U.S. patent application number 14/558402 was filed with the patent office on 2015-11-12 for snps of apolipoprotein b and modulation of their expression.
The applicant listed for this patent is GENZYME CORPORATION. Invention is credited to Rosanne M. Crooke, Mark J. Graham, Steven Mah.
Application Number | 20150322429 14/558402 |
Document ID | / |
Family ID | 35506289 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150322429 |
Kind Code |
A1 |
Crooke; Rosanne M. ; et
al. |
November 12, 2015 |
SNPs OF APOLIPOPROTEIN B AND MODULATION OF THEIR EXPRESSION
Abstract
Compounds, compositions and methods are provided for modulating
the expression of apolipoprotein B. The compositions comprise
oligonucleotides, targeted to nucleic acid encoding apolipoprotein
B. Methods of using these compounds for modulation of
apolipoprotein B expression and for diagnosis and treatment of
diseases and conditions associated with expression of
apolipoprotein B are provided.
Inventors: |
Crooke; Rosanne M.;
(Carlsbad, CA) ; Graham; Mark J.; (Carlsbad,
CA) ; Mah; Steven; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENZYME CORPORATION |
CAMBRIDGE |
MA |
US |
|
|
Family ID: |
35506289 |
Appl. No.: |
14/558402 |
Filed: |
December 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13016917 |
Jan 28, 2011 |
8916694 |
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14558402 |
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11124020 |
May 5, 2005 |
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13016917 |
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60568409 |
May 5, 2004 |
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Current U.S.
Class: |
514/44A ;
435/375; 536/24.5 |
Current CPC
Class: |
C12N 2310/321 20130101;
C12N 2310/341 20130101; C12Q 2600/106 20130101; C12N 2310/11
20130101; C12N 2310/111 20130101; C12Q 1/6883 20130101; C12N
2310/315 20130101; C12N 2320/30 20130101; A61P 3/00 20180101; C12Q
2600/136 20130101; C12N 2310/3341 20130101; C12Q 2600/156 20130101;
C12N 15/113 20130101; C12Q 2600/158 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. An antisense compound 15 to 30 nucleobases in length which
specifically hybridizes with an allelic variant of a nucleic acid
of SEQ ID NO: 1 encoding human apolipoprotein B, wherein said
compound inhibits the expression of apolipoprotein B mRNA by at
least 10% and wherein said compound is targeted to a region that
includes a cytosine (C) at position 27735 of SEQ ID NO: 1.
2. The antisense compound of claim 1 comprising a guanine (G) at
position 15711 of SEQ ID NO: 2.
3. The antisense compound of claim 1 which is 20 nucleobases in
length.
4. The antisense compound of claim 1 comprising an
oligonucleotide.
5. The antisense compound of claim 4 comprising a DNA
oligonucleotide.
6. The antisense compound of claim 4 comprising an RNA
oligonucleotide.
7. The antisense compound of claim 4 comprising a chimeric
oligonucleotide.
8. The antisense compound of claim 4 wherein at least a portion of
said compound hybridizes with RNA to form an oligonucleotide-RNA
duplex.
9. The antisense compound of claim 1 having at least 80%, at least
85%, at least 90% or, at least 95% or at least 99% complementarity
with said nucleic acid molecule encoding apolipoprotein B.
10. The antisense compound of claim 1 having one, two or more types
of modifications, wherein the modification comprises a modified
internucleoside linkage, sugar moiety, or nucleobase.
11. The antisense compound of claim 1 having at least one
2'-O-methoxyethyl sugar moiety.
12. The antisense compound of claim 1 having at least one
phosphorothioate internucleoside linkage.
13. The antisense compound of claim 1 wherein at least one cytosine
is a 5-methylcytosine.
14. A method of inhibiting the expression of apolipoprotein B in a
cell or tissue comprising contacting said cell or tissue with an
antisense compound so that expression of apolipoprotein B is
inhibited, wherein the antisense compound is an antisense compound
15 to 30 nucleobases in length which specifically hybridizes with
an allelic variant of a nucleic acid of SEQ ID NO: 1 encoding human
apolipoprotein B, wherein said compound inhibits the expression of
apolipoprotein B mRNA by at least 10% and wherein said compound is
targeted to a region that includes a cytosine (C) at position 27735
of SEQ ID NO: 1.
