior to extraction. Tissue sample concentrations were
calculated using calibration curves, with a lower limit of
quantitation (LLOQ) of approximately 1.14 .mu.g/g. Half-lives were
then calculated using WinNonlin software (PHARSIGHT).
[0508] The results are presented in Tables 29 and 30, expressed as
.mu.g/g liver tissue. The half-life of each oligonucleotide is
presented in Table 31. Antisense oligonucleotides with half-lives
within 11-34 days were chosen for further studies.
TABLE-US-00029 TABLE 29 Full-length oligonucleotide concentration
(.mu.g/g) in the liver of CD1 mice ISIS NO. 3 days 28 days 56 days
304299 180 56 8 304309 317 254 106 420915 248 126 34 420951 173 109
49 425655 191 113 33 425656 256 73 29 425679 201 73 27 425695 315
194 65 425736 219 110 47 425737 190 40 9 425755 211 120 47
TABLE-US-00030 TABLE 30 Total oligonucleotide concentration
(.mu.g/g) in the liver of CD1 mice ISIS NO. 3 days 28 days 56 days
304299 268 168 38 304309 389 354 152 420915 314 229 83 420951 262
196 131 425655 298 217 87 425656 328 135 85 425679 333 161 103
425695 364 263 143 425736 298 211 140 425737 266 117 31 425755 337
227 140
TABLE-US-00031 TABLE 31 Half-life of oligonucleotide (days) in the
liver of CD1 mice ISIS Half-life NO. (days) 304299 12 304309 33
420915 19 420951 29 425655 21 425656 17 425679 18 425695 23 425736
24 425737 12 425755 24
Example 13: Tolerability of Antisense Oligonucleotides Targeting
Human Transthyretin in Sprague-Dawley Rats
[0509] Sprague-Dawley rats were treated with ISIS antisense
oligonucleotides selected from studies described in Examples 11 and
12 and evaluated for changes in the levels of various metabolic
markers.
Treatment
[0510] The body weights, complete blood count and different blood
count, as well as the urine protein/creatinine ratio of the rats
were evaluated pre-dose. Groups of four Sprague-Dawley rats each
were injected subcutaneously twice a week with 50 mg/kg of ISIS
304299, ISIS 304309, ISIS 420915, ISIS 420951, ISIS 425655, ISIS
425656, ISIS 425679, ISIS 425695, ISIS 425736, ISIS 425737, and
ISIS 425755. Three days after the last dose at each time point,
body weights were taken, mice were euthanized and organs and plasma
were harvested for further analysis.
Body and Organ Weights
[0511] The body weights of the rats were measured pre-dose and at
the end of the treatment period. The body weights are presented in
Table 32, and are expressed as percent increase over the PBS
control weight taken before the start of treatment. Liver, spleen
and kidney weights were measured at the end of the study, and are
also presented in Table 32 as a percentage change over the
respective organ weights of the PBS control.
TABLE-US-00032 TABLE 32 Change in body and organ weights of
Sprague-Dawley rats after antisense oligonucleotide treatment (%)
Body weight Liver Spleen Kidney PBS 1.6 1.0 1.0 1.0 ISIS 304299 1.2
1.7 4.9 1.6 ISIS 304309 1.1 1.6 4.3 1.4 ISIS 420915 1.4 1.4 3.3 1.3
ISIS 420951 1.1 1.4 5.0 1.5 ISIS 425655 1.2 1.5 3.4 1.3 ISIS 425656
1.2 1.5 2.9 1.2 ISIS 425679 1.0 1.9 6.4 1.7 ISIS 425695 1.2 1.6 3.3
1.3 ISIS 425736 1.3 1.5 2.9 1.2 ISIS 425737 1.2 1.7 4.0 1.5 ISIS
425755 1.0 1.5 5.4 1.5
[0512] As shown in Tables 32, certain compounds showed a less than
a 4-fold increase in spleen weight.
Liver Function
[0513] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Table 33 expressed in IU/L. Plasma
levels of bilirubin and albumin were also measured using the same
clinical chemistry analyzer and the results are also presented in
Table 33.
TABLE-US-00033 TABLE 33 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of Sprague-Dawley rats
ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (g/dL) PBS 55 138
0.15 3.3 ISIS 304299 69 154 0.15 2.7 ISIS 304309 80 138 0.11 2.9
ISIS 420915 43 95 0.11 3.0 ISIS 420951 353 511 0.32 2.6 ISIS 425655
312 497 0.47 2.6 ISIS 425656 277 335 0.20 3.0 ISIS 425679 537 659
0.38 2.7 ISIS 425695 228 445 0.23 2.3 ISIS 425736 362 553 0.32 2.9
ISIS 425737 55 79 0.09 1.9 ISIS 425755 271 303 0.41 2.8
Kidney Function
[0514] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) and
creatinine were measured using an automated clinical chemistry
analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are
presented in Table 34, expressed in mg/dL. The ratio of total urine
protein to creatinine was also evaluated and presented in Table
35.
TABLE-US-00034 TABLE 34 Effect of antisense oligonucleotide
treatment on metabolic markers (mg/dL) in the kidney of
Sprague-Dawley rats BUN Creatinine PBS 20 0.26 ISIS 304299 30 0.40
ISIS 304309 24 0.33 ISIS 420915 20 0.26 ISIS 420951 37 0.47 ISIS
425655 28 0.40 ISIS 425656 25 0.34 ISIS 425679 46 0.42 ISIS 425695
30 0.37 ISIS 425736 26 0.37 ISIS 425737 30 0.36 ISIS 425755 29
0.36
TABLE-US-00035 TABLE 35 Effect of antisense oligonucleotide
treatment on total urine protein/creatinine in the kidney of
Sprague-Dawley rats Pre- dose Week 6 PBS 0.82 0.95 ISIS 304299 0.95
7.57 ISIS 304309 1.10 5.20 ISIS 420915 0.91 5.30 ISIS 420951 0.90
5.02 ISIS 425655 0.78 6.03 ISIS 425656 0.86 9.37 ISIS 425679 0.91
7.80 ISIS 425695 0.89 5.71 ISIS 425736 1.00 5.85 ISIS 425737 0.86
43.76 ISIS 425755 0.78 3.70
[0515] As shown in Tables 34 and 35, certain compounds demonstrated
a less than 7-fold increase in the total urine protein/creatinine
in the kidney of these rats. Furthermore, certain compounds
demonstrated a less than 6-fold increase in the total urine
protein/creatinine in the kidney of these rats.
Hematology Assays
[0516] Blood obtained from all rat groups were sent to Antech
Diagnostics for hematocrit (HCT), mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin
concentration (MCHC) measurements and analyses, as well as
measurements of the differential blood cell counts, such as that of
WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets,
and total hemoglobin content. The results are presented in Tables
36 and 37. Percentages given in the tables indicate the percent
change in total blood cell count compared to the PBS control.
TABLE-US-00036 TABLE 36 Effect of antisense oligonucleotide
treatment on complete blood cell count (%) compared to the PBS
control in Sprague-Dawley rats ISIS NO. WBC RBC Hemoglobin HCT MCV
MCH MCHC 304299 +4 -5 -3 +2 +11 +5 -5 304309 -10 -8 -11 -12 -4 -3
+1 420915 -9 -16 -20 -17 +1 -3 -3 420951 +5 -5 -8 -5 +1 -2 -3
425655 +22 -17 -18 -19 -2 0 +2 425656 -1 -13 -19 -16 -3 -6 -2
425679 +49 -42 -32 -28 +26 +19 -5 425695 -2 -25 -31 -29 -4 -8 -3
425736 +18 +1 -3 +2 0 -4 -4 425737 -15 -20 -18 -20 +2 +3 +1 425755
+35 -31 -27 -23 +14 +8 -4
TABLE-US-00037 TABLE 37 Effect of antisense oligonucleotide
treatment on complete blood cell count (%) compared to the PBS
control in Sprague-Dawley rats ISIS NO. Neutrophils Lymphocytes
Monocytes Platelet 304299 -61 +15 -10 -41 304309 -35 +8 +10 -37
420915 -23 +6 0 -29 420951 -62 +15 +10 -67 425655 +23 -8 +80 -13
425656 -14 0 +70 -15 425679 -4 -1 +60 -75 425695 +68 -20 +80 -5
425736 0 -2 +70 -1 425737 -6 +1 +20 -21 425755 -18 +3 +70 -58
Example 14: Pharmacokinetic Studies of Antisense Oligonucleotide
Concentration in Sprague-Dawley Rat Liver and Kidney
[0517] Sprague Dawley rats were treated with ISIS antisense
oligonucleotides from studies described in Example 13 and the
oligonucleotide half-life as well as the elapsed time for
oligonucleotide degradation and elimination from the liver and
kidney was evaluated.
Treatment
[0518] Groups of four Sprague Dawley rats each were injected
subcutaneously twice a week for 2 weeks with 20 mg/kg of ISIS
304299, ISIS 304309, ISIS 420915, ISIS 420951, ISIS 425655, ISIS
425656, ISIS 425679, ISIS 425695, ISIS 425736, ISIS 425737, and
ISIS 425755. Three days after the last dose, the rats were
sacrificed and livers and kidneys were collected for analysis.
Measurement of Oligonucleotide Concentration
[0519] The concentration of the full-length oligonucleotide as well
as the total oligonucleotide concentration (including the degraded
form) was measured. The method used is a modification of previously
published methods (Leeds et al., 1996; Geary et al., 1999) which
consist of a phenol-chloroform (liquid-liquid) extraction followed
by a solid phase extraction. An internal standard (ISIS 355868, a
27-mer 2'40-methoxyethyl modified phosphorothioate oligonucleotide,
GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166)
was added prior to extraction. Tissue sample concentrations were
calculated using calibration curves, with a lower limit of
quantitation (LLOQ) of approximately 1.14 .mu.g/g. The results are
presented in Tables 38 and 39, expressed as .mu.gig liver or kidney
tissue. The kidney to liver ratio of full length oligonucleotide
was also calculated and presented in Table 38.
TABLE-US-00038 TABLE 38 Full-length oligonucleotide concentration
(.mu.g/g) and ratio in the liver and kidney of Sprague-Dawley rats
Kidney/Liver ISIS NO. Liver Kidney Ratio 304299 165 487 2.9 304309
344 606 1.8 420915 171 680 4.0 420951 214 389 1.8 425655 242 466
1.9 425656 286 595 2.1 425679 290 334 1.2 425695 266 566 2.1 425736
245 571 2.3 425737 167 477 2.9 425755 218 379 1.7
TABLE-US-00039 TABLE 39 Total oligonucleotide concentration
(.mu.g/g) in the liver and kidney of Sprague-Dawley rats ISIS NO.
Liver Kidney 304299 208 653 304309 409 803 420915 196 844 420951
348 879 425655 340 764 425656 329 703 425679 461 710 425695 369 843
425736 282 738 425737 195 587 425755 351 886
Example 15: In Vivo Dose-Dependent Inhibition of Human
Transthyretin in Transgenic Mice
[0520] Transgenic mice containing the human transthyretin gene were
dosed in increasing doses of ISIS oligonucleotides selected from
studies described in Example 14 to evaluate the effect of
dose-dependent inhibition of human transthyretin in these mice.
Treatment
[0521] Groups of four mice, two male and two female, each were
injected subcutaneously twice a week for 4 weeks with 4 mg/kg, 10
mg/kg or 25 mg/kg of ISIS 304299, ISIS 420915, ISIS 420951, ISIS
425679, ISIS 425736, ISIS 425737, or ISIS 425755. One group of four
mice, two male and two female, was injected subcutaneously twice a
week for 4 weeks with 25 mg/kg of the control oligonucleotide, ISIS
141923. One control group of four mice, two male and two female,
was injected subcutaneously twice a week for 4 weeks with PBS.
Plasma samples were taken from each group at days 0, 7, 14, 21 and
28. Two days after the last dose, the mice were euthanized and
organs were harvested for further analysis.
RNA Analysis
[0522] RNA was extracted from liver tissue for real-time PCR
analysis of transthyretin using primer probe set RTS3029. Results
are presented as percent inhibition of human transthyretin,
relative to PBS control. As shown in Table 40, treatment with ISIS
antisense oligonucleotides resulted in significant dose-dependent
reduction of human transthyretin mRNA in comparison to the PBS
control. Treatment with the control oligonucleotide, ISIS 141923
did not result in significant reduction of transthyretin, as
expected.
TABLE-US-00040 TABLE 40 Inhibition of human transthyretin mRNA in
the hTTR transgenic mice liver relative to the PBS control ISIS
Dose % NO. (mg/kg) inhibition 304299 25 73 10 60 4 9 420915 25 78
10 57 4 43 420951 25 91 10 85 4 52 425679 25 94 10 88 4 42 425736
25 49 10 54 4 15 425737 25 82 10 59 4 21 425755 25 91 10 79 4 24
141923 25 0
[0523] Protein Analysis
[0524] Human transthyretin protein levels were measured in
transgenic mice plasma by ELISA using an anti-transthyretin
polyclonal antibody (Abcam Ab37774) and a sheep anti-TTR horse
radish peroxidase detection antibody (Abcam cat. no. 35217). The
color reaction was developed by the ImmunoPure.RTM. TMB Substrate
Kit and absorbance measured at 450 nm using a microtiter plate
spectrophotometer. Plasma samples were taken predose and on days 7,
14, 21 and 28. The results are presented in Table 41 expressed as
percentage inhibition compared to the predose levels and
demonstrate a time-dependent and dose-dependent reduction in
protein levels on treatment with ISIS oligonucleotides.
TABLE-US-00041 TABLE 41 Inhibition of human transthyretin protein
in transgenic mice plasma relative to pre-dose levels ISIS NO. Day
0 Day 7 Day 14 Day 21 Day 28 141923 25 0 0 20 77 41 304299 25 0 44
85 100 88 10 0 0 8 93 78 4 0 0 0 57 0 420915 25 0 0 67 86 91 10 0
21 39 70 71 4 0 25 0 0 0 420951 25 0 83 96 100 100 10 0 35 66 91 86
4 0 7 26 0 0 425679 25 0 93 97 96 98 10 0 38 80 96 95 4 0 0 0 0 0
425736 25 0 56 76 82 92 10 0 0 33 37 66 4 0 0 0 0 0 425737 25 0 90
96 99 98 10 0 51 80 88 89 4 0 29 21 37 31 425755 25 0 88 96 98 99
10 0 52 76 90 88 4 0 29 22 36 26
Body Weight and Organ Weight
[0525] The body weights of the mice were measured pre-dose and at
the end of the treatment period. The body weights are presented in
Table 42 and are expressed as percent increase over the PBS control
weight taken before the start of treatment. Liver, spleen and
kidney weights were measured at the end of the study, and are also
presented in Table 42 as a percentage change over the respective
organ weights of the PBS control.
TABLE-US-00042 TABLE 42 Change in body and organ weights of
transgenic mice after antisense oligonucleotide treatment (%) Dose
Body (mg/kg) weight Liver Spleen Kidney PBS +13 0 0 0 ISIS 25 +17
+16 +3 -2 304299 10 +14 +10 -13 -4 4 +17 +2 +17 -2 ISIS 25 +18 +12
-6 -6 420915 10 +16 +6 -4 -5 4 +15 +4 +8 -2 ISIS 25 +22 +23 +32 -2
420951 10 +16 +11 +10 -3 4 +24 +7 +19 +5 ISIS 25 +24 +33 +40 -1
425679 10 +14 +5 +9 -2 4 +19 +7 +10 0 ISIS 25 +16 +15 0 -5 425736
10 +28 +8 -12 -6 4 +20 +10 -9 -2 ISIS 25 +16 +13 0 -2 425737 10 +19
+6 +18 -3 4 +19 +5 +4 +1 ISIS 25 +21 +25 +34 -5 425755 10 +17 +10
+13 -4 4 +22 +3 +27 +4 ISIS 25 +20 +8 -3 -4 141923
Liver Function
[0526] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Table 43 expressed in IU/L. Plasma
levels of bilirubin were also measured using the same clinical
chemistry analyzer; results are also presented in Table 43 and
expressed in mg/dL.
TABLE-US-00043 TABLE 43 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of transgenic mice Dose
ALT AST TBIL (mg/kg) (IU/L) (IU/L) (mg/dL) PBS -- 48 112 0.20 ISIS
25 42 93 0.14 304299 10 37 56 0.18 4 35 71 0.15 ISIS 25 63 181 0.22
420915 10 46 132 0.22 4 35 114 0.22 ISIS 25 63 85 0.17 420951 10 42
107 0.21 4 31 74 0.19 ISIS 25 156 150 0.13 425679 10 93 148 0.23 4
38 119 0.22 ISIS 25 37 78 0.21 425736 10 33 62 0.20 4 46 228 0.23
ISIS 25 55 121 0.20 425737 10 41 94 0.18 4 32 73 0.14 ISIS 25 74
160 0.17 425755 10 31 80 0.16 4 45 122 0.21 ISIS 25 66 141 0.17
141923
Kidney Function
[0527] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) were
measured using an automated clinical chemistry analyzer (Hitachi
Olympus AU400e, Melville, N.Y.). Results are presented in Table 44,
expressed in mg/dL.
TABLE-US-00044 TABLE 44 Effect of antisense oligonucleotide
treatment on BUN (mg/dL) in the kidney of transgenic mice Dose
(mg/kg) BUN PBS 22 ISIS 25 22 304299 10 22 4 22 ISIS 25 24 420915
10 25 4 20 ISIS 25 24 420951 10 25 4 26 ISIS 25 26 425679 10 24 4
22 ISIS 25 20 425736 10 22 4 22 ISIS 25 21 425737 10 19 4 23 ISIS
25 23 425755 10 21 4 20 ISIS 25 21 141923
Example 16: In Vivo Inhibition of Human Transthyretin in Human
Transthyretin-Transgenic Mice
[0528] Antisense oligonucleotides with 5-10-5 MOE motifs, ISIS
304313, ISIS 420913, ISIS 420919, ISIS 420921, ISIS 420922, ISIS
420937, ISIS 420944, ISIS 420947, ISIS 420949, ISIS 420950, ISIS
420951, ISIS 420952, ISIS 420953, ISIS 420955, ISIS 420957, and
ISIS 420959 from Table 4. These antisense oligonucleotides
exhibited 65% inhibition or more of transthyretin mRNA were
selected and tested in transgenic mice containing the human
transthyretin gene. Additional oligonucleotides with overlapping
sequences to ISIS 420951 (GTTTTATTGTCTCTGCCTGG (SEQ ID NO: 116)),
and with various motifs were also designed to test in the
transgenic mice. These additional oligonucleotides were ISIS 450518
(TTTTATTGTCTCTGCCTG (SEQ ID NO: 5-8-5 MOE (SEQ ID NO: 167)), ISIS
450519 (GTTTTATTGTCTCTGCCTGG, 6-8-6 MOE (SEQ ID NO: 116)), ISIS
450520 (GTTTTATTGTCTCTGCCTGG, 3-10-7 MOE (SEQ ID NO: 116)), ISIS
450521 (GTTTTATTGTCTCTGCCTGG, 7-10-3 MOE (SEQ ID NO: 116)), ISIS
450522 (GTTTTATTGTCTCTGCCTGG, 2-10-8 MOE (SEQ ID NO: 116)), and
ISIS 450523 (GTTTTATTGTCTCTGCCTGG, 8-10-2 MOE (SEQ ID NO:
116)).
Treatment
[0529] Groups of four hTTR transgenic mice each, two male and two
female, were administered subcutaneously twice per week for four
weeks with 25 mg/kg of ISIS 304313, ISIS 420913, ISIS 420919, ISIS
420921, ISIS 420922, ISIS 420937, ISIS 420944, ISIS 420947, ISIS
420949, ISIS 420950, ISIS 420951, ISIS 420952, ISIS 420953, ISIS
420955, ISIS 420957, ISIS 420959, ISIS 425518, ISIS 425519, ISIS
425520, ISIS 425521, ISIS 425522, or ISIS 425523. A control group
four hTTR transgenic mice, two male and two female, were injected
subcutaneously with PBS twice per week for four weeks. Blood
samples were collected from all groups on days 0, 14 and 28 for
plasma transthyretin level analysis. The mice were sacrificed two
days after the last dose and livers were harvested for target mRNA
analysis.
RNA Analysis
[0530] RNA was extracted from liver tissue for real-time PCR
analysis of transthyretin using primer probe set RTS3029. Results
are presented as percent inhibition of human transthyretin,
relative to PBS control. As shown in Table 45, treatment with ISIS
antisense oligonucleotides resulted in significant reduction of
human transthyretin mRNA in comparison to the PBS control.
TABLE-US-00045 TABLE 45 Inhibition of human transthyretin mRNA in
the hTTR transgenic mice liver relative to the PBS control ISIS %
NO. inhibition 304313 68 420913 83 420919 64 420921 70 420922 82
420937 46 420944 58 420947 62 420949 87 420950 94 420952 95 420953
93 420955 93 420957 90 420959 73 450518 80 450519 87 450520 85
450521 94 450522 73 450523 94 420951 94
Protein Analysis
[0531] Human transthyretin protein levels were measured in
transgenic mice plasma by ELISA using an anti-transthyretin
transthyretin polyclonal antibody (Abcam Ab37774) and a sheep
anti-TTR horse radish peroxidase detection antibody (Abcam cat. no.
35217). The color reaction was developed by the ImmunoPure.RTM. TMB
Substrate Kit and absorbance measured at 450 nm using a microtiter
plate spectrophotometer. Plasma samples were taken predose and on
days 7, 14 and 28. The results are presented in Table 46 expressed
as percentage inhibition compared to the pre-dose levels and
demonstrate a time-dependent reduction in protein levels on
treatment with ISIS oligonucleotides.
TABLE-US-00046 TABLE 46 Inhibition of human transthyretin protein
in the hTTR transgenic mice plasma relative to pre-dose levels Day
0 Day 14 Day 28 PBS 0 0 0 ISIS 304313 0 62 77 ISIS 420913 0 91 97
ISIS 420919 0 70 82 ISIS 420921 0 83 87 ISIS 420922 0 95 97 ISIS
420937 0 37 59 ISIS 420944 0 57 72 ISIS 420947 0 57 65 ISIS 420949
0 93 99 ISIS 420950 0 97 100 ISIS 420952 0 98 100 ISIS 420953 0 99
100 ISIS 420955 0 89 100 ISIS 420957 0 92 94 ISIS 420959 0 69 87
ISIS 450518 0 80 97 ISIS 450519 0 94 100 ISIS 450520 0 83 100 ISIS
450521 0 100 100 ISIS 450522 0 93 97 ISIS 450523 0 100 100 ISIS
420951 0 99 100
Liver Function
[0532] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Table 47 expressed in IU/L. Plasma
levels of bilirubin were also measured using the same clinical
chemistry analyzer; results are also presented in Table 47 and
expressed in mg/dL.