15. (canceled)
16. (canceled)
17. A method of treating an animal having a disease or condition
associated with apolipoprotein B comprising administering to said
animal a therapeutically or prophylactically effective amount of an
antisense compound so that expression of apolipoprotein B is
inhibited, wherein the antisense compound is an antisense compound
15 to 30 nucleobases in length which specifically hybridizes with
an allelic variant of a nucleic acid of SEQ ID NO: 1 encoding human
apolipoprotein B, wherein said compound inhibits the expression of
apolipoprotein B mRNA by at least 10% and wherein said compound is
targeted to a region that includes a cytosine (C) at position 27735
of SEQ ID NO: 1.
18. The method of claim 17 wherein the disease or condition is a
disorder of lipid metabolism.
19. The antisense compound of claim 7, wherein the antisense
compound is a chimeric oligonucleotide consisting of: a gap segment
consisting of from 10 to 18 linked 2'-deoxynucleosides; a 5' wing
segment consisting of from 1 to 5 linked nucleosides; and a 3' wing
segment consisting of from 1 to 5 linked nucleosides; wherein each
nucleoside of the 5' wing segment is a sugar-modified nucleoside,
each nucleoside of the 3' wing segment is a sugar-modified
nucleoside, and each internucleoside linkage is a phosphorothioate
internucleoside linkage.
20. The antisense compound of claim 19, wherein the sugar
modification is a 2'-O-methoxyethyl sugar modification.
21. The antisense compound of claim 7 wherein the antisense
compound is a chimeric oligonucleotide consisting of: a gap segment
consisting of ten linked 2'-deoxynucleosides; a 5' wing segment
consisting of five linked nucleosides; and a 3' wing segment
consisting of five linked nucleosides; wherein each nucleoside of
the 5' wing segment is a 2'-O-methoxyethyl nucleoside, each
nucleoside of the 3' wing segment is a 2'-O-methoxyethyl
nucleoside, and each internucleoside linkage is a phosphorothioate
internucleoside linkage.
22. The antisense compound of claim 21 comprising at least one
cytosine, wherein the cytosine is a 5-methylcytosine.
23. A pharmaceutical composition comprising the antisense compound
of claim 1 and pharmaceutically acceptable excipient.
24. A pharmaceutical composition comprising the antisense compound
of claim 19 and pharmaceutically acceptable excipient.
25. A pharmaceutical composition comprising the antisense compound
of claim 21 and pharmaceutically acceptable excipient.
26. A pharmaceutical composition comprising the antisense compound
of claim 22 and pharmaceutically acceptable excipient.
27. The antisense compound of claim 19, wherein the gap segment is
flanked on both the 5' and 3' sides by wing segments of the same
length.
28. The antisense compound of claim 19, wherein the gap segment is
flanked on both the 5' and 3' sides by wing segments of different
lengths.
29. The antisense compound of claim 27, wherein the antisense
compound is 20 nucleobases in length, and wherein: (i) the gap
segment is 8 nucleotides in length, and the 5' and 3' wing segments
are each 6 nucleotides in length; (ii) the gap segment is 10
nucleotides in length, and the 5' and 3' wing segments are each 5
nucleotides in length; (iii) the gap segment is 12 nucleotides in
length, and the 5' and 3' wing segments are each 4 nucleotides in
length; (iv) the gap segment is 14 nucleotides in length, and the
5' and 3' wing segments are each 3 nucleotides in length; (v) the
gap segment is 16 nucleotides in length, and the 5' and 3' wing
segments are each 2 nucleotides in length; or (vi) the gap segment
is 18 nucleotides in length, and the 5' and 3' wing segments are
each 1 nucleotide in length.
30. The antisense compound of claim 28, wherein the antisense
compound is 20 nucleobases in length, and wherein the gap segment
is 10 nucleotides in length, flanked on one side by a wing segment
6 nucleotides in length and flanked on the other side by a wing
segment 4 nucleotides in length.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 13/016,917 filed Jan. 28, 2011, which is a continuation
application of U.S. Ser. No. 11/124,020 filed May 5, 2005
(abandoned), which claims the benefit of priority of U.S.
Provisional Application Ser. No. 60/568,409 filed May 5, 2004, each
of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The present specification is being filed with a computer
readable form (CRF) copy of the Sequence Listing. The CRF entitled
10103-068-999_SEQLIST.txt, which was created on Dec. 1, 2014 and is
129,715 bytes in size, is identical to the paper copy of the
Sequence Listing and is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention provides compositions and methods for
modulating the expression of variants of apolipoprotein B (apo B).
In particular, this invention relates to antisense compounds,
particularly oligonucleotide compounds, which, in preferred
embodiments, hybridize with nucleic acid molecules encoding
apolipoprotein B and containing SNPs.
BACKGROUND OF THE INVENTION
[0004] Natural genetic sequence variability exists between
individuals in any and every population. Subtle alteration(s) in
the primary nucleotide sequence of a gene encoding a
pharmaceutically-important protein may be manifested as significant
variation in expression, structure and/or function of the protein.
Such alterations may explain the different response of individuals
to therapy with a particular drug.
[0005] Variability in genetic sequence is particularly likely to
cause a variable response to therapy when the therapeutic is an
antisense compound that modulates the expression of protein through
specific hybridization to the genetic sequence. In this case,
changes in the sequence of the DNA or RNA can have a direct effect
on the ability of such a compound to specifically hybridize.
[0006] Identification of polymorphisms among various populations is
desirable to tailor design of suitable antisense therapeutics,
select antisense therapeutics to administer to a particular
population, and also predict responsiveness to therapeutics.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to antisense compounds,
especially nucleic acid and nucleic acid-like oligomers, which are
targeted to a nucleic acid encoding apolipoprotein B, and which
modulate the expression of apolipoprotein B. Pharmaceutical and
other compositions comprising the compounds of the invention are
also provided. Further provided are methods of screening for
modulators of apolipoprotein B and methods of modulating the
expression of apolipoprotein B in cells, tissues or animals
comprising contacting said cells, tissues or animals with one or
more of the compounds or compositions of the invention. Methods of
treating an animal, particularly a human, suspected of having or
being prone to a disease or condition associated with expression of
apolipoprotein B are also set forth herein. Such methods comprise
administering a therapeutically or prophylactically effective
amount of one or more of the compounds or compositions of the
invention to the person in need of treatment.
DETAILED DESCRIPTION OF THE INVENTION
A. Overview of the Invention
[0008] The present invention employs antisense compounds,
preferably oligonucleotides and similar species for use in
modulating the function or effect of nucleic acid molecules
encoding apolipoprotein B. This is accomplished by providing
oligonucleotides which specifically hybridize with one or more
nucleic acid molecules encoding apolipoprotein B. As used herein,
the terms "target nucleic acid" and "nucleic acid molecule encoding
apolipoprotein B" have been used for convenience to encompass DNA
encoding apolipoprotein B, RNA (including pre-mRNA and mRNA or
portions thereof) transcribed from such DNA, and also cDNA derived
from such RNA. The hybridization of a compound of this invention
with its target nucleic acid is generally referred to as
"antisense". Consequently, the preferred mechanism believed to be
included in the practice of some preferred embodiments of the
invention is referred to herein as "antisense inhibition." Such
antisense inhibition is typically based upon hydrogen bonding-based
hybridization of oligonucleotide strands or segments such that at
least one strand or segment is cleaved, degraded, or otherwise
rendered inoperable. In this regard, it is presently preferred to
target specific nucleic acid molecules and their functions for such
antisense inhibition.