TABLE-US-00047 TABLE 47 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of transgenic mice ALT
AST Bilirubin (IU/L) (IU/L) (mg/dL) PBS 34 88 0.20 ISIS 304313 42
79 0.16 ISIS 420913 35 67 0.17 ISIS 420919 63 177 0.20 ISIS 420921
47 103 0.15 ISIS 420922 42 128 0.16 ISIS 420937 33 160 0.15 ISIS
420944 38 84 0.15 ISIS 420947 42 120 0.17 ISIS 420949 46 125 0.15
ISIS 420950 73 106 0.15 ISIS 420952 151 271 0.19 ISIS 420953 982
452 0.16 ISIS 420955 47 80 0.15 ISIS 420957 53 133 0.18 ISIS 420959
31 89 0.11 ISIS 450518 103 200 0.20 ISIS 450519 64 81 0.12 ISIS
450520 350 270 0.12 ISIS 450521 104 226 0.13 ISIS 450522 109 201
0.14 ISIS 450523 80 170 0.19 ISIS 420951 67 100 0.09
Kidney Function
[0533] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) were
measured using an automated clinical chemistry analyzer (Hitachi
Olympus AU400e, Melville, N.Y.). Results are presented in Table 48,
expressed in mg/dL.
TABLE-US-00048 TABLE 48 Effect of antisense oligonucleotide
treatment on BUN (mg/dL) in the kidney of transgenic mice PBS 35
ISIS 304313 29 ISIS 420913 30 ISIS 420919 29 ISIS 420921 29 ISIS
420922 27 ISIS 420937 29 ISIS 420944 27 ISIS 420947 26 ISIS 420949
25 ISIS 420950 34 ISIS 420952 23 ISIS 420953 34 ISIS 420955 24 ISIS
420957 23 ISIS 420959 29 ISIS 450518 28 ISIS 450519 25 ISIS 450520
29 ISIS 450521 24 ISIS 450522 29 ISIS 450523 27 ISIS 420951 25
Example 17: Tolerability of Antisense Oligonucleotides Targeting
Human Transthyretin in CD1 Mice
[0534] CD1 mice were treated with ISIS antisense oligonucleotides
from Example 16 and evaluated for changes in the levels of various
metabolic markers.
Treatment
[0535] Groups of eight CD1 mice each were injected subcutaneously
twice a week with 50 mg/kg of ISIS 304313, ISIS 420913, ISIS
420919, ISIS 420921, ISIS 420922, ISIS 420937, ISIS 420944, ISIS
420947, ISIS 420949, ISIS 420950, ISIS 420951, ISIS 420952, ISIS
420953, ISIS 420955, ISIS 420957, ISIS 420959, ISIS 425518, ISIS
425519, ISIS 425520, ISIS 425521, ISIS 425522, or ISIS 425523.
Three days after the last dose at each time point, body weights
were taken, mice were euthanized and organs and plasma were
harvested for further analysis.
Body and Organ Weights
[0536] The body weights of the mice were measured pre-dose and at
the end of each treatment period (two weeks and six weeks). The
body weights are presented in Table 49 and are expressed as percent
increase over the PBS control weight taken before the start of
treatment. Liver, spleen and kidney weights were measured at the
end of the study, and are also presented in Table 49 as a
percentage change over the respective organ weights of the PBS
control.
TABLE-US-00049 TABLE 49 Change in body and organ weights of CD1
mice after antisense oligonucleotide treatment (%) at week 6 Body
weight Liver Spleen Kidney PBS 1.3 1.0 1.0 1.0 ISIS 304313 1.2 1.2
1.4 1.2 ISIS 420913 1.2 1.2 1.3 1.1 ISIS 420919 1.3 1.2 1.9 1.1
ISIS 420921 1.1 1.1 2.2 1.1 ISIS 420922 1.1 1.0 1.6 0.9 ISIS 420937
1.1 1.0 1.2 1.0 ISIS 420944 1.1 1.1 2.0 1.0 ISIS 420947 1.3 1.2 1.7
1.0 ISIS 420949 1.3 1.2 1.8 1.1 ISIS 420950 1.3 1.0 1.7 1.0 ISIS
420952 1.4 1.3 2.1 0.9 ISIS 420953 1.3 1.5 2.2 1.0 ISIS 420955 1.2
1.2 2.2 1.0 ISIS 420957 1.1 1.1 1.8 1.1 ISIS 420959 1.3 1.2 3.2 1.1
ISIS 450518 1.4 1.3 1.8 1.1 ISIS 450519 1.3 1.5 2.4 1.0 ISIS 450520
1.4 1.4 2.2 1.0 ISIS 450521 1.2 1.2 1.9 1.1 ISIS 450522 1.3 1.5 2.3
1.1 ISIS 450523 1.2 1.3 2.4 1.1 ISIS 420951 1.3 1.2 1.9 1.0
Liver Function
[0537] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Table 50 expressed in IU/L. Plasma
levels of bilirubin and albumin were also measured using the same
clinical chemistry analyzer and the results are also presented in
Table 50.
TABLE-US-00050 TABLE 50 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of CD1 mice ALT AST
TBIL PBS 34 88 0.20 ISIS 304313 42 79 0.16 ISIS 420913 35 67 0.17
ISIS 420919 63 177 0.20 ISIS 420921 47 103 0.15 ISIS 420922 42 128
0.16 ISIS 420937 33 160 0.15 ISIS 420944 38 84 0.15 ISIS 420947 42
120 0.17 ISIS 420949 46 125 0.15 ISIS 420950 73 106 0.15 ISIS
420952 151 271 0.19 ISIS 420953 982 452 0.16 ISIS 420955 47 80 0.15
ISIS 420957 53 133 0.18 ISIS 420959 31 89 0.11 ISIS 450518 103 200
0.20 ISIS 450519 64 81 0.12 ISIS 450520 350 270 0.12 ISIS 450521
104 226 0.13 ISIS 450522 109 201 0.14 ISIS 450523 80 170 0.19 ISIS
420951 67 100 0.09
Kidney Function
[0538] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) and
creatinine were measured using an automated clinical chemistry
analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are
presented in Table 51, expressed in mg/dL.
TABLE-US-00051 TABLE 51 Effect of antisense oligonucleotide
treatment on BUN (mg/dL) in the kidney of CD1 mice BUN PBS 35 ISIS
304313 29 ISIS 420913 30 ISIS 420919 29 ISIS 420921 29 ISIS 420922
27 ISIS 420937 29 ISIS 420944 27 ISIS 420947 26 ISIS 420949 25 ISIS
420950 34 ISIS 420952 23 ISIS 420953 34 ISIS 420955 24 ISIS 420957
23 ISIS 420959 29 ISIS 450518 28 ISIS 450519 25 ISIS 450520 29 ISIS
450521 24 ISIS 450522 29 ISIS 450523 27 ISIS 420951 25
Hematology Assays
[0539] Blood obtained from all mice groups were sent to Antech
Diagnostics for hematocrit (HCT), mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin
concentration (MCHC) measurements and analyses, as well as
measurements of the differential blood cell counts, such as that of
WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets,
and total hemoglobin content. The results are presented in Table 52
and 53. Percentages given in the tables indicate the percent change
in total blood cell count compared to the PBS control.
TABLE-US-00052 TABLE 52 Effect of antisense oligonucleotide
treatment on complete blood cell count (%) compared to the PBS
control in CD1 mice WBC RBC Hemoglobin HCT MCV MCH MCHC ISIS 304313
+80 -5 -7 -9 -4 -2 +4 ISIS 420913 -10 -1 -3 -5 -4 -2 +3 ISIS 420919
+26 -2 -7 -9 -7 -5 +4 ISIS 420921 +60 -9 -12 -15 -6 -3 +5 ISIS
420922 +18 -6 -11 -16 -11 -6 +6 ISIS 420937 +42 -3 -4 -7 -5 -1 +5
ISIS 420944 +49 -5 -9 -13 -8 -4 +6 ISIS 420947 +36 -2 -2 -5 -3 0 +4
ISIS 420949 +61 -4 -6 -9 -7 -3 +5 ISIS 420950 +56 -14 -16 -19 -7 -3
+6 ISIS 420952 +36 -20 -24 -25 -7 -5 +4 ISIS 420953 +105 -21 -24
-26 -6 -4 +4 ISIS 420955 +107 -14 -19 -21 -9 -5 +6 ISIS 420957 +79
-5 -10 -13 -9 -6 +5 ISIS 420959 +92 -8 -14 -18 -11 -7 +6 ISIS
450518 +138 -5 -10 -12 -7 -4 +4 ISIS 450519 +118 -17 -21 -24 -9 -5
+6 ISIS 450520 +151 -18 -21 -23 -7 -4 +4 ISIS 450521 +118 -15 -21
-23 -11 -7 +5 ISIS 450522 +63 -22 -28 -31 -12 -8 +6 ISIS 450523
+116 -22 -27 -29 -11 -7 +6 ISIS 420951 +54 -15 -21 -24 -10 -6
+5
TABLE-US-00053 TABLE 53 Effect of antisense oligonucleotide
treatment on differential blood cell count (%) compared to the PBS
control in CD1 mice Neutrophils Lymphocytes Monocytes Platelets
ISIS 304313 -54 +49 -45 +36 ISIS 420913 -46 +39 -21 -2 ISIS 420919
-57 +49 -21 +19 ISIS 420921 -55 +47 -24 +25 ISIS 420922 -53 +46 -31
+24 ISIS 420937 -63 +57 -48 +20 ISIS 420944 -40 +37 -28 +18 ISIS
420947 -55 +49 -38 -9 ISIS 420949 -30 +24 +7 +17 ISIS 420950 -50
+40 0 +6 ISIS 420952 -34 +33 -28 +13 ISIS 420953 -37 +35 -34 +11
ISIS 420955 -37 +34 -21 +30 ISIS 420957 -71 +61 -28 +16 ISIS 420959
-52 +45 -24 -1 ISIS 450518 -56 +49 -28 +18 ISIS 450519 -18 +11 +41
+55 ISIS 450520 -41 +34 0 +7 ISIS 450521 -41 +36 -14 +21 ISIS
450522 -41 +31 +17 +58 ISIS 450523 -28 +19 +31 +51 ISIS 420951 -28
+24 0 +26
Example 18: Tolerability of Antisense Oligonucleotides Targeting
Human Transthyretin in Sprague-Dawley Rats
[0540] ISIS oligonucleotides selected from studies described in
Example 17 were also tested in Sprague-Dawley rats and evaluated
for changes in the levels of various metabolic markers.
Treatment
[0541] The body weights, complete blood count and different blood
count, as well as the urine protein/creatinine ratio of the rats
were evaluated pre-dose. Groups of four Sprague-Dawley rats each
were injected subcutaneously twice a week with 50 mg/kg of ISIS
420913, ISIS 420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS
420957, and ISIS 420959. Three days after the last dose at each
time point, body weights were taken, mice were euthanized and
organs and plasma were harvested for further analysis.
Body and Organ Weights
[0542] The body weights of the rats were measured pre-dose and at
the end of the treatment period. The body weights are presented in
Table 54, and are expressed as percent increase over the PBS
control weight taken before the start of treatment. Liver, spleen
and kidney weights were measured at the end of the study, and are
also presented in Table 54 as a percentage change over the
respective organ weights of the PBS control.
TABLE-US-00054 TABLE 54 Change in body and organ weights of
Sprague-Dawley rats after antisense oligonucleotide treatment (%)
Body weight Liver Spleen Kidney PBS 2.1 1.0 1.0 1.0 ISIS 420913 1.5
1.5 4.7 1.1 ISIS 420921 1.6 1.5 4.2 1.3 ISIS 420922 1.3 1.5 4.4 1.4
ISIS 420950 1.4 1.5 6.4 1.7 ISIS 420955 1.5 1.6 5.9 1.4 ISIS 420957
1.4 1.4 6.8 1.3 ISIS 420959 1.5 1.4 5.5 1.4
[0543] As shown in Table 54, the compounds demonstrated a less than
10-fold increase in organ weight of these rats. Furthermore,
certain compounds demonstrated a less than 7-fold increase in organ
weight of these rats. While certain compounds demonstrated a less
than 6-fold increase in organ weight of these rats. Certain
compounds demonstrated a less than 5-fold increase in organ weight
of these rats.
Liver Function
[0544] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Table 55 expressed in IU/L. Plasma
levels of bilirubin and albumin were also measured using the same
clinical chemistry analyzer and the results are also presented in
Table 55.
TABLE-US-00055 TABLE 55 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of Sprague-Dawley rats
ALT AST TBIL Albumin (IU/L (IU/L) (mg/dL) (g/dL) PBS 26 66 0.09 4.5
ISIS 420913 38 95 0.08 3.3 ISIS 420921 65 151 0.11 3.2 ISIS 420922
40 121 0.11 4.0 ISIS 420950 398 327 0.19 4.0 ISIS 420955 78 241
0.18 4.1 ISIS 420957 84 244 0.14 3.7 ISIS 420959 82 405 0.17
4.6
Kidney Function
[0545] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) and
creatinine were measured using an automated clinical chemistry
analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are
presented in Table 56, expressed in mg/dL. The ratio of total urine
protein to creatinine was also evaluated and presented in Table
56.
TABLE-US-00056 TABLE 56 Effect of antisense oligonucleotide
treatment on metabolic markers (mg/dL) in the kidney of
Sprague-Dawley rats BUN Creatinine PBS 14 0.05 ISIS 420913 22 0.09
ISIS 420921 23 0.07 ISIS 420922 21 0.08 ISIS 420950 20 0.11 ISIS
420955 22 0.06 ISIS 420957 23 0.18 ISIS 420959 24 0.17
TABLE-US-00057 TABLE 57 Effect of antisense oligonucleotide
treatment on total urine protein/creatinine in the kidney of
Sprague-Dawley rats Urine protein/creatinine ratio PBS 1.50 ISIS
420913 19.51 ISIS 420921 5.07 ISIS 420922 4.72 ISIS 420950 5.61
ISIS 420955 5.57 ISIS 420957 5.40 ISIS 420959 4.39
Hematology Assays
[0546] Blood obtained from all rat groups were sent to Antech
Diagnostics for hematocrit (HCT), mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin
concentration (MCHC) measurements and analyses, as well as
measurements of the differential blood cell counts, such as that of
WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets,
and total hemoglobin content. The results are presented in Tables
58 and 59. Percents given in the tables indicate the percent change
in total blood cell count compared to the PBS control.
TABLE-US-00058 TABLE 58 Effect of antisense oligonucleotide
treatment on complete blood cell count (%) compared to the PBS
control in Sprague-Dawley rats WBC RBC Hemoglobin HCT MCV MCH MCHC
PBS 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ISIS 420913 1.7 0.9 0.9 0.9 0.9 0.9
1.0 ISIS 420921 1.6 0.9 0.9 0.9 1.0 1.0 1.0 ISIS 420922 1.6 0.9 0.9
0.8 1.0 1.0 1.0 ISIS 420950 2.2 0.7 0.7 0.7 1.0 1.0 1.0 ISIS 420955
1.9 0.7 0.8 0.7 1.1 1.2 1.0 ISIS 420957 3.1 0.8 0.8 0.8 1.0 1.0 1.0
ISIS 420959 2.2 0.8 0.8 0.8 1.0 1.0 1.0
TABLE-US-00059 TABLE 59 Effect of antisense oligonucleotide
treatment on differential blood cell count (%) compared to the PBS
control in Sprague-Dawley rats Neutrophils Lymphocytes Monocytes
Platelet PBS 1.0 1.0 1.0 1.0 ISIS 420913 0.5 1.1 1.7 0.7 ISIS
420921 0.7 1.0 1.6 0.6 ISIS 420922 0.5 1.1 1.3 0.7 ISIS 420950 0.8
1.0 2.3 0.7 ISIS 420955 0.5 1.0 2.4 0.7 ISIS 420957 0.7 1.0 1.6 0.3
ISIS 420959 0.5 1.1 1.3 n.d.
Example 19: Pharmacokinetic Studies of Half-Life of Antisense
Oligonucleotide Concentration in Sprague-Dawley Rat Liver and
Kidney
[0547] Sprague Dawley rats were treated with ISIS antisense
oligonucleotides targeting from studies described in Example 18 and
the oligonucleotide half-life as well as the elapsed time for
oligonucleotide degradation and elimination from the liver and
kidney was evaluated.
Treatment
[0548] Groups of four Sprague Dawley rats each were injected
subcutaneously twice a week for 2 weeks with 20 mg/kg of ISIS
420913, ISIS 420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS
420957, and ISIS 420959. Three days after the last dose, the rats
were sacrificed and livers and kidneys were collected for
analysis.
Measurement of Oligonucleotide Concentration
[0549] The concentration of the full-length oligonucleotide as well
as the total oligonucleotide concentration (including the degraded
form) was measured. The method used is a modification of previously
published methods (Leeds et al., 1996; Geary et al., 1999) which
consist of a phenol-chloroform (liquid-liquid) extraction followed
by a solid phase extraction. An internal standard (ISIS 355868, a
27-mer 2'-O-methoxyethyl modified phosphorothioate oligonucleotide,
GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166)
was added prior to extraction. Tissue sample concentrations were
calculated using calibration curves, with a lower limit of
quantitation (LLOQ) of approximately 1.14 .mu.g/g. The results are
presented in Tables 60 and 61, expressed as .mu.g/g liver or kidney
tissue. The kidney to liver ratio of oligonucleotide concentration
was also calculated and presented in Tables 60 and 61.
TABLE-US-00060 TABLE 60 Full-length oligonucleotide concentration
(.mu.g/g) and ratio in the liver and kidney of Sprague-Dawley rats
Kidney/Liver ISIS NO. Liver Kidney ratio 420913 154 285 1.9 420921
147 293 2.0 420922 226 497 2.2 420950 161 411 2.6 420955 152 383
2.5 420957 235 453 1.9 420959 187 513 2.7
TABLE-US-00061 TABLE 61 Total oligonucleotide concentration
(.mu.g/g) in the liver and kidney of Sprague-Dawley rats ISIS
Kidney/Liver NO. Liver Kidney ratio 420913 180 310 1.7 420921 159
305 1.9 420922 238 544 2.3 420950 168 466 2.8 420955 156 442 2.8
420957 244 551 2.3 420959 202 534 2.6
Example 20: In Vivo Dose-Dependent Inhibition of Human
Transthyretin in Transgenic Mice
[0550] ISIS 420913, ISIS 420921, ISIS 420922, ISIS 420957 and ISIS
420959, which exhibited good efficacy and tolerability, as
demonstrated in Examples 16-19, were chosen for the study of
dose-dependent target knockdown in transgenic mice containing the
human transthyretin gene. ISIS 420950 and ISIS 420955, which
demonstrated 90% or more target knockdown, but which also
demonstrated toxicity in CD1 mice (Examples 16-19) were also chosen
for this study for comparison.
Treatment
[0551] Groups of four mice, two male and two female, each were
injected subcutaneously twice a week for 4 weeks with 4 mg/kg, 10
mg/kg or 25 mg/kg of ISIS 420913, ISIS 420921, ISIS 420922, ISIS
420950, ISIS 420955, ISIS 420957, or ISIS 420959. One group of four
mice, two male and two female, was injected subcutaneously twice a
week for 4 weeks with 25 mg/kg of the control oligonucleotide, ISIS
141923. One control group of four mice, two male and two female,
was injected subcutaneously twice a week for 4 weeks with PBS.
Plasma samples were taken from each group at days 0, 14 and 28. Two
days after the last dose, the mice were euthanized and organs were
harvested for further analysis.
RNA Analysis
[0552] RNA was extracted from liver tissue for real-time PCR
analysis of transthyretin using primer probe set RTS3029. Results
are presented as percent inhibition of human transthyretin,
relative to PBS control. As shown in Table 62, treatment with ISIS
antisense oligonucleotides resulted in significant dose-dependent
reduction of human transthyretin mRNA in comparison to the PBS
control. Treatment with the control oligonucleotide, ISIS 141923
did not result in significant reduction of transthyretin, as
expected.
TABLE-US-00062 TABLE 62 Inhibition of human transthyretin mRNA in
the hTTR transgenic mice liver relative to the PBS control ISIS
Dose % NO. (mg/kg) inhibition 420913 25 78 10 65 4 32 420921 25 76
10 64 4 13 420922 25 80 10 53 4 21 420950 25 92 10 77 4 57 420955
25 88 10 56 4 23 420957 25 85 10 72 4 32 420959 25 75 10 26 4 11
141923 25 0
Protein Analysis
[0553] Human transthyretin protein levels were measured in
transgenic mice plasma by ELISA using an anti-transthyretin
transthyretin polyclonal antibody (Abcam Ab37774) and a sheep
anti-TTR horse radish peroxidase detection antibody (Abcam cat. no.
35217). The color reaction was developed by the ImmunoPure.RTM. TMB
Substrate Kit and absorbance measured at 450 nm using a microtiter
plate spectrophotometer. Plasma samples were taken predose and on
days 7, 14, 21 and 28. The results are presented in Table 63
expressed as percentage inhibition compared to the predose levels
and demonstrate a time-dependent and dose-dependent reduction in
protein levels on treatment with ISIS oligonucleotides.
TABLE-US-00063 TABLE 63 Inhibition of human transthyretin protein
in the hTTR transgenic mice plasma relative to predose levels ISIS
Dose NO. (mg/kg) d0 d14 d28 420913 25 0 73 93 10 0 27 96 4 0 25 54
420921 25 0 73 90 10 0 63 79 4 0 42 67 420922 25 0 63 96 10 0 57 89
4 0 38 77 420950 25 0 95 97 10 0 71 96 4 0 29 53 420955 25 0 84 96
10 0 53 91 4 0 20 30 420957 25 0 83 93 10 0 51 66 4 0 32 49 420959
25 0 74 80 10 0 31 58 4 0 0 0 141923 25 0 22 0
Body Weight and Organ Weight
[0554] The body weights of the mice were measured pre-dose and at
the end of the treatment period. The body weights are presented in
Table 64 and are expressed as percent increase over the PBS control
weight taken before the start of treatment. Liver, spleen and
kidney weights were measured at the end of the study, and are also
presented in Table 64 as a percentage change over the respective
organ weights of the PBS control.