[0009] The functions of DNA to be interfered with can include
replication and transcription. Replication and transcription, for
example, can be from an endogenous cellular template, a vector, a
plasmid construct or otherwise. The functions of RNA to be
interfered with can include functions such as translocation of the
RNA to a site of protein translation, translocation of the RNA to
sites within the cell which are distant from the site of RNA
synthesis, translation of protein from the RNA, splicing of the RNA
to yield one or more RNA species, and catalytic activity or complex
formation involving the RNA which may be engaged in or facilitated
by the RNA. One preferred result of such interference with target
nucleic acid function is modulation of the expression of
apolipoprotein B. In the context of the present invention,
"modulation" and "modulation of expression" mean either an increase
(stimulation) or a decrease (inhibition) in the amount or levels of
a nucleic acid molecule encoding the gene, e.g., DNA or RNA.
Inhibition is often the preferred form of modulation of expression
and mRNA is often a preferred target nucleic acid.
[0010] In the context of this invention, "hybridization" means the
pairing of complementary strands of oligomeric compounds. In the
present invention, the preferred mechanism of pairing involves
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases (nucleobases) of the strands of oligomeric
compounds. For example, adenine and thymine are complementary
nucleobases which pair through the formation of hydrogen bonds.
Hybridization can occur under varying circumstances.
[0011] An antisense compound is specifically hybridizable when
binding of the compound to the target nucleic acid interferes with
the normal function of the target nucleic acid to cause a loss of
activity, and there is a sufficient degree of complementarity to
avoid non-specific binding of the antisense compound to non-target
nucleic acid sequences under conditions in which specific binding
is desired, i.e., under physiological conditions in the case of in
vivo assays or therapeutic treatment, and under conditions in which
assays are performed in the case of in vitro assays.
[0012] In the present invention the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which a compound of the invention will hybridize to its target
sequence, but to a minimal number of other sequences. Stringent
conditions are sequence-dependent and will be different in
different circumstances and in the context of this invention,
"stringent conditions" under which oligomeric compounds hybridize
to a target sequence are determined by the nature and composition
of the oligomeric compounds and the assays in which they are being
investigated.
[0013] "Complementary," as used herein, refers to the capacity for
precise pairing between two nucleobases of an oligomeric compound.
For example, if a nucleobase at a certain position of an
oligonucleotide (an oligomeric compound), is capable of hydrogen
bonding with a nucleobase at a certain position of a target nucleic
acid, said target nucleic acid being a DNA, RNA, or oligonucleotide
molecule, then the position of hydrogen bonding between the
oligonucleotide and the target nucleic acid is considered to be a
complementary position. The oligonucleotide and the further DNA,
RNA, or oligonucleotide molecule are complementary to each other
when a sufficient number of complementary positions in each
molecule are occupied by nucleobases which can hydrogen bond with
each other. Thus, "specifically hybridizable" and "complementary"
are terms which are used to indicate a sufficient degree of precise
pairing or complementarity over a sufficient number of nucleobases
such that stable and specific binding occurs between the
oligonucleotide and a target nucleic acid.
[0014] It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. Moreover, an
oligonucleotide may hybridize over one or more segments such that
intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure or hairpin structure).
It is preferred that the antisense compounds of the present
invention comprise at least 70%, or at least 75%, or at least 80%,
or at least 85% sequence complementarity to a target region within
the target nucleic acid, more preferably that they comprise at
least 90% sequence complementarity and even more preferably
comprise at least 95% or at least 99% sequence complementarity to
the target region within the target nucleic acid sequence to which
they are targeted. For example, an antisense compound in which 18
of 20 nucleobases of the antisense compound are complementary to a
target region, and would therefore specifically hybridize, would
represent 90 percent complementarity. In this example, the
remaining noncomplementary nucleobases may be clustered or
interspersed with complementary nucleobases and need not be
contiguous to each other or to complementary nucleobases. 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).
[0015] Percent homology, sequence identity or complementarity, can
be determined by, for example, the Gap program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, Madison Wis.), using default settings,
which uses the algorithm of Smith and Waterman (Adv. Appl. Math.,
1981, 2, 482-489). In some preferred embodiments, homology,
sequence identity or complementarity, between the oligomeric and
target is between about 50% to about 60%. In some embodiments,
homology, sequence identity or complementarity, is between about
60% to about 70%. In preferred embodiments, homology, sequence
identity or complementarity, is between about 70% and about 80%. In
more preferred embodiments, homology, sequence identity or
complementarity, is between about 80% and about 90%. In some
preferred embodiments, homology, sequence identity or
complementarity, is about 90%, about 92%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99% or about 100%.
B. Compounds of the Invention
[0016] According to the present invention, antisense compounds
include antisense oligomeric compounds, antisense oligonucleotides,
siRNAs, external guide sequence (EGS) oligonucleotides, alternate
splicers, primers, probes, and other oligomeric compounds which
hybridize to at least a portion of the target nucleic acid. As
such, these compounds may be introduced in the form of
single-stranded, double-stranded, circular or hairpin oligomeric
compounds and may contain structural elements such as internal or
terminal bulges or loops. Once introduced to a system, the
compounds of the invention may elicit the action of one or more
enzymes or structural proteins to effect modification of the target
nucleic acid.
[0017] One non-limiting example of such an enzyme is RNAse H, a
cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. It is known in the art that single-stranded antisense
compounds which are "DNA-like" elicit RNAse H. Activation of RNase
H, therefore, results in cleavage of the RNA target, thereby
greatly enhancing the efficiency of oligonucleotide-mediated
inhibition of gene expression. Similar roles have been postulated
for other ribonucleases such as those in the RNase III and
ribonuclease L family of enzymes.
[0018] While the preferred form of antisense compound is a
single-stranded antisense oligonucleotide, in many species the
introduction of double-stranded structures, such as double-stranded
RNA (dsRNA) molecules, has been shown to induce potent and specific
antisense-mediated reduction of the function of a gene or its
associated gene products. This phenomenon occurs in both plants and
animals and is believed to have an evolutionary connection to viral
defense and transposon silencing.
[0019] The first evidence that dsRNA, also known as small
interfering RNAs (siRNAs) could lead to gene silencing in animals
came in 1995 from work in the nematode, Caenorhabditis elegans (Guo
and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have
shown that the primary interference effects of dsRNA are
posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,
1998, 95, 15502-15507). The posttranscriptional antisense mechanism
defined in Caenorhabditis elegans resulting from exposure to
double-stranded RNA (dsRNA) has since been designated RNA
interference (RNAi). This term has been generalized to mean
antisense-mediated gene silencing involving the introduction of
dsRNA leading to the sequence-specific reduction of endogenous
targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811).
Recently, it has been shown that it is, in fact, the
single-stranded RNA oligomers of antisense polarity of the dsRNAs
which are the potent inducers of RNAi (Tijsterman et al., Science,
2002, 295, 694-697).
[0020] The antisense compounds of the present invention also
include modified compounds in which a different base is present at
one or more of the nucleotide positions in the compound. For
example, if the first nucleotide is an adenosine, modified
compounds may be produced which contain thymidine, guanosine or
cytidine at this position. This may be done at any of the positions
of the antisense compound. These compounds are then tested using
the methods described herein to determine their ability to inhibit
expression of apolipoprotein B mRNA.
[0021] In the context of this invention, the term "oligomeric
compound" refers to a polymer or oligomer comprising a plurality of
monomeric units. In the context of this invention, the term
"oligonucleotide" refers to an oligomer or polymer of ribonucleic
acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras,
analogs and homologs thereof. This term includes oligonucleotides
composed of naturally occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages as well as oligonucleotides
having non-naturally occurring portions which function similarly.
Such modified or substituted oligonucleotides are often preferred
over native forms because of desirable properties such as, for
example, enhanced cellular uptake, enhanced affinity for a target
nucleic acid and increased stability in the presence of
nucleases.
[0022] While oligonucleotides are a preferred form of the antisense
compounds of this invention, the present invention comprehends
other families of antisense compounds as well, including but not
limited to oligonucleotide analogs and mimetics such as those
described herein.
[0023] The antisense compounds in accordance with this invention
preferably comprise from about 8 to about 80 nucleobases (i.e. from
about 8 to about 80 linked nucleosides). One of ordinary skill in
the art will appreciate that the invention embodies compounds of 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, or 80 nucleobases in length.