TABLE-US-00064 TABLE 64 Change in body and organ weights of
transgenic mice after antisense oligonucleotide treatment (%) Body
weight Liver Spleen Kidney PBS 6.4 0.0 0.0 0.0 ISIS 25 8.1 0.3 11.4
4.1 420913 10 10.6 -8.6 14.3 13.6 4 7.4 3.7 5.0 12.0 ISIS 25 10.5
8.8 25.6 -0.1 420921 10 9.7 5.7 10.8 4.0 4 8.7 -4.4 16.0 11.0 ISIS
25 8.4 5.6 18.0 1.7 420922 10 9.2 -1.7 27.1 6.3 4 8.1 -2.1 -11.4
5.1 ISIS 25 12.8 14.3 22.8 1.7 420950 10 8.4 4.3 -2.8 0.6 4 9.1 0.4
14.2 1.5 ISIS 25 10.1 14.6 17.7 -4.4 420955 10 11.8 5.6 -0.3 1.4 4
7.9 4.7 -12.3 4.5 ISIS 25 12.8 6.4 33.1 2.8 420957 10 14.5 13.9
-6.3 9.7 4 7.4 -5.4 12.2 6.2 ISIS 25 10.0 2.4 72.7 23.3 420959 10
7.2 -5.4 40.2 9.8 4 4.1 -4.4 27.8 -6.6 ISIS 25 9.2 -1.3 20.4 -5.5
141923
Liver Function
[0555] To evaluate the effect of ISIS oligonucleotides on hepatic
function, plasma concentrations of transaminases were measured
using an automated clinical chemistry analyzer (Hitachi Olympus
AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine
transaminase) and AST (aspartate transaminase) were measured and
the results are presented in Table 65 expressed in IU/L. Plasma
levels of bilirubin were also measured using the same clinical
chemistry analyzer; results are also presented in Table 65 and
expressed in mg/dL.
TABLE-US-00065 TABLE 65 Effect of antisense oligonucleotide
treatment on metabolic markers in the liver of transgenic mice Dose
ALT AST TBIL (mg/kg) (IU/L) (IU/L) (mg/dL) PBS 47 63 0.16 ISIS 25
42 69 0.13 420913 10 49 90 0.17 4 42 59 0.18 ISIS 25 56 96 0.12
420921 10 51 68 0.22 4 42 75 0.14 ISIS 25 50 76 0.12 420922 10 40
170 0.14 4 37 48 0.13 ISIS 25 74 116 0.14 420950 10 37 67 0.13 4 34
64 0.11 ISIS 25 46 117 0.15 420955 10 54 76 0.16 4 50 153 0.17 ISIS
25 40 73 0.13 420957 10 36 63 0.20 4 37 61 0.12 ISIS 25 51 92 0.19
420959 10 48 69 0.13 4 37 67 0.13 ISIS 25 44 79 0.12 141923
Kidney Function
[0556] To evaluate the effect of ISIS oligonucleotides on kidney
function, plasma concentrations of blood urea nitrogen (BUN) were
measured using an automated clinical chemistry analyzer (Hitachi
Olympus AU400e, Melville, N.Y.). Results are presented in Table 66,
expressed in mg/dL.
TABLE-US-00066 TABLE 66 Effect of antisense oligonucleotide
treatment on BUN (mg/dL) in the kidney of transgenic mice Dose
(mg/kg) BUN PBS -- 23 ISIS 25 24 420913 10 22 4 20 ISIS 25 24
420921 10 22 4 23 ISIS 25 23 420922 10 22 4 24 ISIS 25 22 420950 10
26 4 23 ISIS 25 23 420955 10 24 4 25 ISIS 25 20 420957 10 22 4 20
ISIS 25 25 420959 10 22 4 22 ISIS 25 19 141923
Example 21: Dose Response Confirmation of Antisense
Oligonucleotides Targeting Human Transthyretin in Cynomolgus Monkey
Primary Hepatocytes
[0557] Gapmers showing tolerability in CD1 mice and Sprague Dawley
rats (studies described in Examples 17-19) as well as potency in
transgenic mice (studies described in Examples 16 and 20) were
selected and tested at various doses in primary hepatocytes of
cynomolgus monkeys. Cells were plated at a density of 35,000 cells
per well and transfected using electroporation with 156.25 nM,
312.5 nM, 625 nM, 1,250 nM 2,500 nM, 5,000 nM, 10,000 nM and 20,000
nM concentrations of antisense oligonucleotide, as specified in
Table 67. After a treatment period of approximately 16 hours, RNA
was isolated from the cells and transthyretin mRNA levels were
measured by quantitative real-time PCR. Human transthyretin primer
probe set RTS1396 was used to measure mRNA levels. Transthyretin
mRNA levels were adjusted according to total RNA content, as
measured by RIBOGREEN.RTM.. Results are presented as percent
inhibition of transthyretin, relative to untreated control cells.
As illustrated in Table 67, transthyretin mRNA levels were reduced
in a dose-dependent manner in hepatocytes treated with all the ISIS
oligonucleotides, which are cross-reactive with rhesus monkey
transthyretin gene, designated herein as SEQ ID NO: 4 (exons 1-4
extracted from GENBANK Accession No. NW 001105671.1).
TABLE-US-00067 TABLE 67 Dose-dependent antisense inhibition of
human transthyretin in cynomolgus monkey primary hepatocytes using
electroporation 156.25 312.5 625 1250 2500 5000 10000 20000
IC.sub.50 Target Start ISIS No. nM nM nM nM nM nM nM nM (.mu.M)
Site 304299 0 0 25 42 89 95 98 99 1.4 504 420913 0 0 42 49 84 96 98
98 1.2 502 420915 0 8 46 58 84 94 97 99 1 505 420921 0 0 26 30 53
74 94 97 2 512 420922 4 0 13 29 38 69 87 97 2.9 513 420950 23 27 60
71 88 94 98 98 0.6 577 420955 19 0 25 50 74 86 93 97 1.4 582 420957
0 0 15 34 65 72 87 94 2.2 584 420959 3 12 10 37 71 88 94 94 1.5
586
Example 22: Measurement of Viscosity of ISIS Antisense
Oligonucleotides Targeting Human Transthyretin
[0558] The viscosity of antisense oligonucleotides from studies
described in Example 21 was measured with the aim of screening out
antisense oligonucleotides which have a viscosity more than 40 cP.
Oligonucleotides having a viscosity greater than 40 cP would be too
viscous to be administered to any subject.
[0559] ISIS oligonucleotides (32-35 mg) were weighed into a glass
vial, 120 .mu.L of water was added and the antisense
oligonucleotide was dissolved into solution by heating the vial at
50.degree. C. Part of (75 .mu.L) the pre-heated sample was pipetted
to a micro-viscometer (Cambridge). The temperature of the
micro-viscometer was set to 25.degree. C. and the viscosity of the
sample was measured. Another part (20 .mu.L) of the pre-heated
sample was pipetted into 10 mL of water for UV reading at 260 nM at
85.degree. C. (Cary UV instrument). The results are presented in
Table 68 and indicate that all the antisense oligonucleotides
solutions are optimal in their viscosity under the criterion stated
above.
TABLE-US-00068 TABLE 68 Viscosity and concentration of ISIS
antisense oligonucleotides targeting human transthyretin ISIS
Viscosity Concentration No. (cP) (mg/mL) 304299 9.9 169 420913 6.5
178 420915 8.4 227 420921 8.2 234 420922 5.3 191 420950 12.5 297
420955 15.7 259 420957 12.9 233 420959 18.7 276
Example 23: Measurement of Half-Life of Antisense Oligonucleotide
in CD1 Mouse Liver
[0560] CD1 mice were treated with ISIS antisense oligonucleotides
from studies described in Example 22 and the oligonucleotide
half-life as well as the elapsed time for oligonucleotide
degradation and elimination from the liver was evaluated.
Treatment
[0561] Groups of twelve CD1 mice each were injected subcutaneously
twice per week for 2 weeks with 50 mg/kg of ISIS 420913, ISIS
420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS 420957, and
ISIS 420959. Four mice from each group were sacrificed 3 days, 28
days and 56 days following the final dose. Livers were harvested
for analysis.
Measurement of Oligonucleotide Concentration
[0562] The concentration of the full-length oligonucleotide as well
as the total oligonucleotide concentration (including the degraded
form) was measured. The method used is a modification of previously
published methods (Leeds et al., 1996; Geary et al., 1999) which
consist of a phenol-chloroform (liquid-liquid) extraction followed
by a solid phase extraction. An internal standard (ISIS 355868, a
27-mer 2'-O-methoxyethyl modified phosphorothioate oligonucleotide,
GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166)
was added prior to extraction. Tissue sample concentrations were
calculated using calibration curves, with a lower limit of
quantitation (LLOQ) of approximately 1.14 .mu.g/g. Half-lives were
then calculated using WinNonlin software (PHARSIGHT).
[0563] The results are presented in Tables 69, expressed as .mu.gig
liver tissue. The half-life of each oligonucleotide is presented in
Table 70.
TABLE-US-00069 TABLE 69 Full-length oligonucleotide concentration
(.mu.g/g) in the liver of CD1 mice ISIS No. 3 days 28 days 56 days
420913 243 109 33 420921 225 49 6 420922 310 129 53 420950 254 88
62 420955 308 137 79 420957 325 129 49 420959 258 97 37
TABLE-US-00070 TABLE 70 Half-life of oligonucleotide (days) in the
liver of CD1 mice ISIS Half-life No. (days) 420913 18.5 420921 10.0
420922 20.7 420950 26.4 420955 27.2 420957 19.5 420959 18.9
Example 24: Effect of ISIS Antisense Oligonucleotides Targeting
Human Transthyretin in Cynomolgus Monkeys
[0564] Cynomolgus monkeys were treated with ISIS antisense
oligonucleotides from studies described in Examples 21, 22 and 23.
Antisense oligonucleotide efficacy and tolerability, as well as
their pharmacokinetic profile in the liver and kidney, were
evaluated.
Treatment
[0565] Prior to the study, the monkeys were kept in quarantine for
a 30-day time period, during which standard panels of serum
chemistry and hematology, examination of fecal samples for ova and
parasites, and a tuberculosis test, were conducted to screen out
abnormal or ailing monkeys. Nine groups of four randomly assigned
male cynomolgus monkeys each were injected subcutaneously thrice
per week for the first week, and subsequently twice a week for the
next 11 weeks, with 25 mg/kg of ISIS 304299, ISIS 420915, ISIS
420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS 420957, or ISIS
420959. A control group of 4 cynomolgus monkeys was injected with
PBS subcutaneously thrice per week for the first week, and
subsequently twice a week for the next 11 weeks. Blood samples were
collected 5 days before the treatment as well as on various days of
the study period and analyzed. The animals were fasted for at least
13 hours (overnight) prior to blood collection. Terminal sacrifices
of all groups were conducted on day 86, which was 48 hours after
the last dose.
[0566] During the study period, the monkeys were observed daily for
signs of illness or distress. Any animal showing adverse effects to
the treatment was removed and referred to the veterinarian and
Study Director. All the animals treated with ISIS 420955 were
removed from the study on day 31 due to symptoms of illness
displayed by 2 monkeys in the group. Similarly, one monkey each
from groups treated with ISIS 420957 and ISIS 420950 was removed
from the study on days 44 and 76, respectively, due to signs of
illness.
Inhibition Studies
RNA Analysis
[0567] On day 86, RNA was extracted from liver tissue for real-time
PCR analysis of transthyretin using primer probe set RTS3029.
Results are presented as percent inhibition of transthyretin,
relative to PBS control, normalized to cyclophilin. Similar results
were obtained on normalization with RIBOGREEN.RTM.. As shown in
Table 71, treatment with ISIS antisense oligonucleotides resulted
in significant reduction of transthyretin mRNA in comparison to the
PBS control. Specifically, treatment with ISIS 420915 caused
greater inhibition of TTR mRNA than treatment with ISIS 304299,
even though the two oligonucleotides differ from each other by a
single base-pair shift. The data for animals treated with ISIS
420955 was taken at day 31.
TABLE-US-00071 TABLE 71 Inhibition of transthyretin mRNA in the
cynomolgus monkey liver relative to the PBS control % ISIS No
inhibition 304299 59 420915 78 420921 54 420922 61 420950 91
420955* 79 420957 64 420959 55 (*Data of day 31)
Protein Analysis
[0568] The monkeys were fasted overnight prior to blood collection.
Approximately 1 mL of blood was collected from all available
animals and placed in tubes containing the potassium salt of EDTA.
The tubes were centrifuged (3000 rpm for 10 min at room
temperature) to obtain plasma. Transthyretin protein levels were
measured in the plasma using a clinical analyzer. Plasma samples
were taken predose (on day -5) and on days 1, 9, 16, 23, 30, 44,
58, 72, and 86. The results are presented in Table 72 expressed as
percentage inhibition compared to the predose levels and
demonstrate a time-dependent reduction in protein levels with
treatment with ISIS oligonucleotides. The final plasma TTR levels
are presented in Table 73 and demonstrate the strong correlation
between TTR protein level reduction and TTR mRNA inhibition (Table
71). Specifically, treatment with ISIS 420915 caused greater
inhibition of TTR plasma protein than treatment with ISIS 304299
(76% inhibition vs. 47% inhibition), even though the two
oligonucleotides differ from each other by a single base-pair
shift.
TABLE-US-00072 TABLE 72 Time course of transthyretin protein level
reduction in the cynomolgus monkey plasma relative to predose
levels ISIS Day Day Day Day Day Day Day Day Day No. 0 9 16 23 30 44
58 72 86 304299 4 15 21 23 26 27 31 38 47 420915 2 8 23 34 42 54 63
70 76 420921 5 11 21 31 23 27 30 40 50 420922 0 17 37 42 49 49 50
49 54 420950 0 39 63 68 72 79 85 82 87 420955 0 42 63 80 81 n/a n/a
n/a n/a 420957 2 18 28 26 26 35 35 41 50 420959 0 25 29 28 32 38 42
43 50 n/a = study was terminated on day 31 for animals treated with
ISIS 420955; therefore data for subsequent days is not
available.
TABLE-US-00073 TABLE 73 Day 86 transthyretin protein level
reduction in the cynomolgus monkey plasma relative to predose
levels ISIS % No. reduction 304299 47 420915 76 420921 50 420922 54
420950 87 420957 50 420959 50
[0569] RBP4 protein levels were also measured in the plasma using
an ELISA kit. Plasma samples were taken predose (on day -5) and on
days 9, 16, 23, 30, 44, 58, 72, and 86. The results are presented
in Table 74 expressed as percentage inhibition compared to the
predose levels. Some of the ISIS oligonucleotides (ISIS 420915,
ISIS 420922, ISIS 420950, ISIS 420955 and ISIS 420959) demonstrate
a time-dependent reduction in protein levels, concomitant with TTR
reduction. The final plasma RBP4 levels are presented in Table 75
and also demonstrate the strong correlation between RBP4 and TTR
protein level reductions (Table 73) on treatment with the
above-mentioned oligonucleotides. Specifically, treatment with ISIS
420915 caused greater inhibition of RBP4 plasma protein than
treatment with ISIS 304299 (63% inhibition vs. 19% inhibition),
even though the two oligonucleotides differ from each other by a
single base-pair shift.
TABLE-US-00074 TABLE 74 Time course of RBP4 protein level reduction
in the cynomolgus monkey plasma relative to predose levels ISIS Day
Day Day Day Day Day Day Day No. 9 16 23 30 44 58 72 86 304299 0 6
10 4 1 9 13 19 420915 5 22 22 30 38 47 54 63 420921 0 0 0 0 0 0 6
24 420922 4 19 16 34 33 29 15 32 420950 30 44 46 47 52 54 47 48
420955 6 36 53 65 n/a n/a n/a n/a 420957 0 10 0 0 0 0 3 27 420959
18 22 14 17 19 25 22 34 n/a = study was terminated on day 31 for
animals treated with ISIS 420955; therefore data for subsequent
days is not available.
TABLE-US-00075 TABLE 75 Day 86 RBP4 protein level reduction in the
cynomolgus monkey plasma relative to predose levels ISIS % No.
reduction 304299 19 420915 63 420921 24 420922 32 420950 48 420957
27 420959 34
Tolerability Studies
Body and Organ Weight Measurements
[0570] To evaluate the effect of ISIS oligonucleotides on the
overall health of the animals, body and organ weights were measured
at day 86. The data for animals treated with ISIS 420955 was taken
at day 31. Body weights were measured and compared to that at
pre-dose levels. Organ weights were measured and treatment group
weights were compared to the corresponding PBS control weights. The
data is presented in Table 76.
TABLE-US-00076 TABLE 76 Final body and organ weight % changes in
the cynomolgus monkey relative to predose levels ISIS Body Liver
Kidney Spleen No. weight weight weight weight 304299 +6 +27 +37 +53
420915 +6 +37 +26 +41 420921 +4 +42 +43 +22 420922 +4 +45 +39 +63
420950 0 +204 +166 +297 420955* -3 +36 +81 +70 420957 -6 +55 +184
+109 420959 0 +57 +101 +112 (*Data of day 31)
Liver Function
[0571] To evaluate the effect of ISIS oligonucleotides on hepatic
function, blood samples were collected from all the study groups.
The blood samples were collected in tubes without any anticoagulant
for serum separation. The tubes were kept at room temperature for
90 min and then centrifuged (3000 rpm for 10 min at room
temperature) to obtain serum. Concentrations of transaminases were
measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co.,
Japan). Plasma concentrations of ALT (alanine transaminase) and AST
(aspartate transaminase) were measured on day 86 and the results
are presented in Table 77, expressed in IU/L. Alkaline phosphatase,
which is synthesized in increased amounts by damaged liver cells,
is also a marker of liver disease and was similarly measured.
C-reactive protein (CRP), which is synthesized in the liver and
which serves as a marker of inflammation, was also similarly
measured on day 86. Both alkaline phosphatase and CRP data are also
presented in Table 77. Bilirubin is also a liver metabolic marker
and was similarly measured and is presented in Table 77, expressed
in mg/dL.
TABLE-US-00077 TABLE 77 Effect of antisense oligonucleotide
treatment on liver metabolic markers in cynomolgus monkey plasma
AST ALT ALP CRP Bilirubin (IU/L) (IU/L) (IU/L) (mg/L) (mg/dL) PBS
60 54 955 2.4 0.24 ISIS 304299 81 101 747 3.3 0.17 ISIS 420915 68
62 672 1.6 0.15 ISIS 420921 98 107 832 3.2 0.14 ISIS 420922 94 96
907 2.4 0.15 ISIS 420950 132 94 1032 12.9 0.11 ISIS 420957 100 73
868 23.5 0.15 ISIS 420959 70 63 811 16.0 0.13
Kidney Function
[0572] To evaluate the effect of ISIS oligonucleotides on kidney
function, blood samples were collected from all the study groups.
The blood samples were collected in tubes without any anticoagulant
for serum separation. The tubes were kept at room temperature for
90 min and then centrifuged (3000 rpm for 10 min at room
temperature) to obtain serum. Concentrations of BUN and creatinine
were measured at day 86 using a Toshiba 200FR NEO chemistry
analyzer (Toshiba Co., Japan). Results are presented in Table 78,
expressed in mg/dL.
[0573] Urine samples were collected by drainage from special
stainless-steel cage pans on day 5 before the study, and
subsequently on days 25 and 84. Urine total protein to creatinine
ratio was measured using a Toshiba 200FR NEO chemistry analyzer
(Toshiba Co., Japan) and the results are presented in Table 79.
TABLE-US-00078 TABLE 78 Effect of antisense oligonucleotide
treatment on plasma BUN and creatinine levels (mg/dL) in cynomolgus
monkeys BUN Creatinine PBS 28 0.86 ISIS 304299 27 0.85 ISIS 420915
25 0.90 ISIS 420921 33 0.99 ISIS 420922 28 0.86 ISIS 420950 36 0.97
ISIS 420957 35 0.86 ISIS 420959 27 0.89
TABLE-US-00079 TABLE 79 Effect of antisense oligonucleotide
treatment on urine protein to creatine ratio in cynomolgus monkeys
Day -5 Day 25 Day 84 PBS 0.003 0.01 0.00 ISIS 304299 0.000 0.01
0.00 ISIS 420915 0.003 0.00 0.00 ISIS 420921 0.033 0.13 0.09 ISIS
420922 0.010 0.05 0.02 ISIS 420950 0.008 0.29 0.21 ISIS 420955
0.000 0.61 n/a ISIS 420957 0.000 0.48 0.36 ISIS 420959 0.005 0.08
0.03 n/a = study was terminated on day 31 for animals treated with
ISIS 420955; therefore data for subsequent days is not
available.
Hematology
[0574] To evaluate any inflammatory effect of ISIS oligonucleotides
in cynomolgus monkeys, blood samples were approximately 0.5 mL of
blood was collected from each of the available study animals in
tubes containing the potassium salt of EDTA. Samples were analyzed
for red blood cell (RBC) count, white blood cells (WBC) count,
individual white blood cell percentages, such as that of monocytes,
neutrophils, lymphocytes, as well as for platelet count and
hematocrit (%), using an ADVIA120 hematology analyzer (Bayer, USA).
The data is presented in Table 80.
TABLE-US-00080 TABLE 80 Effect of antisense oligonucleotide
treatment on hematologic parameters in cynomolgus monkeys WBC RBC
Platelet Hematocrit Lymphocytes Neutrophil Monocytes
(.times.10.sup.3/.mu.L) (.times.10.sup.6/.mu.L) (.times.1000/.mu.L)
(%) (%) (%) (%) PBS 9.6 5.3 415 40 62 35 1.8 ISIS 304299 11.6 5.2
395 38 68 26 3.1 ISIS 420915 10.3 5.1 382 36 72 22 3.5 ISIS 420921
9.8 5.2 385 36 60 34 2.5 ISIS 420922 11.6 5.2 396 37 62 29 5.4 ISIS
420950 13.7 4.4 260 33 51 34 7.8 ISIS 420957 18.6 4.7 298 33 52 35
9.1 ISIS 420959 7.7 4.8 306 32 62 29 5.5
Analysis of Factors of Inflammation
[0575] To evaluate the effect of ISIS oligonucleotides on factors
involved in inflammation, blood was collected on day 86 from all
available animals for complement C.sub.3 analysis, as well as for
measurement of cytokine levels. For complement C.sub.3 analysis,
the blood samples were collected in tubes without anticoagulant for
serum separation. The tubes were kept at room temperature for 90
min and then centrifuged (3000 rpm for 10 min at room temperature)
to obtain serum. Complement C.sub.3 was measured using an automatic
analyzer (Toshiba 200 FR NEO chemistry analyzer, Toshiba co.,
Japan). The data is presented in Table 81, expressed in mg/dL.
[0576] For cytokine level analyses, blood was collected in tubes
containing EDTA for plasma separation. The tubes were then
centrifuged (3000 rpm for 10 min at room temperature) to obtain
plasma. Plasma samples were sent to Aushon Biosystems Inc.
(Billerica, Mass.) for measurement of chemokine and cytokine
levels. Levels of TNF-.alpha. were measured using the respective
primate antibodies and levels of MIP-1.alpha., MCP-1, and
MIP-1.beta. were measured using the respective cross-reacting human
antibodies. Measurements were taken 5 days before the start of
treatment and on days 3 and 86. The results are presented in Tables
82-85.
TABLE-US-00081 TABLE 81 Effect of antisense oligonucleotide
treatment on complement C3 (mg/dL) in cynomolgus monkeys C3 PBS 133
ISIS 304299 96 ISIS 420915 104 ISIS 420921 91 ISIS 420922 102 ISIS
420950 70 ISIS 420957 69 ISIS 420959 95
TABLE-US-00082 TABLE 82 Effect of antisense oligonucleotide
treatment on MCP-1 (pg/mL) in cynomolgus monkeys Day -5 Day 3 Day
86 PBS 232 362 206 ISIS 304299 219 292 427 ISIS 420915 204 342 400
ISIS 420921 281 407 2120 ISIS 420922 215 482 838 ISIS 420950 170
370 3355 ISIS 420957 208 308 3485 ISIS 420959 237 715 2035
TABLE-US-00083 TABLE 83 Effect of antisense oligonucleotide
treatment on TNF-.alpha. (pg/mL) in cynomolgus monkeys Day -5 Day 3
Day 86 PBS 60 46 16 ISIS 304299 46 35 24 ISIS 420915 113 83 30 ISIS
420921 57 50 56 ISIS 420922 30 59 46 ISIS 420950 48 54 266 ISIS
420957 29 33 87 ISIS 420959 22 77 74
TABLE-US-00084 TABLE 84 Effect of antisense oligonucleotide
treatment on MIP-1.alpha. (pg/mL) in cynomolgus monkeys Day -5 Day
3 Day 86 PBS 6 7 7 ISIS 304299 6 7 9 ISIS 420915 5 5 10 ISIS 420921
8 11 9 ISIS 420922 9 8 5 ISIS 420950 7 9 5 ISIS 420957 6 6 6 ISIS
420959 9 6 5
TABLE-US-00085 TABLE 85 Effect of antisense oligonucleotide
treatment on MIP-1.beta. (pg/mL) in cynomolgus monkeys Day -5 Day 3
Day 86 PBS 13 19 42 ISIS 304299 17 23 54 ISIS 420915 15 27 72 ISIS
420921 23 43 112 ISIS 420922 9 41 70 ISIS 420950 8 25 126 ISIS
420957 16 27 182 ISIS 420959 36 46 117
Coagulation Tests
[0577] To evaluate the effect of ISIS oligonucleotides on factors
involved in the coagulation pathway, the standard tests for
coagulation were employed. PT and aPTT were measured using platelet
poor plasma (PPP) from the monkeys over a period of 48 hrs. PT and
aPTT values are provided in Tables 86 and 87 and expressed in
seconds. Fibrinogen levels on the plasma were also quantitated over
a period of 48 hrs and the data is presented in Table 88. As shown
in Tables 86-88, PT, aPTT and fibrinogen were not significantly
altered in monkeys treated with ISIS oligonucleotides compared to
the PBS control.
TABLE-US-00086 TABLE 86 Effect of antisense oligonucleotide
treatment on PT (sec) 0 hr 1 hr 4 hr 8 hr 24 hr 48 hr PBS 10.08
10.38 10.10 10.33 9.83 9.40 ISIS 304299 10.38 10.30 10.48 10.20
9.95 9.53 ISIS 420915 10.15 10.13 10.38 9.93 9.75 9.48 ISIS 420921
10.28 10.13 10.43 10.18 9.80 9.55 ISIS 420922 9.95 10.00 10.05 9.70
9.48 9.28 ISIS 420950 10.30 10.47 10.57 10.27 9.63 9.50 ISIS 420957
10.63 10.47 10.60 10.77 10.33 10.27 ISIS 420959 10.08 10.10 10.20
10.15 9.80 9.55
TABLE-US-00087 TABLE 87 Effect of antisense oligonucleotide
treatment on aPTT (sec) 0 hr 1 hr 4 hr 8 hr 24 hr 48 hr PBS 19.40
19.70 20.13 20.20 19.43 17.30 ISIS 304299 21.83 24.35 27.05 25.73
22.40 18.78 ISIS 420915 20.05 22.83 23.83 24.00 21.78 17.90 ISIS
420921 24.15 26.68 31.78 31.90 27.80 22.15 ISIS 420922 25.28 29.48
34.83 33.90 29.13 25.08 ISIS 420950 28.13 31.40 35.40 35.40 31.40
28.37 ISIS 420957 29.13 33.27 39.13 37.40 36.50 29.93 ISIS 420959
22.45 24.73 29.18 28.38 25.50 20.65
TABLE-US-00088 TABLE 88 Effect of antisense oligonucleotide
treatment on fibrinogen (mg/dL) 0 hr 1 hr 4 hr 8 hr 24 hr 48 hr PBS
212 203 240 247 282 272 ISIS 304299 175 172 198 207 227 200 ISIS
420915 213 196 204 258 257 215 ISIS 420921 208 209 230 237 301 249
ISIS 420922 278 277 335 338 400 304 ISIS 420950 293 295 348 376 390
296 ISIS 420957 280 299 344 330 434 328 ISIS 420959 276 211 354 326
414 320
Thyroid Panel Analysis
[0578] To evaluate the effect of ISIS oligonucleotides on thyroid
hormones, monkeys were fasted overnight and 3.5 mL of blood was
drawn from each of the available study animals 5 days prior to the
start of treatment and on days 51 and 86. Collected blood samples
were kept in tubes without anticoagulant for serum separation. The
tubes were kept for 90 min at room temperature, after which they
were centrifuged (3000 rpm for 10 min at room temperature) to
obtain serum. Serum samples were sent to the Biomarkers Core
Laboratory of Emory University (Atlanta, Ga.) for thyroid panel
analysis. The results for thyroid stimulating hormone (TSH) are
provided in Table 89 and expressed .mu.L/mL. The results for total
and free T3 hormone are provided in Tables 90 and 91. The results
for total and free T4 hormone are provided in Tables 92 and 93.
Overall, the thyroid panel analysis showed that all the animals
remained within acceptable hormone levels even though transthyretin
expression levels were reduced, demonstrating that the
transthyretin antisense oligonucleotides did not affect hormone
levels.
TABLE-US-00089 TABLE 89 Effect of antisense oligonucleotide
treatment on TSH (.mu.L/mL) Day -5 Day 51 Day 86 PBS 0.8 0.7 1.0
ISIS 304299 1.4 1.0 2.2 ISIS 420915 1.4 1.5 2.5 ISIS 420921 0.7 0.6
1.0 ISIS 420922 1.0 1.2 1.9 ISIS 420950 0.6 2.2 5.4 ISIS 420957 0.6
2.6 4.9 ISIS 420959 0.9 1.6 4.7
TABLE-US-00090 TABLE 90 Effect of antisense oligonucleotide
treatment on total T3 (ng/dL) Day -5 Day 51 Day 86 PBS 177 248 140
ISIS 304299 202 226 176 ISIS 420915 156 206 156 ISIS 420921 217 204
137 ISIS 420922 188 177 131 ISIS 420950 260 208 105 ISIS 420957 266
160 78 ISIS 420959 299 219 137
TABLE-US-00091 TABLE 91 Effect of antisense oligonucleotide
treatment on free T3 (pg/mL) Day -5 Day 51 Day 86 PBS 7.7 5.8 5.2
ISIS 304299 9.2 6.0 4.7 ISIS 420915 8.9 5.6 4.5 ISIS 420921 10.2
4.8 4.0 ISIS 420922 8.9 5.4 3.7 ISIS 420950 7.2 3.8 2.1 ISIS 420957
8.8 4.0 2.4 ISIS 420959 8.3 4.9 3.3
TABLE-US-00092 TABLE 92 Effect of antisense oligonucleotide
treatment on total T4 (ng/dL) Day -5 Day 51 Day 86 PBS 5.8 4.9 4.4
ISIS 304299 8.1 5.5 6.1 ISIS 420915 8.3 5.7 5.5 ISIS 420921 7.6 6.1
5.6 ISIS 420922 7.3 6.1 5.8 ISIS 420950 6.1 6.3 5.7 ISIS 420957 6.3
4.4 5.0 ISIS 420959 7.9 5.9 8.1
TABLE-US-00093 TABLE 93 Effect of antisense oligonucleotide
treatment on free T4 (pg/mL) Day -5 Day 51 Day 86 PBS 3.4 2.4 1.7
ISIS 304299 3.2 2.5 1.7 ISIS 420915 5.0 1.8 1.7 ISIS 420921 2.6 1.5
1.5 ISIS 420922 3.5 1.6 1.5 ISIS 420950 2.5 1.2 1.1 ISIS 420957 2.4
1.2 1.2 ISIS 420959 3.8 1.4 1.5
Pharmacokinetic Studies
Measurement of Oligonucleotide Concentration
[0579] The concentration of the full-length oligonucleotide as well
as the total oligonucleotide concentration (including the degraded
form) was measured. The method used is a modification of previously
published methods (Leeds et al., 1996; Geary et al., 1999) which
consist of a phenol-chloroform (liquid-liquid) extraction followed
by a solid phase extraction. An internal standard (ISIS 355868, a
27-mer 2'-O-methoxyethyl modified phosphorothioate oligonucleotide,
GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166)
was added prior to extraction. Tissue sample concentrations were
calculated using calibration curves, with a lower limit of
quantitation (LLOQ) of approximately 1.14 .mu.gig. The ratio of the
concentrations in the kidney versus the liver was calculated. The
results are presented in Tables 94 and 95, expressed as .mu.gig
tissue.
TABLE-US-00094 TABLE 94 Full-length oligonucleotide concentration
(.mu.g/g) in the liver of cynomolgus monkey ISIS No. Kidney Liver
Kidney/Liver ratio 304299 2179 739 2.9 420915 2439 1064 2.3 420921
4617 1521 3.0 420922 3957 1126 3.5 420950 3921 1082 3.6 420955 2444
1111 2.2 420957 3619 1230 2.9 420959 3918 1158 3.4
TABLE-US-00095 TABLE 95 Total oligonucleotide concentration
(.mu.g/g) in the liver of cynomolgus monkey ISIS No. Kidney Liver
Kidney/Liver ratio 304299 3098 992 3.1 420915 3024 1266 2.4 420921
6100 1974 3.1 420922 4861 1411 3.4 420950 6003 1553 3.9 420955 2763
1208 2.3 420957 5420 1582 3.4 420959 5498 1501 3.7
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 177 <210> SEQ ID NO 1 <211> LENGTH: 650
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (27)..(470) <400> SEQUENCE: 1 acagaagtcc actcattctt
ggcagg atg gct tct cat cgt ctg ctc ctc ctc 53 Met Ala Ser His Arg
Leu Leu Leu Leu 1 5 tgc ctt gct gga ctg gta ttt gtg tct gag gct ggc
cct acg ggc acc 101 Cys Leu Ala Gly Leu Val Phe Val Ser Glu Ala Gly
Pro Thr Gly Thr 10 15 20 25 ggt gaa tcc aag tgt cct ctg atg gtc aaa
gtt cta gat gct gtc cga 149 Gly Glu Ser Lys Cys Pro Leu Met Val Lys
Val Leu Asp Ala Val Arg 30 35 40 ggc agt cct gcc atc aat gtg gcc
gtg cat gtg ttc aga aag gct gct 197 Gly Ser Pro Ala Ile Asn Val Ala
Val His Val Phe Arg Lys Ala Ala 45 50 55 gat gac acc tgg gag cca
ttt gcc tct ggg aaa acc agt gag tct gga 245 Asp Asp Thr Trp Glu Pro
Phe Ala Ser Gly Lys Thr Ser Glu Ser Gly 60 65 70 gag ctg cat ggg
ctc aca act gag gag gaa ttt gta gaa ggg ata tac 293 Glu Leu His Gly
Leu Thr Thr Glu Glu Glu Phe Val Glu Gly Ile Tyr 75 80 85 aaa gtg
gaa ata gac acc aaa tct tac tgg aag gca ctt ggc atc tcc 341 Lys Val
Glu Ile Asp Thr Lys Ser Tyr Trp Lys Ala Leu Gly Ile Ser 90 95 100
105 cca ttc cat gag cat gca gag gtg gta ttc aca gcc aac gac tcc ggc
389 Pro Phe His Glu His Ala Glu Val Val Phe Thr Ala Asn Asp Ser Gly
110 115 120 ccc cgc cgc tac acc att gcc gcc ctg ctg agc ccc tac tcc
tat tcc 437 Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr Ser
Tyr Ser 125 130 135 acc acg gct gtc gtc acc aat ccc aag gaa tga
gggacttctc ctccagtgga 490 Thr Thr Ala Val Val Thr Asn Pro Lys Glu
140 145 cctgaaggac gagggatggg atttcatgta accaagagta ttccattttt
actaaagcag 550 tgttttcacc tcatatgcta tgttagaagt ccaggcagag
acaataaaac attcctgtga 610 aaggcacttt tcattccaaa aaaaaaaaaa
aaaaaaaaaa 650 <210> SEQ ID NO 2 <211> LENGTH: 8054
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 ttgttgaccc atggatccat caagtgcaaa cattttctaa
tgcactatat ttaagcctgt 60 gcagctagat gtcattcaac atgaaataca
ttattacaac ttgcatctgt ctaaaatctt 120 gcatctaaaa tgagagacaa
aaaatctata aaaatggaaa acatgcatag aaatatgtga 180 gggaggaaaa
aattaccccc aagaatgtta gtgcacgcag tcacacaggg agaagactat 240
ttttgttttg ttttgattgt tttgttttgt tttggttgtt ttgttttggt gacctaactg
300 gtcaaatgac ctattaagaa tatttcatag aacgaatgtt ccgatgctct
aatctctcta 360 gacaaggttc atatttgtat gggttactta ttctctcttt
gttgactaag tcaataatca 420 gaatcagcag gtttgcagtc agattggcag
ggataagcag cctagctcag gagaagtgag 480 tataaaagcc ccaggctggg
agcagccatc acagaagtcc actcattctt ggcaggatgg 540 cttctcatcg
tctgctcctc ctctgccttg ctggactggt atttgtgtct gaggctggcc 600
ctacggtgag tgtttctgtg acatcccatt cctacattta agattcacgc taaatgaagt
660 agaagtgact ccttccagct ttgccaacca gcttttatta ctagggcaag
ggtacccagc 720 atctattttt aatataatta attcaaactt caaaaagaat
gaagttccac tgagcttact 780 gagctgggac ttgaactctg agcattctac
ctcattgctt tggtgcatta ggtttgtaat 840 atctggtacc tctgtttcct
cagatagatg atagaaataa agatatgata ttaaggaagc 900 tgttaatact
gaattttcag aaaagtatcc ctccataaaa tgtatttggg ggacaaactg 960
caggagatta tattctggcc ctatagttat tcaaaacgta tttattgatt aatctttaaa
1020 aggcttagtg aacaatattc tagtcagata tctaattctt aaatcctcta
gaagaattaa 1080 ctaatactat aaaatgggtc tggatgtagt tctgacatta
ttttataaca actggtaaga 1140 gggagtgact atagcaacaa ctaaaatgat
ctcaggaaaa cctgtttggc cctatgtatg 1200 gtacattaca tcttttcagt
aattccactc aaatggagac ttttaacaaa gcaactgttc 1260 tcaggggacc
tattttctcc cttaaaattc attatacaca tccctggttg atagcagtgt 1320
gtctggaggc agaaaccatt cttgctttgg aaacaattac gtctgtgtta tactgagtag
1380 ggaagctcat taattgtcga cacttacgtt cctgataatg ggatcagtgt
gtaattcttg 1440 tttcgctcca gatttctaat accacaaaga ataaatcctt
tcactctgat caattttgtt 1500 aacttctcac gtgtcttctc tacacccagg
gcaccggtga atccaagtgt cctctgatgg 1560 tcaaagttct agatgctgtc
cgaggcagtc ctgccatcaa tgtggccgtg catgtgttca 1620 gaaaggctgc
tgatgacacc tgggagccat ttgcctctgg gtaagttgcc aaagaaccct 1680
cccacaggac ttggttttat cttcccgttt gcccctcact tggtagagag aggctcacat
1740 catctgctaa agaatttaca agtagattga aaaacgtagg cagaggtcaa
gtatgccctc 1800 tgaaggatgc cctctttttg ttttgcttag ctaggaagtg
accaggaacc tgagcatcat 1860 ttaggggcag acagtagaga aaagaaggaa
tcagaactcc tctcctctag ctgtggtttg 1920 caaccctttt gggtcacaga
acactttatg taggtgatga aaagtaaaca ttctatgccc 1980 agaaaaaatg
cacagataca cacacataca aaatcatata tgtgatttta ggagtttcac 2040
agattccctg gtgtccctgg gtaacaccaa agctaagtgt ccttgtctta gaattttagg
2100 aaaaggtata atgtgtatta acccattaac aaaaggaaag gaattcagaa
atattattaa 2160 ccaggcatct gtctgtagtt aatatggatc acccaaaacc
caaggctttt gcctaatgaa 2220 cactttgggg cacctactgt gtgcaaggct
gggggctgtc aagctcagtt aaaaaaaaaa 2280 agatagaaga gatggatcca
tgaggcaaag tacagcccca ggctaatccc acgatcaccc 2340 gacttcatgt
ccaagagtgg cttctcacct tcattagcca gttcacaatt ttcatggagt 2400
ttttctacct gcactagcaa aaacttcaag gaaaatacat attaataaat ctaagcaaag
2460 tgaccagaag acagagcaat caggagaccc tttgcatcca gcagaagagg
aactgctaag 2520 tatttacatc tccacagaga agaatttctg ttgggtttta
attgaacccc aagaaccaca 2580 tgattcttca accattattg ggaagatcat
tttcttaggt ctggttttaa ctggcttttt 2640 atttgggaat tcatttatgt
ttatataaaa tgccaagcat aacatgaaaa gtggttacag 2700 gactattcta
agggagagac agaatggaca ccaaaaatat tccaatgttc ttgtgaatct 2760
tttccttgca ccaggacaaa aaaaaaaaga agtgaaaaga agaaaggagg aggggcataa
2820 tcagagtcag taaagacaac tgctattttt atctatcgta gctgttgcag
tcaaatggga 2880 agcaatttcc aacattcaac tatggagctg gtacttacat
ggaaatagaa gttgcctagt 2940 gtttgttgct ggcaaagagt tatcagagag
gttaaatata taaaagggaa aagagtcaga 3000 tacaggttct tcttcctact
ttaggttttc cactgtgtgt gcaaatgata ctccctggtg 3060 gtgtgcagat
gcctcaaagc tatcctcaca ccacaaggga gaggagcgag atcctgctgt 3120
cctggagaag tgcagagtta gaacagctgt ggccacttgc atccaatcat caatcttgaa
3180 tcacagggac tctttcttaa gtaaacatta tacctggccg ggcacggtgg
ctcacgcctg 3240 taatcccagc actttgggat gccaaagtgg gcatatcatc
tgaggtcagg agttcaagac 3300 cagcctggcc aacatggcaa aactccgtct
ttatgaaaaa tacaaaaatt agccaggcat 3360 ggtggcaggc gcctgtaatc
ccagctaatt gggaggctga ggctggagaa tcccttgaat 3420 ctaggaggca
gaggttgcag tgagctgaga tcgtgccatt gcactccagc ctgggtgaca 3480
agagtaaaac tctgtctcaa aaaaaaaaaa ttatacctac attctcttct tatcagagaa
3540 aaaaatctac agtgagcttt tcaaaaagtt tttacaaact ttttgccatt
taatttcagt 3600 taggagtttt ccctacttct gacttagttg aggggaaatg
ttcataacat gtttataaca 3660 tgtttatgtg tgttagttgg tgggggtgta
ttactttgcc atgccatttg tttcctccat 3720 gcgtaactta atccagactt
tcacacctta taggaaaacc agtgagtctg gagagctgca 3780 tgggctcaca
actgaggagg aatttgtaga agggatatac aaagtggaaa tagacaccaa 3840
atcttactgg aaggcacttg gcatctcccc attccatgag catgcagagg tgagtataca
3900 gaccttcgag ggttgttttg gttttggttt ttgcttttgg cattccagga
aatgcacagt 3960 tttactcagt gtaccacaga aatgtcctaa ggaaggtgat
gaatgaccaa aggttccctt 4020 tcctattata caagaaaaaa ttcacaacac
tctgagaagc aaatttcttt ttgactttga 4080 tgaaaatcca cttagtaaca
tgacttgaac ttacatgaaa ctactcatag tctattcatt 4140 ccactttata
tgaatattga tgtatctgct gttgaaataa tagtttatga ggcagccctc 4200
cagaccccac gtagagtgta tgtaacaaga gatgcaccat tttatttctc gaaaacccgt
4260 aacattcttc attccaaaac acatctggct tctcggaggt ctggacaagt
gattcttggc 4320 aacacatacc tatagagaca ataaaatcaa agtaataatg
gcaacacaat agataacatt 4380 taccaagcat acaccatgtg gcagacacaa
ttataagtgt tttccatatt taacctactt 4440 aatcctcagg aataagccac
tgaggtcagt cctattatta tccccatctt atagatgaag 4500 aaaatgaggc
accaggaagt caaataactt gtcaaaggtc acaagactag gaaatacaca 4560
agtagaaatg tttacaatta aggcccaggc tgggtttgcc ctcagttctg ctatgcctcg
4620 cattatgccc caggaaactt tttcccttgt gaaagccaag cttaaaaaaa
gaaaagccac 4680 atttgtaacg tgctctgttc ccctgcctat ggtgaggatc
ttcaaacagt tatacatgga 4740 cccagtcccc ctgccttctc cttaatttct
taagtcattt gaaacagatg gctgtcatgg 4800 aaatagaatc cagacatgtt
ggtcagagtt aaagatcaac taattccatc aaaaatagct 4860 cggcatgaaa
gggaactatt ctctggctta gtcatggatg agactttcaa ttgctataaa 4920
gtggttcctt tattagacaa tgttaccagg gaaacaacag gggtttgttt gacttctggg
4980 gcccacaagt caacaagaga gccccatcta ccaaggagca tgtccctgac
tacccctcag 5040 ccagcagcaa gacatggacc ccagtcaggg caggagcagg
gtttcggcgg cgcccagcac 5100 aagacattgc ccctagagtc tcagccccta
ccctcgagta atagatctgc ctacctgaga 5160 ctgttgtttg cccaagagct
gggtctcagc ctgatgggaa ccatataaaa aggttcactg 5220 acatactgcc
cacatgttgt tctctttcat tagatcttag cttccttgtc tgctcttcat 5280
tcttgcagta ttcattcaac aaacattaaa aaaaaaaaaa agcattctat gtgtggaaca
5340 ctctgctaga tgctgtggat ttagaaatga aaatacatcc cgacccttgg
aatggaaggg 5400 aaaggactga agtaagacag attaagcagg accgtcagcc
cagcttgaag cccagataaa 5460 tacggagaac aagagagagc gagtagtgag
agatgagtcc caatgcctca ctttggtgac 5520 gggtgcgtgg tgggcttcat
gcagcttctt ctgataaatg cctccttcag aactggtcaa 5580 ctctaccttg
gccagtgacc caggtggtca tagtagattt accaagggaa aatggaaact 5640
tttattagga gctcttaggc ctcttcactt catggatttt tttttccttt ttttttgaga
5700 tggagttttg ccctgtcacc caggctggaa tgcagtggtg caatctcagc
tcactgcaac 5760 ctccgcctcc caggttcaag caattctcct gcctcagcct
cccgagtagc tgggactaca 5820 ggtgtgcgcc accacaccag gctaattttt
gtattttttg taaagacagg ttttcaccac 5880 gttggccagg ctggtctgaa
ctccagacct caggtgattc acctgtctca gcctcccaaa 5940 gtgctgggat
tacaggtgtg agccaccgtg cccggctact tcatggattt ttgattacag 6000
attatgcctc ttacaatttt taagaagaat caagtgggct gaaggtcaat gtcaccataa
6060 gacaaaagac atttttatta gttgattcta gggaattggc cttaagggga
gccctttctt 6120 cctaagagat tcttaggtga ttctcacttc ctcttgcccc
agtattattt ttgtttttgg 6180 tatggctcac tcagatcctt ttttcctcct
atccctaagt aatccgggtt tctttttccc 6240 atatttagaa caaaatgtat
ttatgcagag tgtgtccaaa cctcaaccca aggcctgtat 6300 acaaaataaa
tcaaattaaa cacatcttta ctgtcttcta cctctttcct gacctcaata 6360
tatcccaact tgcctcactc tgagaaccaa ggctgtccca gcacctgagt cgcagatatt
6420 ctactgattt gacagaactg tgtgactatc tggaacagca ttttgatcca
caatttgccc 6480 agttacaaag cttaaatgag ctctagtgca tgcatatata
tttcaaaatt ccaccatgat 6540 cttccacact ctgtattgta aatagagccc
tgtaatgctt ttacttcgta tttcattgct 6600 tgttatacat aaaaatatac
ttttcttctt catgttagaa aatgcaaaga ataggagggt 6660 gggggaatct
ctgggcttgg agacaggaga cttgccttcc tactatggtt ccatcagaat 6720
gtagactggg acaatacaat aattcaagtc tggtttgctc atctgtaaat tgggaagaat
6780 gtttccagct ccagaatgct aaatctctaa gtctgtggtt ggcagccact
attgcagcag 6840 ctcttcaatg actcaatgca gttttgcatt ctccctacct
tttttttcta aaaccaataa 6900 aatagataca gcctttaggc tttctgggat
ttcccttagt caagctaggg tcatcctgac 6960 tttcggcgtg aatttgcaaa
acaagacctg actctgtact cctgctctaa ggactgtgca 7020 tggttccaaa
ggcttagctt gccagcatat ttgagctttt tccttctgtt caaactgttc 7080
caaaatataa aagaataaaa ttaattaagt tggcactgga cttccggtgg tcagtcatgt
7140 gtgtcatctg tcacgttttt cgggctctgg tggaaatgga tctgtctgtc