[0024] In one preferred embodiment, the antisense compounds of the
invention are 12 to 50 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies compounds of
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, or 50 nucleobases in length.
[0025] In another preferred embodiment, the antisense compounds of
the invention are 15 to 30 nucleobases in length. One having
ordinary skill in the art will appreciate that this embodies
compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30 nucleobases in length.
[0026] Particularly preferred compounds are oligonucleotides from
about 12 to about 50 nucleobases, even more preferably those
comprising from about 15 to about 30 nucleobases.
[0027] Antisense compounds 8-80 nucleobases in length comprising a
stretch of at least eight (8) consecutive nucleobases selected from
within the illustrative antisense compounds are considered to be
suitable antisense compounds as well.
[0028] Exemplary preferred antisense compounds include
oligonucleotide sequences that comprise at least the 8 consecutive
nucleobases from the 5'-terminus of one of the illustrative
preferred antisense compounds (the remaining nucleobases being a
consecutive stretch of the same oligonucleotide beginning
immediately upstream of the 5'-terminus of the antisense compound
which is specifically hybridizable to the target nucleic acid and
continuing until the oligonucleotide contains about 8 to about 80
nucleobases). Similarly preferred antisense compounds are
represented by oligonucleotide sequences that comprise at least the
8 consecutive nucleobases from the 3'-terminus of one of the
illustrative preferred antisense compounds (the remaining
nucleobases being a consecutive stretch of the same oligonucleotide
beginning immediately downstream of the 3'-terminus of the
antisense compound which is specifically hybridizable to the target
nucleic acid and continuing until the oligonucleotide contains
about 8 to about 80 nucleobases). It is also understood that
preferred antisense compounds may be represented by oligonucleotide
sequences that comprise at least 8 consecutive nucleobases from an
internal portion of the sequence of an illustrative preferred
antisense compound, and may extend in either or both directions
until the oligonucleotide contains about 8 to about 80
nucleobases.
[0029] One having skill in the art armed with the preferred
antisense compounds illustrated herein will be able, without undue
experimentation, to identify further preferred antisense
compounds.
C. Targets of the Invention
[0030] "Targeting" an antisense compound to a particular nucleic
acid molecule, in the context of this invention, can be a multistep
process. The process usually begins with the identification of a
target nucleic acid whose function is to be modulated. This target
nucleic acid may be, for example, a cellular gene (or mRNA
transcribed from the gene) whose expression is associated with a
particular disorder or disease state, or a nucleic acid molecule
from an infectious agent. In the present invention, the target
nucleic acid encodes apolipoprotein B.
[0031] The targeting process usually also includes determination of
at least one target region, segment, or site within the target
nucleic acid for the antisense interaction to occur such that the
desired effect, e.g., modulation of expression, will result. Within
the context of the present invention, the term "region" is defined
as a portion of the target nucleic acid having at least one
identifiable structure, function, or characteristic. Within regions
of target nucleic acids are segments. "Segments" are defined as
smaller or sub-portions of regions within a target nucleic acid.
"Sites," as used in the present invention, are defined as positions
within a target nucleic acid.
[0032] Since, as is known in the art, the translation initiation
codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in
the corresponding DNA molecule), the translation initiation codon
is also referred to as the "AUG codon," the "start codon" or the
"AUG start codon". A minority of genes has a translation initiation
codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA,
5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the
terms "translation initiation codon" and "start codon" can
encompass many codon sequences, even though the initiator amino
acid in each instance is typically methionine (in eukaryotes) or
formylmethionine (in prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA transcribed from a gene encoding
apolipoprotein B, regardless of the sequence(s) of such codons. It
is also known in the art that a translation termination codon (or
"stop codon") of a gene may have one of three sequences, i.e.,
5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are
5'-TAA, 5'-TAG and 5'-TGA, respectively).