ttctctcata 7200 ggtggtattc acagccaacg actccggccc ccgccgctac
accattgccg ccctgctgag 7260 cccctactcc tattccacca cggctgtcgt
caccaatccc aaggaatgag ggacttctcc 7320 tccagtggac ctgaaggacg
agggatggga tttcatgtaa ccaagagtat tccattttta 7380 ctaaagcagt
gttttcacct catatgctat gttagaagtc caggcagaga caataaaaca 7440
ttcctgtgaa aggcactttt cattccactt taacttgatt ttttaaattc ccttattgtc
7500 ccttccaaaa aaaagagaat caaaatttta caaagaatca aaggaattct
agaaagtatc 7560 tgggcagaac gctaggagag atccaaattt ccattgtctt
gcaagcaaag cacgtattaa 7620 atatgatctg cagccattaa aaagacacat
tctgtaaatg agagagcctt attttcctgt 7680 aaccttcagc aaatagcaaa
agacacattc caagggccca cttctttact gtgggcattt 7740 cttttttttt
ctttttttct tttttccttt tttgagacaa agtctcactc tgttgcccag 7800
gctagaatgc agtggtgtaa tctcagctca ctgcaacctc tgcttcctgg gttcaagcga
7860 ttctcctgcc tcagcctccc aagtaactgg gattacaggc gcatgccacc
acgcctagct 7920 catttttgta tttttagtag agatgggatt ttgccatgtt
ggctaggctg gtctacgaac 7980 tcctgacctc aggtgatcca cctgcctcag
cctcccaaag tgctgggatt acaggcatga 8040 gccactacac ccgg 8054
<210> SEQ ID NO 3 <400> SEQUENCE: 3 000 <210> SEQ
ID NO 4 <211> LENGTH: 10001 <212> TYPE: DNA <213>
ORGANISM: Macaca mulatta <400> SEQUENCE: 4 gactctattc
ctagttatgg tctcaactac atttgctcat tgctgtgagg ggtgagccca 60
cctcccggag tcctctcctg cacattccta tgttcctgaa aggactttcc atcccttcca
120 ctactccctg aaaactcctg tgcttcatga tttcttgttg aattttttct
aatctgactc 180 tatcagttat gggaatgttc cctcaactct tagtgctcca
gactggactc gctcttggca 240 tgtatttgtc caaaatattt gtctgctcta
tgtcttctac atatttgtct tataaggaac 300 aaacacctga tttgtttatc
catgaacaaa gccacacatg ctagtgcaca cgcacataca 360 cacacacaca
tacacacaga ggattctgta cgtgattaat gaatcatcaa atcatcataa 420
tttctggact tgtattaata cgtcggccag gaggaaaaga atccgttgtc aatcatggct
480 tctggttctc acggtcatct ctactttctt ccagcaagtt tgattctgtc
aaacaccagc 540 tggcagcttt gttccggcat gcccagtgca ggagagtcag
taaagaagat ttcattctct 600 gtatttcagc gtcgtcaatg ccaggttgaa
atacgctatt ctggcccagc tcagtggctc 660 acacgtgtaa tcccagcact
ttggaaggcc aaggcaggca gatcgcttga gcccaggaat 720 tcgagaccag
cctgggcaag aggctgaggt gggaggatga cctgagcccg ggaggtcaag 780
gctgcagcca gctgtgatca tgtcactgca ctcaagccag ggcgtcggag tgagaccgtg
840 tcaaaaaaaa aggaaggaaa gaaggaagga aggaaggaag gaaggaagga
aggaaggaag 900 gaaggaagga aggaagggag ggagggaggg agggagggag
gaaggaagga aggaagtatg 960 taaggaagga aggaaggaag gaagggaggg
acggagggcg ggagggaagg aagatgccat 1020 tcttagattg aagtggacct
tatgtgggca gaacacacac acacacacac acattgtgga 1080 gaaattgctg
actaagcaaa gcttccaaat gactgagttt ggctaaaacg taggctttta 1140
aaaatgtgag cactgccaag ggtttttcct tgttgaccca tggatccatc aagtgcaaac
1200 attttctaat gcactatatt taagcctgtg cagccaaatg tcattcaaca
tgaaatgcat 1260 tattacaact tgcatctgtc taaaatcttg catcaaaaat
gaaagacaaa aatgtataaa 1320 aatggaaaac atgcatagaa atatgtgagg
gaggaaaaaa atatccccag gaatgttagt 1380 gcacggagtc acacagggag
aagactattt ttgttttgtt ttgattgttt tgttctggtg 1440 acctcactgg
tcaaatgacc tattaagaat attttgtaga gctaatgttc cgatgctcta 1500
atctctctag acaaggttca tatttgtatg ggttacttat tctctctttg ttgactaagt
1560 caataatcag aatcagcagg tttggagtca gattggcagg gataagcagc
ctagctcagg 1620 agaagtgggt ataaaagccc caggctggga gcagccatca
cagaagtcca ctcgttcttg 1680 gcaggatggc ttctcatcgt ctgctcctcc
tctgcctcgc tggactggta tttgtgtctg 1740 aggctggtcc tacggtgagt
gtttctgtga catctcgctc ctacatttaa aatgcacgct 1800 aaatgaggta
ggagtgactc cttccagctt tgccaagcag cttttgttac tagggcaagg 1860
gtacccagca tctattttta atatcattta ttcaaacttc aaaaagaatg aagttccact
1920 gagcttactg agctgggact tgaactctgg ggattctgcc tcattgcttt
ggtgcattgg 1980 gtttgtaatg tctggtatct ccacttcctc agatcgatga
tagagataaa gatatgatat 2040 taaggaagct gttaatcctg aattttcaga
aaagtatccc tttataaaat gtatttgggg 2100 ggcaaactgc atgagattat
attctggccc tatagctaat caaaatgtat ttaccgatta 2160 atctttaaaa
ggcttagtga acaatatttt agtcagatat ctaattctta aatactctag 2220
aaggattaac taatctataa aatgggtctg gatatagtct gacataattt tataacaacc
2280 ggtaagaggg agtgactaga gcaacaacta aaatcatctc aggaaaaact
gttttggtcc 2340 tatgtatggt acgttacatc ttttcagtaa ttccattcaa
atggagaagt ttaacaaggc 2400 aactgttctc agggggccta ttctctccct
taaaattcat tatacacatc cctggttgat 2460 agcagtgtgt ctggaggcag
aaaccattct tgctttggaa acaattatgt ctgtgttata 2520 ctgagtaggg
aagctcatta actgtcaaca cttatgttct tcataatgga atcagtgtgt 2580
aattcttgtt ttgttccaga tttctaacac cataaagaat aaatcctttc actctgatca
2640 atgttgttaa cttctcactt gtcttctcta tacccagggc gttgatgaat
ccaagtgtcc 2700 tctgatggtc aaagttctag atgccgtccg aggcagtcct
gccgtcaatg tggctgtgaa 2760 cgtgttcaaa aaggctgctg atgagacctg
ggcgccattt gcctctgggt aagaaccctc 2820 ccacgggact tggttttatc
ttcccattgg cccctcagtt ggtagacaga ggctcacatc 2880 atctgctaaa
gaatttacaa gtagattgaa aaacgtgagc agaggtcaag tatgccctct 2940
gaagatgccc tctttttgtt ttgcttagct aggaagggac caggaacctg agcatcattt
3000 aggggcagac agtagagaaa agaaggaatc agaactcttc ccctctagct
gtggtgtgca 3060 acccttttgg gtcacagacc actttatgta ggtgataaaa
actaaagatt ctatgcccag 3120 aaaaaatgta cagatacaca cacacaaaac
catatatgtg attttaggag tttcacagat 3180 tccctggtgt ccctgggtaa
caccaaaggt gagagtcctc gtcttagaat tttaggaaag 3240 aggtgcaatg
tgtattaacc cactaacgaa aggaaaggga ttcagaaata ttattgacta 3300
ggcatctgtc tgtagttcat ttggatcacc ccaaacccag ggcttttgcc taatgaacac
3360 tttggggcac ctactgtgtg cagggctgga ggctgtcaag ctcagttaaa
acaaatgtaa 3420 aaaaagacag aagaaatgga tccatgaggc aaagtacagc
cccagactaa tcccatgatc 3480 acccaacttc atgtgcaaga gtgacttcta
accttcatga gccagtttac aattttcatg 3540 gagtttttct acctacacta
caaaaactcc aaggaaaata tatattaata aacctaagcg 3600 aattgaccga
aagacagagc aataaggaga cccttcgcac ccagcagaag aggaactgtt 3660
aagtacttac ttctcctcag agaagaattt ctgttgtatt ttaattgaac cccaagaacc
3720 acacgattct tcaaccatta ttggtaagat cattttctta ggtctggttt
taactgactt 3780 tttatttggt aattcattta tgtttatata aaatgccaag
cataacatga aaagtggtta 3840 caggactatt ctaagggaga gagaaaatgg
ataccaaaaa tattccaatg ttcttatgaa 3900 tcttttccct tgcgtcagga
caaaaaaaaa aaaaatgtaa agaagaaagg aggagatgca 3960 caatcagagt
cagtaaagac aactgctatt tttatctgtc atagctgttg cagtctaatg 4020
ggaagcaatt tccaacattc aactatggag ctggtactta catggaaata gaagttgcct
4080 agtgtttgtt gctggcaaag agttatcaga gaggttaaat atataaaagg
gaagagtcag 4140 atacaggttc ttcttcctac tttaggtttt ccactgtgtg
tgcaaatgac cctccctggt 4200 ggtgtgcaga tgctctgaaa ggtatcctca
caccacaagg cagaggagcg agaccctgct 4260 gtcctggaga agtgcagagt
tagaacagct gtggccactt ccagggatgg tcacaacatc 4320 ccatctaatc
atcaatcttg aacaacaagg actctttctt aagaaaacat tatacccagc 4380
cgggcgcggt ggctcacacc tgtaaatctc agcgctttgg gaggctgaaa tgggcatatc
4440 atctgaggtc gagagctcaa gaccaacctg gccaacatgg caaaactccg
tctctatgaa 4500 aaatacaaaa attagccagg catggtggca ggcacctgta
atcccggcta ctcgggaggc 4560 tgagactgga gaatcccttg aacctggagg
cagaggttgc agtgagctga gatcacgcca 4620 ctgcactcca gcctgggtga
ctagagtaaa actctgtctc aaaaataaaa aaaaatttaa 4680 aaaattatac
ctacattctc ttcttatcag agaaaaatat ctacagtgag cttttcaaaa 4740
gtttttacaa actttttgcc atttaatttc agacagttat gagttttccc tacttctgac
4800 ttagttgagg ggaaatgtat ataacacatt tatgtgtgtt gtgtatataa
cacatataac 4860 acgtttatgt gtgttggtgg gggtattact ttgccatgcc
atttgtttcc tccatgccta 4920 acttaaccca gactttcaca ccttatagga
aaaccagtga gtctggagag ctgcatgggc 4980 tcacaactga ggaggaattt
gtagaaggga tatacaaagt ggaaatagac accaaatctt 5040 actggaagtc
acttggcatc tccccattcc atgagcatgc agaggtgagt atataaacct 5100
tcgagggttg ttttggtttt ggtttttgct tttggcatcc caggaaatgc acagttttac
5160 ttagcatacc acagaaatgt cctaaagaag gtgatgaatg accaaaggtt
ccctctcctc 5220 ttatacaaga acaaattcac aacactctga gaagcacatt
tctttttgac tttgaggaaa 5280 acccatttag taacatgact tgaacttaca
tgacactatt catagtctac tcattccatt 5340 ttatatgaat attgatgtat
ttgccgttga aataacatgt ttatgaggca gacctccaga 5400 ccccacgtag
agtgtatgaa acaagagatg caccatttta tttctctaaa acctgtaaca 5460
ttcttcattc caaaacacat ctggctcctc ggaggtttgg acaagtgatt cttggcaaca
5520 catacataga gagacaataa aatcaaagta ataatggcaa cacaacagat
aacatttact 5580 aagcatacac catgtggcag acacaattat aagtgttttc
tatatttaac ctacttcatc 5640 ctcagggaca agccactgag gtcagtccta
ttattatccc catctcatag atgaagcaag 5700 tgaggcacca ggaagtcaaa
taacttgtca aaggtcacaa ggctaggaaa cacacaagta 5760 gagatgttta
caaacaaggc ccaggctggg tttgccctca attctgctct gcctcgcatt 5820
gcgacccagg aaatttgttc ccctgtgaaa agccaagctt aaaaaaagaa aagccacatt
5880 tgtaacgtgc tctgttcccc tgcctatggt gaggatcttc gaacagttat
acagctccct 5940 gtcttcccct tgtcttaatt tcttcagtca tttgaaacag
atggctgtca tggaaataga 6000 atccagacat gttggtcaga gttaaagatc
aactaattcc atcaaaaata gctcagcatg 6060 aaagggaact attctctggc
ttagtcatgg atgagacttt caattgctat aaagtggttc 6120 ctttattagg
caatgttacc agggaaacaa taggggcttg tttgacttct ggggcccaca 6180
agtcaccaag ggagccccat ctaccaagaa gcatgtccct gactaccccg cagccaggca
6240 gcaagacacg gacgccggtc agggcagcag cagggtttca gtggtgccca
gcacaagaca 6300 ttgctcctag agtctcagcc cctacccttg agtagtagaa
ctgcctacct gagaccgttg 6360 tttgcctaag acctgggtct cagcctgatg
ggaaccatct aaaaagttca tcgccatact 6420 gcccacgtgt tgttctcttt
cattagatct cagcttcctt gactgctctt cactcttgtt 6480 tattcattca
acaaacattt aaaaaataaa agcattctat gtgtggaaca ctctgataga 6540
ggctggagat tcagaaatga aaatacatcc ctacccttgg aatggagggg aaaggactga
6600 agtaagacag actaggcagg cccgtcagcc cagcttgaag cccagataaa
tatggagaac 6660 aagagtgagt gagtagtgag agatgagtcc cagtgcctca
ctttggtgac tggtgcatgg 6720 tgggcttcat gcagcttctt ctgataaatg
cctccttcag aaccggtcac ctctaccttg 6780 gccagtgacc caagtggtca
tattagattt accaagggaa attggaaact tttattagga 6840 gctcttaggc
ctcttcactt catggaattt tttctttttt cttttttttt tgagatggag 6900
ttttgctctg tcacccaggc tggaatgcag tggtgcaatc ttggctcact gcaaccttta
6960 cttcccaggt tcaagcaatt cttctgcctc agcctcccga gtagctggga
ctacaggtgc 7020 acgccaccat acccagctaa gttttgtatt tttcatagag
acagggtttc gccatgttgg 7080 ccaggctggt ctcgaactcc agacctcagg
tgattcaccc acctcagcct cccaaagtgc 7140 tggcattaca ggtgtgagcc
agtacaccca gctacttcat ggatttttga tcacagatta 7200 tgcctcttat
aatttttaag aagaatcaag tgggctgaag gtcaatgtca ccataagata 7260
aaagatattt ttattagttg attctaggga gttggcctta aggggagccc tttcttctta
7320 agagattctt ggccgggcgc ggtggctcaa gcctgtaatc ccagcacttt
gggaggccga 7380 gacgggtgga tcatgaggtc aggagatcga gaccatcctg
gtaacacggt gaaaccccgt 7440 ctctactaaa aaatacaaaa aaactagccg
ggtgagttgg cgggcgcctg tagtcccagc 7500 tactcgggag gctgaggcag
gagaatggcg taaacccggg aggcggagct tgcagtgagc 7560 tgagatctgg
ccactgcact ccagcccggg tgacagagcg agactccgtc tcaaaaaaaa 7620
aaaaaaaaaa aaagagattc ttaggtgatt ctcacttcct cttgccccaa tattattttt
7680 gtttttggta tggctcactc agctcctttt tccctcctat ccctaagtaa
tccgggtttc 7740 ttttccccat atttggaaca aaatgtattt atgcagagtg
tgtccaaaac tcaacccaag 7800 gcccgtatac aaaataaatc aaattaaaca
catctttact gtcttctacc tctttcctga 7860 cctcaattta tcccaacttg
cctcactctg agaatcaagc ctgtcccagc acatgagttg 7920 cagatactct
actgaatttg acagaactgt gtgactatct ggaacagcat tttgatccac 7980
aatttgccca gttacaaagc ttaaatgaag tctagtgcat gcatgtatat ttcaaaattc
8040 caccatgctc ttccacactc tgtattgtaa atagagccct gaaatgcttt
tggttcatat 8100 ttcattgctt gctatacata aaaatatact ttttcttctt
catgttagaa aatgcaaaga 8160 atagtggggt gggggaatct ctgggcttgg
agacaggaga cttaccttcc tactgtggtt 8220 ccatcagtat gtagactggg
gcaatacaat aattcaagtc tggtttgctc atctgtaaaa 8280 tgggaagaat
gtttccagct ccagaatgct aagtctctaa gtctgtggtt ggcagccact 8340
attgcagcag cttttcaatg actcagtgca tttttccatt ctccccacct tttttttttc
8400 taaaaccaac aaaatagata cagcctttag gctctctggg atttccctta
gtcaagctag 8460 ggccatcctg acttttgatg tgaatttgca aaacaagacc
tggttctgta ctcctgctct 8520 aagggctgtg catggttcca aaggcttggc
ttgccagtgt atttgagctt tttccttctg 8580 ttcaaacttc aaaatataaa
agaataaaat taattaagtt ggcactggac ttccggtggt 8640 cagtcatgtg
tgtcatctgt acggttttcg ggctctggtg gaaatggata ctgtctgtct 8700
tctctcatag gtggtattca cagccaacga ttccggcccc cgccactaca ccatcgccgc
8760 cctgctgagc ccctactcct attccaccac ggctgtcgtc accaatccca
aggaatgagg 8820 gacttctcca gaggatctga aggacgaggg atgggatttc
atgtaaccaa gagtattcca 8880 tttttactaa agcagtgttt tcacctcata
agctatgtta ggagtccagg cagagacaat 8940 aaaacattcc tgtgaaaggc
acttttcatt ccattttaac ttgatttttt aaattccctt 9000 attgtccctt
ccaaaaaaac aagaatcaaa attctacaaa gaagcaaagg aattctagaa 9060
cgtatctggg cagaacgcta ggagagatcc aaatttccaa tttattgcaa gcaaagcaca
9120 tattaaatat gatctgcagt catcaaaaag acacattctg taaatgagaa
agccttattt 9180 tcctgtaacc ttcagtgaat agcaaaagac acattctaag
ggcccacttc tttactgtgg 9240 gcatttcttt tcttttcttt tttttttttt
cttttctttt ccttttttga gacaaagtct 9300 cactctgtcg cccaggctag
aatgcagtgg tgtgatctca gctcactgca acctctgctt 9360 ccgggttcaa
gcgattctcc tgcctcagcc tcccaagcag ctgggattac aggcgcccgc 9420
caccacacct ggctaatttt tctacttgta gtagagatgg ggtttcgcca tgttggctag
9480 gctggcctcg aactcctgac ctcaggtgat ccacctgcct cagcctccca
aagtgctggg 9540 attacaggca tgagccacta cacccggccc ctactctggg
catttctttg atttaagaga 9600 agggcagctc caacaagaca cacctgcaga
gactcaggcc atccgatcag ttcaggctag 9660 atccacgctg caatcagcca
ggtcagggac aaaccaaaga accccacaca cccaatttac 9720 ttaggctgat
ccaaaatcca tgtatggaga actcacatgc agcaggcact attttaggtg 9780
atctgaacat aaagaatagg acccagtacc tgcatttatt taaagaactc acaatctttt
9840 gaaaagataa ctgtttcatc atggtttggc aggaggctat ggtacaaggc
acagcaaagg 9900 taagaaggag aaaaaaccaa caccctagag aaatcagaaa
atgactctga ataggtgtca 9960 cttaatctga gtgttggtaa tttgtcagat
agacaaggga a 10001 <210> SEQ ID NO 5 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 5 ccctgctgag cccctactc 19 <210> SEQ ID
NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 6 tccctcattc
cttgggattg 20 <210> SEQ ID NO 7 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 7 attccaccac ggctgtcgtc a 21 <210> SEQ
ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
8 actggttttc ccagaggcaa 20 <210> SEQ ID NO 9 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 9 gactcactgg
ttttcccaga 20 <210> SEQ ID NO 10 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 10 tgaataccac ctctgcatgc 20
<210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 11 ccgtggtgga ataggagtag 20 <210> SEQ
ID NO 12 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
12 agccgtggtg gaataggagt 20 <210> SEQ ID NO 13 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 13 cgacagccgt
ggtggaatag 20 <210> SEQ ID NO 14 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 14 ttggtgacga cagccgtggt 20
<210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 15 gattggtgac gacagccgtg 20 <210> SEQ
ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
16 gggattggtg acgacagccg 20 <210> SEQ ID NO 17 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 17 tgggattggt
gacgacagcc 20 <210> SEQ ID NO 18 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 18 attccttggg attggtgacg 20
<210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 19 cattccttgg gattggtgac 20 <210> SEQ
ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
20 tcattccttg ggattggtga 20 <210> SEQ ID NO 21 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 21 agaagtccct
cattccttgg 20 <210> SEQ ID NO 22 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 22 gtccttcagg tccactggag 20
<210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 23 catccctcgt ccttcaggtc 20 <210> SEQ
ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
24 tacatgaaat cccatccctc 20 <210> SEQ ID NO 25 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 25 cttggttaca
tgaaatccca 20 <210> SEQ ID NO 26 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 26 aatactcttg gttacatgaa 20
<210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 27 ttagtaaaaa tggaatactc 20 <210> SEQ
ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
28 actgctttag taaaaatgga 20 <210> SEQ ID NO 29 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 29 tgaaaacact
gctttagtaa 20 <210> SEQ ID NO 30 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 30 tatgaggtga aaacactgct 20
<210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 31 tggacttcta acatagcata 20 <210> SEQ
ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
32 tctctgcctg gacttctaac 20 <210> SEQ ID NO 33 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 33 ttattgtctc
tgcctggact 20 <210> SEQ ID NO 34 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 34 tgcctttcac aggaatgttt 20
<210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 35 gtgcctttca caggaatgtt 20 <210> SEQ
ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
36 cagaggagga gcagacgatg 20 <210> SEQ ID NO 37 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 37 tctagaactt
tgaccatcag 20 <210> SEQ ID NO 38 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 38 ttttcccaga ggcaaatggc 20
<210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 39 tccagactca ctggttttcc 20 <210> SEQ
ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
40 tatcccttct acaaattcct 20 <210> SEQ ID NO 41 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 41 atttccactt
tgtatatccc 20 <210> SEQ ID NO 42 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 42 tggtgtctat ttccactttg 20
<210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 43 cagtaagatt tggtgtctat 20 <210> SEQ
ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
44 cttccagtaa gatttggtgt 20 <210> SEQ ID NO 45 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 45 ccacctctgc
atgctcatgg 20 <210> SEQ ID NO 46 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 46 tgtgaatacc acctctgcat 20
<210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 47 gctgtgaata ccacctctgc 20 <210> SEQ
ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
48 cgttggctgt gaataccacc 20 <210> SEQ ID NO 49 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 49 cagccgtggt
ggaataggag 20 <210> SEQ ID NO 50 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 50 gacagccgtg gtggaatagg 20
<210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 51 acgacagccg tggtggaata 20 <210> SEQ
ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
52 gacgacagcc gtggtggaat 20 <210> SEQ ID NO 53 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 53 tgacgacagc
cgtggtggaa 20 <210> SEQ ID NO 54 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 54 gtgacgacag ccgtggtgga 20
<210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 55 ggtgacgaca gccgtggtgg 20 <210> SEQ
ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
56 tggtgacgac agccgtggtg 20 <210> SEQ ID NO 57 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 57 attggtgacg
acagccgtgg 20 <210> SEQ ID NO 58 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 58 ggattggtga cgacagccgt 20
<210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 59 ttgggattgg tgacgacagc 20 <210> SEQ
ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
60 cttgggattg gtgacgacag 20 <210> SEQ ID NO 61 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 61 ccttgggatt
ggtgacgaca 20 <210> SEQ ID NO 62 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 62 tccttgggat tggtgacgac 20
<210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 63 ttccttggga ttggtgacga 20 <210> SEQ
ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
64 ctcattcctt gggattggtg 20 <210> SEQ ID NO 65 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 65 cctcattcct
tgggattggt 20 <210> SEQ ID NO 66 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 66 ccctcattcc ttgggattgg 20
<210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 67 tccctcattc cttgggattg 20 <210> SEQ
ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
68 gtccctcatt ccttgggatt 20 <210> SEQ ID NO 69 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 69 agtccctcat
tccttgggat 20 <210> SEQ ID NO 70 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 70 aagtccctca ttccttggga 20
<210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 71 gaagtccctc attccttggg 20 <210> SEQ
ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
72 gagaagtccc tcattccttg 20 <210> SEQ ID NO 73 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 73 ggagaagtcc
ctcattcctt 20 <210> SEQ ID NO 74 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 74 acatgaaatc ccatccctcg 20
<210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 75 ttacatgaaa tcccatccct 20 <210> SEQ
ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
76 gttacatgaa atcccatccc 20 <210> SEQ ID NO 77 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 77 ggttacatga
aatcccatcc 20 <210> SEQ ID NO 78 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 78 tggttacatg aaatcccatc 20
<210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 79 ttggttacat gaaatcccat 20 <210> SEQ
ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
80 tcttggttac atgaaatccc 20 <210> SEQ ID NO 81 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 81 ctcttggtta
catgaaatcc 20 <210> SEQ ID NO 82 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 82 actcttggtt acatgaaatc 20
<210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 83 tactcttggt tacatgaaat 20 <210> SEQ
ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
84 atactcttgg ttacatgaaa 20 <210> SEQ ID NO 85 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 85 gaatactctt
ggttacatga 20 <210> SEQ ID NO 86 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 86 ggaatactct tggttacatg 20
<210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 87 tggaatactc ttggttacat 20 <210> SEQ
ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
88 atggaatact cttggttaca 20 <210> SEQ ID NO 89 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 89 aatggaatac
tcttggttac 20 <210> SEQ ID NO 90 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 90 aaatggaata ctcttggtta 20
<210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 91 aaaatggaat actcttggtt 20 <210> SEQ
ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
92 aaaaatggaa tactcttggt 20 <210> SEQ ID NO 93 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 93 taaaaatgga
atactcttgg 20 <210> SEQ ID NO 94 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 94 gtaaaaatgg aatactcttg 20
<210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 95 agtaaaaatg gaatactctt 20 <210> SEQ
ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
96 tagtaaaaat ggaatactct 20 <210> SEQ ID NO 97 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 97 tttagtaaaa
atggaatact 20 <210> SEQ ID NO 98 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 98 ctttagtaaa aatggaatac 20
<210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 99 gctttagtaa aaatggaata 20 <210> SEQ
ID NO 100 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
100 tgctttagta aaaatggaat 20 <210> SEQ ID NO 101 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 101 ctgctttagt
aaaaatggaa 20 <210> SEQ ID NO 102 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 102 cactgcttta gtaaaaatgg 20
<210> SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 103 acactgcttt agtaaaaatg 20 <210> SEQ
ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
104 aacactgctt tagtaaaaat 20 <210> SEQ ID NO 105 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 105 aaacactgct
ttagtaaaaa 20 <210> SEQ ID NO 106 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 106 aaaacactgc tttagtaaaa 20
<210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 107 gaaaacactg ctttagtaaa 20 <210> SEQ
ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
108 gtgaaaacac tgctttagta 20 <210> SEQ ID NO 109 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 109 ggtgaaaaca
ctgctttagt 20 <210> SEQ ID NO 110 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 110 aggtgaaaac actgctttag 20
<210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 111 gaggtgaaaa cactgcttta 20 <210> SEQ
ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
112 tgaggtgaaa acactgcttt 20 <210> SEQ ID NO 113 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 113 atgaggtgaa
aacactgctt 20 <210> SEQ ID NO 114 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 114 tttattgtct ctgcctggac 20
<210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 115 ttttattgtc tctgcctgga 20 <210> SEQ
ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
116 gttttattgt ctctgcctgg 20 <210> SEQ ID NO 117 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 117 tgttttattg
tctctgcctg 20 <210> SEQ ID NO 118 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 118 atgttttatt gtctctgcct 20
<210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 119 aatgttttat tgtctctgcc 20 <210> SEQ
ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
120 gaatgtttta ttgtctctgc 20 <210> SEQ ID NO 121 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 121 ggaatgtttt
attgtctctg 20 <210> SEQ ID NO 122 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 122 aggaatgttt tattgtctct 20
<210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 123 caggaatgtt ttattgtctc 20 <210> SEQ
ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
124 acaggaatgt tttattgtct 20 <210> SEQ ID NO 125 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 125 gatgtcacag
aaacactcac 20 <210> SEQ ID NO 126 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 126 gcaaagctgg aaggagtcac 20
<210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 127 gaacttcatt ctttttgaag 20 <210> SEQ
ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
128 agcttcctta atatcatatc 20 <210> SEQ ID NO 129 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 129 tatagggcca
gaatataatc 20 <210> SEQ ID NO 130 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 130 actaagcctt ttaaagatta 20
<210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 131 tggaattact gaaaagatgt 20 <210> SEQ
ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
132 accagggatg tgtataatga 20 <210> SEQ ID NO 133 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 133 tccctactca
gtataacaca 20 <210> SEQ ID NO 134 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 134 gatcagagtg aaaggattta 20
<210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 135 gggaagataa aaccaagtcc 20 <210> SEQ
ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
136 taaattcttt agcagatgat 20 <210> SEQ ID NO 137 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 137 aatgatgctc
aggttcctgg 20 <210> SEQ ID NO 138 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 138 ttggtgttac ccagggacac 20
<210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 139 aaagtgttca ttaggcaaaa 20 <210> SEQ
ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
140 ggcattttat ataaacataa 20 <210> SEQ ID NO 141 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 141 aagaacattg
gaatattttt 20 <210> SEQ ID NO 142 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 142 gttggaaatt gcttcccatt 20
<210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 143 agtggaaaac ctaaagtagg 20 <210> SEQ
ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
144 ttcccctcaa ctaagtcaga 20 <210> SEQ ID NO 145 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 145 cctataaggt
gtgaaagtct 20 <210> SEQ ID NO 146 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 146 tgtaagttca agtcatgtta 20
<210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 147 gtgttgccaa gaatcacttg 20 <210> SEQ
ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
148 aaaacactta taattgtgtc 20 <210> SEQ ID NO 149 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 149 ctttgacaag
ttatttgact 20 <210> SEQ ID NO 150 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 150 atccatgact aagccagaga 20
<210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 151 atggttccca tcaggctgag 20 <210> SEQ
ID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
152 gcatttatca gaagaagctg 20 <210> SEQ ID NO 153 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 153 ttgaccttca
gcccacttga 20 <210> SEQ ID NO 154 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 154 aggaagtgag aatcacctaa 20
<210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 155 agaagacagt aaagatgtgt 20 <210> SEQ
ID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
156 aaattgtgga tcaaaatgct 20 <210> SEQ ID NO 157 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 157 aaccagactt
gaattattgt 20 <210> SEQ ID NO 158 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 158 agtggctgcc aaccacagac 20
<210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 159 ggaagtccag tgccaactta 20 <210> SEQ
ID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
160 atccatttcc accagagccc 20 <210> SEQ ID NO 161 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 161 cttgctggac tggtatttgt gtct 24 <210>
SEQ ID NO 162 <211> LENGTH: 25 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer <400> SEQUENCE: 162
agaactttga ccatcagagg acact 25 <210> SEQ ID NO 163
<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Probe <400> SEQUENCE: 163 ccctacgggc accggtgaat
cc 22 <210> SEQ ID NO 164 <211> LENGTH: 612 <212>
TYPE: DNA <213> ORGANISM: Macaca fascicularis <220>
FEATURE: <221> NAME/KEY: CDS <222> LOCATION:
(27)..(470) <400> SEQUENCE: 164 acagaagtcc actcgttctt ggcagg
atg gct tct cat cgt ctg ctc ctt ctc 53 Met Ala Ser His Arg Leu Leu
Leu Leu 1 5 tgc ctc gct gga ctg gta ttt gtg tct gaa gct ggt cct acg
ggc gtt 101 Cys Leu Ala Gly Leu Val Phe Val Ser Glu Ala Gly Pro Thr
Gly Val 10 15 20 25 gat gaa tcc aag tgt cct ctg atg gtc aaa gtt cta
gat gcc gtc cga 149 Asp Glu Ser Lys Cys Pro Leu Met Val Lys Val Leu
Asp Ala Val Arg 30 35 40 ggc agt cct gcc gtc aat gtg gct gtg aac
gtg ttc aaa aag gct gct 197 Gly Ser Pro Ala Val Asn Val Ala Val Asn
Val Phe Lys Lys Ala Ala 45 50 55 gat gag acc tgg gcg cca ttt gcc
tct ggg aaa acc agt gag tct gga 245 Asp Glu Thr Trp Ala Pro Phe Ala
Ser Gly Lys Thr Ser Glu Ser Gly 60 65 70 gag ctg cat ggg ctc aca
act gag gag gaa ttt gta gaa ggg ata tac 293 Glu Leu His Gly Leu Thr
Thr Glu Glu Glu Phe Val Glu Gly Ile Tyr 75 80 85 aaa gtg gaa ata
gac acc aaa tct tac tgg aag tca ctt ggc atc tcc 341 Lys Val Glu Ile
Asp Thr Lys Ser Tyr Trp Lys Ser Leu Gly Ile Ser 90 95 100 105 cca
ttc cat gag cat gca gag gtg gta ttc aca gcc aac gat tcc ggc 389 Pro
Phe His Glu His Ala Glu Val Val Phe Thr Ala Asn Asp Ser Gly 110 115
120 ccc cgc cac tac acc atc gcc cgc ctg ctg agc ccc tac tcc tat tcc
437 Pro Arg His Tyr Thr Ile Ala Arg Leu Leu Ser Pro Tyr Ser Tyr Ser
125 130 135 acc acg gct gtc gtc acc aat ccc aag gaa tga gggacttctc
cagaggatct 490 Thr Thr Ala Val Val Thr Asn Pro Lys Glu 140 145
gaaggacgag ggatgggatt tcatgtaacc aagagtattc catttttact aaagcagtgt
550 tttcacctca taagctatgt taggagtcca ggcagagaca ataaaacatt
cctgtgaaag 610 gc 612 <210> SEQ ID NO 165 <211> LENGTH:
20 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 165 ccttccctga aggttcctcc 20
<210> SEQ ID NO 166 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 166 gcgtttgctc ttcttcttgc gtttttt 27
<210> SEQ ID NO 167 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 167 ttttattgtc tctgcctg 18 <210> SEQ ID
NO 168 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
168 tacaaatggg atgctactgc 20 <210> SEQ ID NO 169 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 169 ggaatcccaa
gcctcaaacg 20 <210> SEQ ID NO 170 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 170 cgtccttcag gtccactgga 20
<210> SEQ ID NO 171 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 171 atatgaggtg aaaacactgc 20 <210> SEQ
ID NO 172 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
172 tattgtctct gcctggactt 20 <210> SEQ ID NO 173 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 173 cacaggaatg
ttttattgtc 20 <210> SEQ ID NO 174 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 174 cctttcacag gaatgtttta 20
<210> SEQ ID NO 175 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 175 gcctttcaca ggaatgtttt 20 <210> SEQ
ID NO 176 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
176 agtgcctttc acaggaatgt 20 <210> SEQ ID NO 177 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 177 aagtgccttt
cacaggaatg 20
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 177
<210> SEQ ID NO 1 <211> LENGTH: 650 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (27)..(470)
<400> SEQUENCE: 1 acagaagtcc actcattctt ggcagg atg gct tct
cat cgt ctg ctc ctc ctc 53 Met Ala Ser His Arg Leu Leu Leu Leu 1 5
tgc ctt gct gga ctg gta ttt gtg tct gag gct ggc cct acg ggc acc 101
Cys Leu Ala Gly Leu Val Phe Val Ser Glu Ala Gly Pro Thr Gly Thr 10
15 20 25 ggt gaa tcc aag tgt cct ctg atg gtc aaa gtt cta gat gct
gtc cga 149 Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val Leu Asp Ala
Val Arg 30 35 40 ggc agt cct gcc atc aat gtg gcc gtg cat gtg ttc
aga aag gct gct 197 Gly Ser Pro Ala Ile Asn Val Ala Val His Val Phe
Arg Lys Ala Ala 45 50 55 gat gac acc tgg gag cca ttt gcc tct ggg
aaa acc agt gag tct gga 245 Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly
Lys Thr Ser Glu Ser Gly 60 65 70 gag ctg cat ggg ctc aca act gag
gag gaa ttt gta gaa ggg ata tac 293 Glu Leu His Gly Leu Thr Thr Glu
Glu Glu Phe Val Glu Gly Ile Tyr 75 80 85 aaa gtg gaa ata gac acc
aaa tct tac tgg aag gca ctt ggc atc tcc 341 Lys Val Glu Ile Asp Thr
Lys Ser Tyr Trp Lys Ala Leu Gly Ile Ser 90 95 100 105 cca ttc cat
gag cat gca gag gtg gta ttc aca gcc aac gac tcc ggc 389 Pro Phe His
Glu His Ala Glu Val Val Phe Thr Ala Asn Asp Ser Gly 110 115 120 ccc
cgc cgc tac acc att gcc gcc ctg ctg agc ccc tac tcc tat tcc 437 Pro
Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr Ser Tyr Ser 125 130
135 acc acg gct gtc gtc acc aat ccc aag gaa tga gggacttctc
ctccagtgga 490 Thr Thr Ala Val Val Thr Asn Pro Lys Glu 140 145
cctgaaggac gagggatggg atttcatgta accaagagta ttccattttt actaaagcag
550 tgttttcacc tcatatgcta tgttagaagt ccaggcagag acaataaaac
attcctgtga 610 aaggcacttt tcattccaaa aaaaaaaaaa aaaaaaaaaa 650
<210> SEQ ID NO 2 <211> LENGTH: 8054 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2
ttgttgaccc atggatccat caagtgcaaa cattttctaa tgcactatat ttaagcctgt
60 gcagctagat gtcattcaac atgaaataca ttattacaac ttgcatctgt
ctaaaatctt 120 gcatctaaaa tgagagacaa aaaatctata aaaatggaaa
acatgcatag aaatatgtga 180 gggaggaaaa aattaccccc aagaatgtta
gtgcacgcag tcacacaggg agaagactat 240 ttttgttttg ttttgattgt
tttgttttgt tttggttgtt ttgttttggt gacctaactg 300 gtcaaatgac
ctattaagaa tatttcatag aacgaatgtt ccgatgctct aatctctcta 360
gacaaggttc atatttgtat gggttactta ttctctcttt gttgactaag tcaataatca
420 gaatcagcag gtttgcagtc agattggcag ggataagcag cctagctcag
gagaagtgag 480 tataaaagcc ccaggctggg agcagccatc acagaagtcc
actcattctt ggcaggatgg 540 cttctcatcg tctgctcctc ctctgccttg
ctggactggt atttgtgtct gaggctggcc 600 ctacggtgag tgtttctgtg
acatcccatt cctacattta agattcacgc taaatgaagt 660 agaagtgact
ccttccagct ttgccaacca gcttttatta ctagggcaag ggtacccagc 720
atctattttt aatataatta attcaaactt caaaaagaat gaagttccac tgagcttact
780 gagctgggac ttgaactctg agcattctac ctcattgctt tggtgcatta
ggtttgtaat 840 atctggtacc tctgtttcct cagatagatg atagaaataa
agatatgata ttaaggaagc 900 tgttaatact gaattttcag aaaagtatcc
ctccataaaa tgtatttggg ggacaaactg 960 caggagatta tattctggcc
ctatagttat tcaaaacgta tttattgatt aatctttaaa 1020 aggcttagtg
aacaatattc tagtcagata tctaattctt aaatcctcta gaagaattaa 1080
ctaatactat aaaatgggtc tggatgtagt tctgacatta ttttataaca actggtaaga
1140 gggagtgact atagcaacaa ctaaaatgat ctcaggaaaa cctgtttggc
cctatgtatg 1200 gtacattaca tcttttcagt aattccactc aaatggagac
ttttaacaaa gcaactgttc 1260 tcaggggacc tattttctcc cttaaaattc
attatacaca tccctggttg atagcagtgt 1320 gtctggaggc agaaaccatt
cttgctttgg aaacaattac gtctgtgtta tactgagtag 1380 ggaagctcat
taattgtcga cacttacgtt cctgataatg ggatcagtgt gtaattcttg 1440
tttcgctcca gatttctaat accacaaaga ataaatcctt tcactctgat caattttgtt
1500 aacttctcac gtgtcttctc tacacccagg gcaccggtga atccaagtgt
cctctgatgg 1560 tcaaagttct agatgctgtc cgaggcagtc ctgccatcaa
tgtggccgtg catgtgttca 1620 gaaaggctgc tgatgacacc tgggagccat
ttgcctctgg gtaagttgcc aaagaaccct 1680 cccacaggac ttggttttat
cttcccgttt gcccctcact tggtagagag aggctcacat 1740 catctgctaa
agaatttaca agtagattga aaaacgtagg cagaggtcaa gtatgccctc 1800
tgaaggatgc cctctttttg ttttgcttag ctaggaagtg accaggaacc tgagcatcat
1860 ttaggggcag acagtagaga aaagaaggaa tcagaactcc tctcctctag
ctgtggtttg 1920 caaccctttt gggtcacaga acactttatg taggtgatga
aaagtaaaca ttctatgccc 1980 agaaaaaatg cacagataca cacacataca
aaatcatata tgtgatttta ggagtttcac 2040 agattccctg gtgtccctgg
gtaacaccaa agctaagtgt ccttgtctta gaattttagg 2100 aaaaggtata
atgtgtatta acccattaac aaaaggaaag gaattcagaa atattattaa 2160
ccaggcatct gtctgtagtt aatatggatc acccaaaacc caaggctttt gcctaatgaa
2220 cactttgggg cacctactgt gtgcaaggct gggggctgtc aagctcagtt
aaaaaaaaaa 2280 agatagaaga gatggatcca tgaggcaaag tacagcccca
ggctaatccc acgatcaccc 2340 gacttcatgt ccaagagtgg cttctcacct
tcattagcca gttcacaatt ttcatggagt 2400 ttttctacct gcactagcaa
aaacttcaag gaaaatacat attaataaat ctaagcaaag 2460 tgaccagaag
acagagcaat caggagaccc tttgcatcca gcagaagagg aactgctaag 2520
tatttacatc tccacagaga agaatttctg ttgggtttta attgaacccc aagaaccaca
2580 tgattcttca accattattg ggaagatcat tttcttaggt ctggttttaa
ctggcttttt 2640 atttgggaat tcatttatgt ttatataaaa tgccaagcat
aacatgaaaa gtggttacag 2700 gactattcta agggagagac agaatggaca
ccaaaaatat tccaatgttc ttgtgaatct 2760 tttccttgca ccaggacaaa
aaaaaaaaga agtgaaaaga agaaaggagg aggggcataa 2820 tcagagtcag
taaagacaac tgctattttt atctatcgta gctgttgcag tcaaatggga 2880
agcaatttcc aacattcaac tatggagctg gtacttacat ggaaatagaa gttgcctagt
2940 gtttgttgct ggcaaagagt tatcagagag gttaaatata taaaagggaa
aagagtcaga 3000 tacaggttct tcttcctact ttaggttttc cactgtgtgt
gcaaatgata ctccctggtg 3060 gtgtgcagat gcctcaaagc tatcctcaca
ccacaaggga gaggagcgag atcctgctgt 3120 cctggagaag tgcagagtta
gaacagctgt ggccacttgc atccaatcat caatcttgaa 3180 tcacagggac
tctttcttaa gtaaacatta tacctggccg ggcacggtgg ctcacgcctg 3240
taatcccagc actttgggat gccaaagtgg gcatatcatc tgaggtcagg agttcaagac
3300 cagcctggcc aacatggcaa aactccgtct ttatgaaaaa tacaaaaatt
agccaggcat 3360 ggtggcaggc gcctgtaatc ccagctaatt gggaggctga
ggctggagaa tcccttgaat 3420 ctaggaggca gaggttgcag tgagctgaga
tcgtgccatt gcactccagc ctgggtgaca 3480 agagtaaaac tctgtctcaa
aaaaaaaaaa ttatacctac attctcttct tatcagagaa 3540 aaaaatctac
agtgagcttt tcaaaaagtt tttacaaact ttttgccatt taatttcagt 3600
taggagtttt ccctacttct gacttagttg aggggaaatg ttcataacat gtttataaca
3660 tgtttatgtg tgttagttgg tgggggtgta ttactttgcc atgccatttg
tttcctccat 3720 gcgtaactta atccagactt tcacacctta taggaaaacc
agtgagtctg gagagctgca 3780 tgggctcaca actgaggagg aatttgtaga
agggatatac aaagtggaaa tagacaccaa 3840 atcttactgg aaggcacttg
gcatctcccc attccatgag catgcagagg tgagtataca 3900 gaccttcgag
ggttgttttg gttttggttt ttgcttttgg cattccagga aatgcacagt 3960
tttactcagt gtaccacaga aatgtcctaa ggaaggtgat gaatgaccaa aggttccctt
4020 tcctattata caagaaaaaa ttcacaacac tctgagaagc aaatttcttt
ttgactttga 4080 tgaaaatcca cttagtaaca tgacttgaac ttacatgaaa
ctactcatag tctattcatt 4140 ccactttata tgaatattga tgtatctgct
gttgaaataa tagtttatga ggcagccctc 4200 cagaccccac gtagagtgta
tgtaacaaga gatgcaccat tttatttctc gaaaacccgt 4260 aacattcttc
attccaaaac acatctggct tctcggaggt ctggacaagt gattcttggc 4320
aacacatacc tatagagaca ataaaatcaa agtaataatg gcaacacaat agataacatt
4380 taccaagcat acaccatgtg gcagacacaa ttataagtgt tttccatatt
taacctactt 4440 aatcctcagg aataagccac tgaggtcagt cctattatta
tccccatctt atagatgaag 4500 aaaatgaggc accaggaagt caaataactt
gtcaaaggtc acaagactag gaaatacaca 4560 agtagaaatg tttacaatta
aggcccaggc tgggtttgcc ctcagttctg ctatgcctcg 4620 cattatgccc
caggaaactt tttcccttgt gaaagccaag cttaaaaaaa gaaaagccac 4680
atttgtaacg tgctctgttc ccctgcctat ggtgaggatc ttcaaacagt tatacatgga
4740 cccagtcccc ctgccttctc cttaatttct taagtcattt gaaacagatg
gctgtcatgg 4800 aaatagaatc cagacatgtt ggtcagagtt aaagatcaac
taattccatc aaaaatagct 4860 cggcatgaaa gggaactatt ctctggctta
gtcatggatg agactttcaa ttgctataaa 4920 gtggttcctt tattagacaa
tgttaccagg gaaacaacag gggtttgttt gacttctggg 4980 gcccacaagt
caacaagaga gccccatcta ccaaggagca tgtccctgac tacccctcag 5040
ccagcagcaa gacatggacc ccagtcaggg caggagcagg gtttcggcgg cgcccagcac
5100 aagacattgc ccctagagtc tcagccccta ccctcgagta atagatctgc
ctacctgaga 5160 ctgttgtttg cccaagagct gggtctcagc ctgatgggaa
ccatataaaa aggttcactg 5220 acatactgcc cacatgttgt tctctttcat
tagatcttag cttccttgtc tgctcttcat 5280 tcttgcagta ttcattcaac
aaacattaaa aaaaaaaaaa agcattctat gtgtggaaca 5340 ctctgctaga
tgctgtggat ttagaaatga aaatacatcc cgacccttgg aatggaaggg 5400
aaaggactga agtaagacag attaagcagg accgtcagcc cagcttgaag cccagataaa
5460 tacggagaac aagagagagc gagtagtgag agatgagtcc caatgcctca
ctttggtgac 5520 gggtgcgtgg tgggcttcat gcagcttctt ctgataaatg
cctccttcag aactggtcaa 5580 ctctaccttg gccagtgacc caggtggtca
tagtagattt accaagggaa aatggaaact 5640 tttattagga gctcttaggc
ctcttcactt catggatttt tttttccttt ttttttgaga 5700 tggagttttg
ccctgtcacc caggctggaa tgcagtggtg caatctcagc tcactgcaac 5760
ctccgcctcc caggttcaag caattctcct gcctcagcct cccgagtagc tgggactaca
5820 ggtgtgcgcc accacaccag gctaattttt gtattttttg taaagacagg
ttttcaccac 5880 gttggccagg ctggtctgaa ctccagacct caggtgattc
acctgtctca gcctcccaaa 5940 gtgctgggat tacaggtgtg agccaccgtg
cccggctact tcatggattt ttgattacag 6000 attatgcctc ttacaatttt
taagaagaat caagtgggct gaaggtcaat gtcaccataa 6060 gacaaaagac
atttttatta gttgattcta gggaattggc cttaagggga gccctttctt 6120
cctaagagat tcttaggtga ttctcacttc ctcttgcccc agtattattt ttgtttttgg
6180 tatggctcac tcagatcctt ttttcctcct atccctaagt aatccgggtt
tctttttccc 6240 atatttagaa caaaatgtat ttatgcagag tgtgtccaaa
cctcaaccca aggcctgtat 6300 acaaaataaa tcaaattaaa cacatcttta
ctgtcttcta cctctttcct gacctcaata 6360 tatcccaact tgcctcactc
tgagaaccaa ggctgtccca gcacctgagt cgcagatatt 6420 ctactgattt
gacagaactg tgtgactatc tggaacagca ttttgatcca caatttgccc 6480
agttacaaag cttaaatgag ctctagtgca tgcatatata tttcaaaatt ccaccatgat
6540 cttccacact ctgtattgta aatagagccc tgtaatgctt ttacttcgta
tttcattgct 6600 tgttatacat aaaaatatac ttttcttctt catgttagaa
aatgcaaaga ataggagggt 6660 gggggaatct ctgggcttgg agacaggaga
cttgccttcc tactatggtt ccatcagaat 6720 gtagactggg acaatacaat
aattcaagtc tggtttgctc atctgtaaat tgggaagaat 6780 gtttccagct
ccagaatgct aaatctctaa gtctgtggtt ggcagccact attgcagcag 6840
ctcttcaatg actcaatgca gttttgcatt ctccctacct tttttttcta aaaccaataa
6900 aatagataca gcctttaggc tttctgggat ttcccttagt caagctaggg
tcatcctgac 6960 tttcggcgtg aatttgcaaa acaagacctg actctgtact
cctgctctaa ggactgtgca 7020 tggttccaaa ggcttagctt gccagcatat
ttgagctttt tccttctgtt caaactgttc 7080 caaaatataa aagaataaaa
ttaattaagt tggcactgga cttccggtgg tcagtcatgt 7140 gtgtcatctg
tcacgttttt cgggctctgg tggaaatgga tctgtctgtc ttctctcata 7200
ggtggtattc acagccaacg actccggccc ccgccgctac accattgccg ccctgctgag
7260 cccctactcc tattccacca cggctgtcgt caccaatccc aaggaatgag
ggacttctcc 7320 tccagtggac ctgaaggacg agggatggga tttcatgtaa
ccaagagtat tccattttta 7380 ctaaagcagt gttttcacct catatgctat
gttagaagtc caggcagaga caataaaaca 7440 ttcctgtgaa aggcactttt
cattccactt taacttgatt ttttaaattc ccttattgtc 7500 ccttccaaaa
aaaagagaat caaaatttta caaagaatca aaggaattct agaaagtatc 7560
tgggcagaac gctaggagag atccaaattt ccattgtctt gcaagcaaag cacgtattaa
7620 atatgatctg cagccattaa aaagacacat tctgtaaatg agagagcctt
attttcctgt 7680 aaccttcagc aaatagcaaa agacacattc caagggccca
cttctttact gtgggcattt 7740 cttttttttt ctttttttct tttttccttt
tttgagacaa agtctcactc tgttgcccag 7800 gctagaatgc agtggtgtaa
tctcagctca ctgcaacctc tgcttcctgg gttcaagcga 7860 ttctcctgcc
tcagcctccc aagtaactgg gattacaggc gcatgccacc acgcctagct 7920
catttttgta tttttagtag agatgggatt ttgccatgtt ggctaggctg gtctacgaac
7980 tcctgacctc aggtgatcca cctgcctcag cctcccaaag tgctgggatt
acaggcatga 8040 gccactacac ccgg 8054 <210> SEQ ID NO 3
<400> SEQUENCE: 3 000 <210> SEQ ID NO 4 <211>
LENGTH: 10001 <212> TYPE: DNA <213> ORGANISM: Macaca
mulatta <400> SEQUENCE: 4 gactctattc ctagttatgg tctcaactac
atttgctcat tgctgtgagg ggtgagccca 60 cctcccggag tcctctcctg
cacattccta tgttcctgaa aggactttcc atcccttcca 120 ctactccctg
aaaactcctg tgcttcatga tttcttgttg aattttttct aatctgactc 180
tatcagttat gggaatgttc cctcaactct tagtgctcca gactggactc gctcttggca
240 tgtatttgtc caaaatattt gtctgctcta tgtcttctac atatttgtct
tataaggaac 300 aaacacctga tttgtttatc catgaacaaa gccacacatg
ctagtgcaca cgcacataca 360 cacacacaca tacacacaga ggattctgta
cgtgattaat gaatcatcaa atcatcataa 420 tttctggact tgtattaata
cgtcggccag gaggaaaaga atccgttgtc aatcatggct 480 tctggttctc
acggtcatct ctactttctt ccagcaagtt tgattctgtc aaacaccagc 540
tggcagcttt gttccggcat gcccagtgca ggagagtcag taaagaagat ttcattctct
600 gtatttcagc gtcgtcaatg ccaggttgaa atacgctatt ctggcccagc
tcagtggctc 660 acacgtgtaa tcccagcact ttggaaggcc aaggcaggca
gatcgcttga gcccaggaat 720 tcgagaccag cctgggcaag aggctgaggt
gggaggatga cctgagcccg ggaggtcaag 780 gctgcagcca gctgtgatca
tgtcactgca ctcaagccag ggcgtcggag tgagaccgtg 840 tcaaaaaaaa
aggaaggaaa gaaggaagga aggaaggaag gaaggaagga aggaaggaag 900
gaaggaagga aggaagggag ggagggaggg agggagggag gaaggaagga aggaagtatg
960 taaggaagga aggaaggaag gaagggaggg acggagggcg ggagggaagg
aagatgccat 1020 tcttagattg aagtggacct tatgtgggca gaacacacac
acacacacac acattgtgga 1080 gaaattgctg actaagcaaa gcttccaaat
gactgagttt ggctaaaacg taggctttta 1140 aaaatgtgag cactgccaag
ggtttttcct tgttgaccca tggatccatc aagtgcaaac 1200 attttctaat
gcactatatt taagcctgtg cagccaaatg tcattcaaca tgaaatgcat 1260
tattacaact tgcatctgtc taaaatcttg catcaaaaat gaaagacaaa aatgtataaa
1320 aatggaaaac atgcatagaa atatgtgagg gaggaaaaaa atatccccag
gaatgttagt 1380 gcacggagtc acacagggag aagactattt ttgttttgtt
ttgattgttt tgttctggtg 1440 acctcactgg tcaaatgacc tattaagaat
attttgtaga gctaatgttc cgatgctcta 1500 atctctctag acaaggttca
tatttgtatg ggttacttat tctctctttg ttgactaagt 1560 caataatcag
aatcagcagg tttggagtca gattggcagg gataagcagc ctagctcagg 1620
agaagtgggt ataaaagccc caggctggga gcagccatca cagaagtcca ctcgttcttg
1680 gcaggatggc ttctcatcgt ctgctcctcc tctgcctcgc tggactggta
tttgtgtctg 1740 aggctggtcc tacggtgagt gtttctgtga catctcgctc
ctacatttaa aatgcacgct 1800 aaatgaggta ggagtgactc cttccagctt
tgccaagcag cttttgttac tagggcaagg 1860 gtacccagca tctattttta
atatcattta ttcaaacttc aaaaagaatg aagttccact 1920 gagcttactg
agctgggact tgaactctgg ggattctgcc tcattgcttt ggtgcattgg 1980
gtttgtaatg tctggtatct ccacttcctc agatcgatga tagagataaa gatatgatat
2040 taaggaagct gttaatcctg aattttcaga aaagtatccc tttataaaat
gtatttgggg 2100 ggcaaactgc atgagattat attctggccc tatagctaat
caaaatgtat ttaccgatta 2160 atctttaaaa ggcttagtga acaatatttt
agtcagatat ctaattctta aatactctag 2220 aaggattaac taatctataa
aatgggtctg gatatagtct gacataattt tataacaacc 2280 ggtaagaggg
agtgactaga gcaacaacta aaatcatctc aggaaaaact gttttggtcc 2340
tatgtatggt acgttacatc ttttcagtaa ttccattcaa atggagaagt ttaacaaggc
2400 aactgttctc agggggccta ttctctccct taaaattcat tatacacatc
cctggttgat 2460 agcagtgtgt ctggaggcag aaaccattct tgctttggaa
acaattatgt ctgtgttata 2520 ctgagtaggg aagctcatta actgtcaaca
cttatgttct tcataatgga atcagtgtgt 2580 aattcttgtt ttgttccaga
tttctaacac cataaagaat aaatcctttc actctgatca 2640 atgttgttaa
cttctcactt gtcttctcta tacccagggc gttgatgaat ccaagtgtcc 2700
tctgatggtc aaagttctag atgccgtccg aggcagtcct gccgtcaatg tggctgtgaa
2760 cgtgttcaaa aaggctgctg atgagacctg ggcgccattt gcctctgggt
aagaaccctc 2820 ccacgggact tggttttatc ttcccattgg cccctcagtt
ggtagacaga ggctcacatc 2880 atctgctaaa gaatttacaa gtagattgaa
aaacgtgagc agaggtcaag tatgccctct 2940 gaagatgccc tctttttgtt
ttgcttagct aggaagggac caggaacctg agcatcattt 3000 aggggcagac
agtagagaaa agaaggaatc agaactcttc ccctctagct gtggtgtgca 3060
acccttttgg gtcacagacc actttatgta ggtgataaaa actaaagatt ctatgcccag
3120 aaaaaatgta cagatacaca cacacaaaac catatatgtg attttaggag
tttcacagat 3180 tccctggtgt ccctgggtaa caccaaaggt gagagtcctc
gtcttagaat tttaggaaag 3240 aggtgcaatg tgtattaacc cactaacgaa
aggaaaggga ttcagaaata ttattgacta 3300 ggcatctgtc tgtagttcat
ttggatcacc ccaaacccag ggcttttgcc taatgaacac 3360 tttggggcac
ctactgtgtg cagggctgga ggctgtcaag ctcagttaaa acaaatgtaa 3420
aaaaagacag aagaaatgga tccatgaggc aaagtacagc cccagactaa tcccatgatc
3480 acccaacttc atgtgcaaga gtgacttcta accttcatga gccagtttac
aattttcatg 3540 gagtttttct acctacacta caaaaactcc aaggaaaata
tatattaata aacctaagcg 3600 aattgaccga aagacagagc aataaggaga
cccttcgcac ccagcagaag aggaactgtt 3660 aagtacttac ttctcctcag
agaagaattt ctgttgtatt ttaattgaac cccaagaacc 3720 acacgattct
tcaaccatta ttggtaagat cattttctta ggtctggttt taactgactt 3780
tttatttggt aattcattta tgtttatata aaatgccaag cataacatga aaagtggtta
3840 caggactatt ctaagggaga gagaaaatgg ataccaaaaa tattccaatg
ttcttatgaa 3900 tcttttccct tgcgtcagga caaaaaaaaa aaaaatgtaa
agaagaaagg aggagatgca 3960 caatcagagt cagtaaagac aactgctatt
tttatctgtc atagctgttg cagtctaatg 4020 ggaagcaatt tccaacattc
aactatggag ctggtactta catggaaata gaagttgcct 4080 agtgtttgtt
gctggcaaag agttatcaga gaggttaaat atataaaagg gaagagtcag 4140
atacaggttc ttcttcctac tttaggtttt ccactgtgtg tgcaaatgac cctccctggt
4200 ggtgtgcaga tgctctgaaa ggtatcctca caccacaagg cagaggagcg
agaccctgct 4260 gtcctggaga agtgcagagt tagaacagct gtggccactt
ccagggatgg tcacaacatc 4320 ccatctaatc atcaatcttg aacaacaagg
actctttctt aagaaaacat tatacccagc 4380
cgggcgcggt ggctcacacc tgtaaatctc agcgctttgg gaggctgaaa tgggcatatc
4440 atctgaggtc gagagctcaa gaccaacctg gccaacatgg caaaactccg
tctctatgaa 4500 aaatacaaaa attagccagg catggtggca ggcacctgta
atcccggcta ctcgggaggc 4560 tgagactgga gaatcccttg aacctggagg
cagaggttgc agtgagctga gatcacgcca 4620 ctgcactcca gcctgggtga
ctagagtaaa actctgtctc aaaaataaaa aaaaatttaa 4680 aaaattatac
ctacattctc ttcttatcag agaaaaatat ctacagtgag cttttcaaaa 4740
gtttttacaa actttttgcc atttaatttc agacagttat gagttttccc tacttctgac
4800 ttagttgagg ggaaatgtat ataacacatt tatgtgtgtt gtgtatataa
cacatataac 4860 acgtttatgt gtgttggtgg gggtattact ttgccatgcc
atttgtttcc tccatgccta 4920 acttaaccca gactttcaca ccttatagga
aaaccagtga gtctggagag ctgcatgggc 4980 tcacaactga ggaggaattt
gtagaaggga tatacaaagt ggaaatagac accaaatctt 5040 actggaagtc
acttggcatc tccccattcc atgagcatgc agaggtgagt atataaacct 5100
tcgagggttg ttttggtttt ggtttttgct tttggcatcc caggaaatgc acagttttac
5160 ttagcatacc acagaaatgt cctaaagaag gtgatgaatg accaaaggtt
ccctctcctc 5220 ttatacaaga acaaattcac aacactctga gaagcacatt
tctttttgac tttgaggaaa 5280 acccatttag taacatgact tgaacttaca
tgacactatt catagtctac tcattccatt 5340 ttatatgaat attgatgtat
ttgccgttga aataacatgt ttatgaggca gacctccaga 5400 ccccacgtag
agtgtatgaa acaagagatg caccatttta tttctctaaa acctgtaaca 5460
ttcttcattc caaaacacat ctggctcctc ggaggtttgg acaagtgatt cttggcaaca
5520 catacataga gagacaataa aatcaaagta ataatggcaa cacaacagat
aacatttact 5580 aagcatacac catgtggcag acacaattat aagtgttttc
tatatttaac ctacttcatc 5640 ctcagggaca agccactgag gtcagtccta
ttattatccc catctcatag atgaagcaag 5700 tgaggcacca ggaagtcaaa
taacttgtca aaggtcacaa ggctaggaaa cacacaagta 5760 gagatgttta
caaacaaggc ccaggctggg tttgccctca attctgctct gcctcgcatt 5820
gcgacccagg aaatttgttc ccctgtgaaa agccaagctt aaaaaaagaa aagccacatt
5880 tgtaacgtgc tctgttcccc tgcctatggt gaggatcttc gaacagttat
acagctccct 5940 gtcttcccct tgtcttaatt tcttcagtca tttgaaacag
atggctgtca tggaaataga 6000 atccagacat gttggtcaga gttaaagatc
aactaattcc atcaaaaata gctcagcatg 6060 aaagggaact attctctggc
ttagtcatgg atgagacttt caattgctat aaagtggttc 6120 ctttattagg
caatgttacc agggaaacaa taggggcttg tttgacttct ggggcccaca 6180
agtcaccaag ggagccccat ctaccaagaa gcatgtccct gactaccccg cagccaggca
6240 gcaagacacg gacgccggtc agggcagcag cagggtttca gtggtgccca
gcacaagaca 6300 ttgctcctag agtctcagcc cctacccttg agtagtagaa
ctgcctacct gagaccgttg 6360 tttgcctaag acctgggtct cagcctgatg
ggaaccatct aaaaagttca tcgccatact 6420 gcccacgtgt tgttctcttt
cattagatct cagcttcctt gactgctctt cactcttgtt 6480 tattcattca
acaaacattt aaaaaataaa agcattctat gtgtggaaca ctctgataga 6540
ggctggagat tcagaaatga aaatacatcc ctacccttgg aatggagggg aaaggactga
6600 agtaagacag actaggcagg cccgtcagcc cagcttgaag cccagataaa
tatggagaac 6660 aagagtgagt gagtagtgag agatgagtcc cagtgcctca
ctttggtgac tggtgcatgg 6720 tgggcttcat gcagcttctt ctgataaatg
cctccttcag aaccggtcac ctctaccttg 6780 gccagtgacc caagtggtca
tattagattt accaagggaa attggaaact tttattagga 6840 gctcttaggc
ctcttcactt catggaattt tttctttttt cttttttttt tgagatggag 6900
ttttgctctg tcacccaggc tggaatgcag tggtgcaatc ttggctcact gcaaccttta
6960 cttcccaggt tcaagcaatt cttctgcctc agcctcccga gtagctggga
ctacaggtgc 7020 acgccaccat acccagctaa gttttgtatt tttcatagag
acagggtttc gccatgttgg 7080 ccaggctggt ctcgaactcc agacctcagg
tgattcaccc acctcagcct cccaaagtgc 7140 tggcattaca ggtgtgagcc
agtacaccca gctacttcat ggatttttga tcacagatta 7200 tgcctcttat
aatttttaag aagaatcaag tgggctgaag gtcaatgtca ccataagata 7260
aaagatattt ttattagttg attctaggga gttggcctta aggggagccc tttcttctta
7320 agagattctt ggccgggcgc ggtggctcaa gcctgtaatc ccagcacttt
gggaggccga 7380 gacgggtgga tcatgaggtc aggagatcga gaccatcctg
gtaacacggt gaaaccccgt 7440 ctctactaaa aaatacaaaa aaactagccg
ggtgagttgg cgggcgcctg tagtcccagc 7500 tactcgggag gctgaggcag
gagaatggcg taaacccggg aggcggagct tgcagtgagc 7560 tgagatctgg
ccactgcact ccagcccggg tgacagagcg agactccgtc tcaaaaaaaa 7620
aaaaaaaaaa aaagagattc ttaggtgatt ctcacttcct cttgccccaa tattattttt
7680 gtttttggta tggctcactc agctcctttt tccctcctat ccctaagtaa
tccgggtttc 7740 ttttccccat atttggaaca aaatgtattt atgcagagtg
tgtccaaaac tcaacccaag 7800 gcccgtatac aaaataaatc aaattaaaca
catctttact gtcttctacc tctttcctga 7860 cctcaattta tcccaacttg
cctcactctg agaatcaagc ctgtcccagc acatgagttg 7920 cagatactct
actgaatttg acagaactgt gtgactatct ggaacagcat tttgatccac 7980
aatttgccca gttacaaagc ttaaatgaag tctagtgcat gcatgtatat ttcaaaattc
8040 caccatgctc ttccacactc tgtattgtaa atagagccct gaaatgcttt
tggttcatat 8100 ttcattgctt gctatacata aaaatatact ttttcttctt
catgttagaa aatgcaaaga 8160 atagtggggt gggggaatct ctgggcttgg
agacaggaga cttaccttcc tactgtggtt 8220 ccatcagtat gtagactggg
gcaatacaat aattcaagtc tggtttgctc atctgtaaaa 8280 tgggaagaat
gtttccagct ccagaatgct aagtctctaa gtctgtggtt ggcagccact 8340
attgcagcag cttttcaatg actcagtgca tttttccatt ctccccacct tttttttttc
8400 taaaaccaac aaaatagata cagcctttag gctctctggg atttccctta
gtcaagctag 8460 ggccatcctg acttttgatg tgaatttgca aaacaagacc
tggttctgta ctcctgctct 8520 aagggctgtg catggttcca aaggcttggc
ttgccagtgt atttgagctt tttccttctg 8580 ttcaaacttc aaaatataaa
agaataaaat taattaagtt ggcactggac ttccggtggt 8640 cagtcatgtg
tgtcatctgt acggttttcg ggctctggtg gaaatggata ctgtctgtct 8700
tctctcatag gtggtattca cagccaacga ttccggcccc cgccactaca ccatcgccgc
8760 cctgctgagc ccctactcct attccaccac ggctgtcgtc accaatccca
aggaatgagg 8820 gacttctcca gaggatctga aggacgaggg atgggatttc
atgtaaccaa gagtattcca 8880 tttttactaa agcagtgttt tcacctcata
agctatgtta ggagtccagg cagagacaat 8940 aaaacattcc tgtgaaaggc
acttttcatt ccattttaac ttgatttttt aaattccctt 9000 attgtccctt
ccaaaaaaac aagaatcaaa attctacaaa gaagcaaagg aattctagaa 9060
cgtatctggg cagaacgcta ggagagatcc aaatttccaa tttattgcaa gcaaagcaca
9120 tattaaatat gatctgcagt catcaaaaag acacattctg taaatgagaa
agccttattt 9180 tcctgtaacc ttcagtgaat agcaaaagac acattctaag
ggcccacttc tttactgtgg 9240 gcatttcttt tcttttcttt tttttttttt
cttttctttt ccttttttga gacaaagtct 9300 cactctgtcg cccaggctag
aatgcagtgg tgtgatctca gctcactgca acctctgctt 9360 ccgggttcaa
gcgattctcc tgcctcagcc tcccaagcag ctgggattac aggcgcccgc 9420
caccacacct ggctaatttt tctacttgta gtagagatgg ggtttcgcca tgttggctag
9480 gctggcctcg aactcctgac ctcaggtgat ccacctgcct cagcctccca
aagtgctggg 9540 attacaggca tgagccacta cacccggccc ctactctggg
catttctttg atttaagaga 9600 agggcagctc caacaagaca cacctgcaga
gactcaggcc atccgatcag ttcaggctag 9660 atccacgctg caatcagcca
ggtcagggac aaaccaaaga accccacaca cccaatttac 9720 ttaggctgat
ccaaaatcca tgtatggaga actcacatgc agcaggcact attttaggtg 9780
atctgaacat aaagaatagg acccagtacc tgcatttatt taaagaactc acaatctttt
9840 gaaaagataa ctgtttcatc atggtttggc aggaggctat ggtacaaggc
acagcaaagg 9900 taagaaggag aaaaaaccaa caccctagag aaatcagaaa
atgactctga ataggtgtca 9960 cttaatctga gtgttggtaa tttgtcagat
agacaaggga a 10001 <210> SEQ ID NO 5 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 5 ccctgctgag cccctactc 19 <210> SEQ ID
NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 6 tccctcattc
cttgggattg 20 <210> SEQ ID NO 7 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Probe
<400> SEQUENCE: 7 attccaccac ggctgtcgtc a 21 <210> SEQ
ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
8 actggttttc ccagaggcaa 20 <210> SEQ ID NO 9 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 9 gactcactgg
ttttcccaga 20 <210> SEQ ID NO 10
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 10
tgaataccac ctctgcatgc 20 <210> SEQ ID NO 11 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 11 ccgtggtgga
ataggagtag 20 <210> SEQ ID NO 12 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 12 agccgtggtg gaataggagt 20
<210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 13 cgacagccgt ggtggaatag 20 <210> SEQ
ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
14 ttggtgacga cagccgtggt 20 <210> SEQ ID NO 15 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 15 gattggtgac
gacagccgtg 20 <210> SEQ ID NO 16 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 16 gggattggtg acgacagccg 20
<210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 17 tgggattggt gacgacagcc 20 <210> SEQ
ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
18 attccttggg attggtgacg 20 <210> SEQ ID NO 19 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 19 cattccttgg
gattggtgac 20 <210> SEQ ID NO 20 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 20 tcattccttg ggattggtga 20
<210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 21 agaagtccct cattccttgg 20 <210> SEQ
ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
22 gtccttcagg tccactggag 20 <210> SEQ ID NO 23 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 23 catccctcgt
ccttcaggtc 20 <210> SEQ ID NO 24 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 24 tacatgaaat cccatccctc 20
<210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 25 cttggttaca tgaaatccca 20 <210> SEQ
ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
26 aatactcttg gttacatgaa 20 <210> SEQ ID NO 27 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 27 ttagtaaaaa
tggaatactc 20 <210> SEQ ID NO 28 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 28 actgctttag taaaaatgga 20
<210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 29 tgaaaacact gctttagtaa 20 <210> SEQ
ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
30 tatgaggtga aaacactgct 20
<210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 31 tggacttcta acatagcata 20 <210> SEQ
ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
32 tctctgcctg gacttctaac 20 <210> SEQ ID NO 33 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 33 ttattgtctc
tgcctggact 20 <210> SEQ ID NO 34 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 34 tgcctttcac aggaatgttt 20
<210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 35 gtgcctttca caggaatgtt 20 <210> SEQ
ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
36 cagaggagga gcagacgatg 20 <210> SEQ ID NO 37 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 37 tctagaactt
tgaccatcag 20 <210> SEQ ID NO 38 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 38 ttttcccaga ggcaaatggc 20
<210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 39 tccagactca ctggttttcc 20 <210> SEQ
ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
40 tatcccttct acaaattcct 20 <210> SEQ ID NO 41 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 41 atttccactt
tgtatatccc 20 <210> SEQ ID NO 42 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 42 tggtgtctat ttccactttg 20
<210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 43 cagtaagatt tggtgtctat 20 <210> SEQ
ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
44 cttccagtaa gatttggtgt 20 <210> SEQ ID NO 45 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 45 ccacctctgc
atgctcatgg 20 <210> SEQ ID NO 46 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 46 tgtgaatacc acctctgcat 20
<210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 47 gctgtgaata ccacctctgc 20 <210> SEQ
ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
48 cgttggctgt gaataccacc 20 <210> SEQ ID NO 49 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 49 cagccgtggt
ggaataggag 20 <210> SEQ ID NO 50 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 50 gacagccgtg gtggaatagg 20
<210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 51 acgacagccg tggtggaata 20
<210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 52 gacgacagcc gtggtggaat 20 <210> SEQ
ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
53 tgacgacagc cgtggtggaa 20 <210> SEQ ID NO 54 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 54 gtgacgacag
ccgtggtgga 20 <210> SEQ ID NO 55 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 55 ggtgacgaca gccgtggtgg 20
<210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 56 tggtgacgac agccgtggtg 20 <210> SEQ
ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
57 attggtgacg acagccgtgg 20 <210> SEQ ID NO 58 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 58 ggattggtga
cgacagccgt 20 <210> SEQ ID NO 59 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 59 ttgggattgg tgacgacagc 20
<210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 60 cttgggattg gtgacgacag 20 <210> SEQ
ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
61 ccttgggatt ggtgacgaca 20 <210> SEQ ID NO 62 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 62 tccttgggat
tggtgacgac 20 <210> SEQ ID NO 63 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 63 ttccttggga ttggtgacga 20
<210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 64 ctcattcctt gggattggtg 20 <210> SEQ
ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
65 cctcattcct tgggattggt 20 <210> SEQ ID NO 66 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 66 ccctcattcc
ttgggattgg 20 <210> SEQ ID NO 67 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 67 tccctcattc cttgggattg 20
<210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 68 gtccctcatt ccttgggatt 20 <210> SEQ
ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
69 agtccctcat tccttgggat 20 <210> SEQ ID NO 70 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 70 aagtccctca
ttccttggga 20 <210> SEQ ID NO 71 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 71 gaagtccctc attccttggg 20
<210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 72 gagaagtccc tcattccttg 20
<210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 73 ggagaagtcc ctcattcctt 20 <210> SEQ
ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
74 acatgaaatc ccatccctcg 20 <210> SEQ ID NO 75 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 75 ttacatgaaa
tcccatccct 20 <210> SEQ ID NO 76 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 76 gttacatgaa atcccatccc 20
<210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 77 ggttacatga aatcccatcc 20 <210> SEQ
ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
78 tggttacatg aaatcccatc 20 <210> SEQ ID NO 79 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 79 ttggttacat
gaaatcccat 20 <210> SEQ ID NO 80 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 80 tcttggttac atgaaatccc 20
<210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 81 ctcttggtta catgaaatcc 20 <210> SEQ
ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
82 actcttggtt acatgaaatc 20 <210> SEQ ID NO 83 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 83 tactcttggt
tacatgaaat 20 <210> SEQ ID NO 84 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 84 atactcttgg ttacatgaaa 20
<210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 85 gaatactctt ggttacatga 20 <210> SEQ
ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
86 ggaatactct tggttacatg 20 <210> SEQ ID NO 87 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 87 tggaatactc
ttggttacat 20 <210> SEQ ID NO 88 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 88 atggaatact cttggttaca 20
<210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 89 aatggaatac tcttggttac 20 <210> SEQ
ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
90 aaatggaata ctcttggtta 20 <210> SEQ ID NO 91 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 91 aaaatggaat
actcttggtt 20 <210> SEQ ID NO 92 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 92 aaaaatggaa tactcttggt 20
<210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 93
taaaaatgga atactcttgg 20 <210> SEQ ID NO 94 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 94 gtaaaaatgg
aatactcttg 20 <210> SEQ ID NO 95 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 95 agtaaaaatg gaatactctt 20
<210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 96 tagtaaaaat ggaatactct 20 <210> SEQ
ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
97 tttagtaaaa atggaatact 20 <210> SEQ ID NO 98 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 98 ctttagtaaa
aatggaatac 20 <210> SEQ ID NO 99 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 99 gctttagtaa aaatggaata 20
<210> SEQ ID NO 100 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 100 tgctttagta aaaatggaat 20 <210> SEQ
ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
101 ctgctttagt aaaaatggaa 20 <210> SEQ ID NO 102 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 102 cactgcttta
gtaaaaatgg 20 <210> SEQ ID NO 103 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 103 acactgcttt agtaaaaatg 20
<210> SEQ ID NO 104 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 104 aacactgctt tagtaaaaat 20 <210> SEQ
ID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
105 aaacactgct ttagtaaaaa 20 <210> SEQ ID NO 106 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 106 aaaacactgc
tttagtaaaa 20 <210> SEQ ID NO 107 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 107 gaaaacactg ctttagtaaa 20
<210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 108 gtgaaaacac tgctttagta 20 <210> SEQ
ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
109 ggtgaaaaca ctgctttagt 20 <210> SEQ ID NO 110 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 110 aggtgaaaac
actgctttag 20 <210> SEQ ID NO 111 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 111 gaggtgaaaa cactgcttta 20
<210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 112 tgaggtgaaa acactgcttt 20 <210> SEQ
ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
113 atgaggtgaa aacactgctt 20 <210> SEQ ID NO 114 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 114
tttattgtct ctgcctggac 20 <210> SEQ ID NO 115 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 115 ttttattgtc
tctgcctgga 20 <210> SEQ ID NO 116 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 116 gttttattgt ctctgcctgg 20
<210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 117 tgttttattg tctctgcctg 20 <210> SEQ
ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
118 atgttttatt gtctctgcct 20 <210> SEQ ID NO 119 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 119 aatgttttat
tgtctctgcc 20 <210> SEQ ID NO 120 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 120 gaatgtttta ttgtctctgc 20
<210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 121 ggaatgtttt attgtctctg 20 <210> SEQ
ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
122 aggaatgttt tattgtctct 20 <210> SEQ ID NO 123 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 123 caggaatgtt
ttattgtctc 20 <210> SEQ ID NO 124 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 124 acaggaatgt tttattgtct 20
<210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 125 gatgtcacag aaacactcac 20 <210> SEQ
ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
126 gcaaagctgg aaggagtcac 20 <210> SEQ ID NO 127 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 127 gaacttcatt
ctttttgaag 20 <210> SEQ ID NO 128 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 128 agcttcctta atatcatatc 20
<210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 129 tatagggcca gaatataatc 20 <210> SEQ
ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
130 actaagcctt ttaaagatta 20 <210> SEQ ID NO 131 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 131 tggaattact
gaaaagatgt 20 <210> SEQ ID NO 132 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 132 accagggatg tgtataatga 20
<210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 133 tccctactca gtataacaca 20 <210> SEQ
ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
134 gatcagagtg aaaggattta 20 <210> SEQ ID NO 135 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide
<400> SEQUENCE: 135 gggaagataa aaccaagtcc 20 <210> SEQ
ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
136 taaattcttt agcagatgat 20 <210> SEQ ID NO 137 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 137 aatgatgctc
aggttcctgg 20 <210> SEQ ID NO 138 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 138 ttggtgttac ccagggacac 20
<210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 139 aaagtgttca ttaggcaaaa 20 <210> SEQ
ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
140 ggcattttat ataaacataa 20 <210> SEQ ID NO 141 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 141 aagaacattg
gaatattttt 20 <210> SEQ ID NO 142 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 142 gttggaaatt gcttcccatt 20
<210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 143 agtggaaaac ctaaagtagg 20 <210> SEQ
ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
144 ttcccctcaa ctaagtcaga 20 <210> SEQ ID NO 145 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 145 cctataaggt
gtgaaagtct 20 <210> SEQ ID NO 146 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 146 tgtaagttca agtcatgtta 20
<210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 147 gtgttgccaa gaatcacttg 20 <210> SEQ
ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
148 aaaacactta taattgtgtc 20 <210> SEQ ID NO 149 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 149 ctttgacaag
ttatttgact 20 <210> SEQ ID NO 150 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 150 atccatgact aagccagaga 20
<210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 151 atggttccca tcaggctgag 20 <210> SEQ
ID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
152 gcatttatca gaagaagctg 20 <210> SEQ ID NO 153 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 153 ttgaccttca
gcccacttga 20 <210> SEQ ID NO 154 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 154 aggaagtgag aatcacctaa 20
<210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 155 agaagacagt aaagatgtgt 20 <210> SEQ
ID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 156 aaattgtgga tcaaaatgct 20 <210> SEQ
ID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
157 aaccagactt gaattattgt 20 <210> SEQ ID NO 158 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 158 agtggctgcc
aaccacagac 20 <210> SEQ ID NO 159 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 159 ggaagtccag tgccaactta 20
<210> SEQ ID NO 160 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 160 atccatttcc accagagccc 20 <210> SEQ
ID NO 161 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer <400> SEQUENCE: 161 cttgctggac
tggtatttgt gtct 24 <210> SEQ ID NO 162 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
<400> SEQUENCE: 162 agaactttga ccatcagagg acact 25
<210> SEQ ID NO 163 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Probe <400> SEQUENCE: 163
ccctacgggc accggtgaat cc 22 <210> SEQ ID NO 164 <211>
LENGTH: 612 <212> TYPE: DNA <213> ORGANISM: Macaca
fascicularis <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (27)..(470) <400> SEQUENCE: 164
acagaagtcc actcgttctt ggcagg atg gct tct cat cgt ctg ctc ctt ctc 53
Met Ala Ser His Arg Leu Leu Leu Leu 1 5 tgc ctc gct gga ctg gta ttt
gtg tct gaa gct ggt cct acg ggc gtt 101 Cys Leu Ala Gly Leu Val Phe
Val Ser Glu Ala Gly Pro Thr Gly Val 10 15 20 25 gat gaa tcc aag tgt
cct ctg atg gtc aaa gtt cta gat gcc gtc cga 149 Asp Glu Ser Lys Cys
Pro Leu Met Val Lys Val Leu Asp Ala Val Arg 30 35 40 ggc agt cct
gcc gtc aat gtg gct gtg aac gtg ttc aaa aag gct gct 197 Gly Ser Pro
Ala Val Asn Val Ala Val Asn Val Phe Lys Lys Ala Ala 45 50 55 gat
gag acc tgg gcg cca ttt gcc tct ggg aaa acc agt gag tct gga 245 Asp
Glu Thr Trp Ala Pro Phe Ala Ser Gly Lys Thr Ser Glu Ser Gly 60 65
70 gag ctg cat ggg ctc aca act gag gag gaa ttt gta gaa ggg ata tac
293 Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe Val Glu Gly Ile Tyr
75 80 85 aaa gtg gaa ata gac acc aaa tct tac tgg aag tca ctt ggc
atc tcc 341 Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys Ser Leu Gly
Ile Ser 90 95 100 105 cca ttc cat gag cat gca gag gtg gta ttc aca
gcc aac gat tcc ggc 389 Pro Phe His Glu His Ala Glu Val Val Phe Thr
Ala Asn Asp Ser Gly 110 115 120 ccc cgc cac tac acc atc gcc cgc ctg
ctg agc ccc tac tcc tat tcc 437 Pro Arg His Tyr Thr Ile Ala Arg Leu
Leu Ser Pro Tyr Ser Tyr Ser 125 130 135 acc acg gct gtc gtc acc aat
ccc aag gaa tga gggacttctc cagaggatct 490 Thr Thr Ala Val Val Thr
Asn Pro Lys Glu 140 145 gaaggacgag ggatgggatt tcatgtaacc aagagtattc
catttttact aaagcagtgt 550 tttcacctca taagctatgt taggagtcca
ggcagagaca ataaaacatt cctgtgaaag 610 gc 612 <210> SEQ ID NO
165 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
165 ccttccctga aggttcctcc 20 <210> SEQ ID NO 166 <211>
LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 166 gcgtttgctc
ttcttcttgc gtttttt 27 <210> SEQ ID NO 167 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 167 ttttattgtc tctgcctg 18
<210> SEQ ID NO 168 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 168 tacaaatggg atgctactgc 20 <210> SEQ
ID NO 169 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
169 ggaatcccaa gcctcaaacg 20 <210> SEQ ID NO 170 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 170 cgtccttcag
gtccactgga 20 <210> SEQ ID NO 171 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 171 atatgaggtg aaaacactgc 20
<210> SEQ ID NO 172 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 172 tattgtctct gcctggactt 20 <210> SEQ
ID NO 173 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 173 cacaggaatg ttttattgtc 20 <210> SEQ
ID NO 174 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
174 cctttcacag gaatgtttta 20 <210> SEQ ID NO 175 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 175 gcctttcaca
ggaatgtttt 20 <210> SEQ ID NO 176 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 176 agtgcctttc acaggaatgt 20
<210> SEQ ID NO 177 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 177 aagtgccttt cacaggaatg 20
* * * * *