[0033] The terms "start codon region" and "translation initiation
codon region" refer to a portion of such an mRNA or gene that
encompasses from about 25 to about 50 contiguous nucleotides in
either direction (i.e., 5' or 3') from a translation initiation
codon. Similarly, the terms "stop codon region" and "translation
termination codon region" refer to a portion of such an mRNA or
gene that encompasses from about 25 to about 50 contiguous
nucleotides in either direction (i.e., 5' or 3') from a translation
termination codon. Consequently, the "start codon region" (or
"translation initiation codon region") and the "stop codon region"
(or "translation termination codon region") are all regions which
may be targeted effectively with the antisense compounds of the
present invention.
[0034] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Within the context of the
present invention, a preferred region is the intragenic region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of a gene.
[0035] Other target regions include the 5' untranslated region
(5'UTR), known in the art to refer to the portion of an mRNA in the
5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA (or corresponding nucleotides on the
gene), and the 3' untranslated region (3 `UTR), known in the art to
refer to the portion of an mRNA in the 3` direction from the
translation termination codon, and thus including nucleotides
between the translation termination codon and 3' end of an mRNA (or
corresponding nucleotides on the gene). The 5' cap site of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap
site. It is also preferred to target the 5' cap region.
[0036] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence,
resulting in exon-exon junctions at the sites where exons are
joined. Targeting exon-exon junctions can be useful in situations
where the overproduction of a normal splice product is implicated
in disease, or where the overproduction of an aberrant splice
product is implicated in disease. Targeting splice sites, i.e.,
intron-exon junctions or exon-intron junctions, may also be
particularly useful in situations where aberrant splicing is
implicated in disease, or where an overproduction of a particular
splice product is implicated in disease. Aberrant fusion junctions
due to rearrangements or deletions are also preferred target sites.
mRNA transcripts produced via the process of splicing of two (or
more) mRNAs from different gene sources are known as "fusion
transcripts". It is also known that introns can be effectively
targeted using antisense compounds targeted to, for example, DNA or
pre-mRNA.
[0037] It is also known in the art that alternative RNA transcripts
can be produced from the same genomic region of DNA. These
alternative transcripts are generally known as "variants". More
specifically, "pre-mRNA variants" are transcripts produced from the
same genomic DNA that differ from other transcripts produced from
the same genomic DNA in either their start or stop position and
contain both intronic and exonic sequence.
[0038] Upon excision of one or more exon or intron regions, or
portions thereof during splicing, pre-mRNA variants produce smaller
"mRNA variants". Consequently, mRNA variants are processed pre-mRNA
variants and each unique pre-mRNA variant must always produce a
unique mRNA variant as a result of splicing. These mRNA variants
are also known as "alternative splice variants". If no splicing of
the pre-mRNA variant occurs then the pre-mRNA variant is identical
to the mRNA variant.
[0039] It is also known in the art that variants can be produced
through the use of alternative signals to start or stop
transcription and that pre-mRNAs and mRNAs can possess more that
one start codon or stop codon. Variants that originate from a
pre-mRNA or mRNA that use alternative start codons are known as
"alternative start variants" of that pre-mRNA or mRNA. Those
transcripts that use an alternative stop codon are known as
"alternative stop variants" of that pre-mRNA or mRNA. One specific
type of alternative stop variant is the "polyA variant" in which
the multiple transcripts produced result from the alternative
selection of one of the "polyA stop signals" by the transcription
machinery, thereby producing transcripts that terminate at unique
polyA sites. Within the context of the invention, the types of
variants described herein are also preferred target nucleic
acids.
[0040] The locations on the target nucleic acid to which the
preferred antisense compounds hybridize are hereinbelow referred to
as "preferred target segments." As used herein the term "preferred
target segment" is defined as at least an 8-nucleobase portion of a
target region to which an active antisense compound is targeted.
While not wishing to be bound by theory, it is presently believed
that these target segments represent portions of the target nucleic
acid which are accessible for hybridization.
[0041] While the specific sequences of certain preferred target
segments are set forth herein, one of skill in the art will
recognize that these serve to illustrate and describe particular
embodiments within the scope of the present invention. Additional
preferred target segments may be identified by one having ordinary
skill.
[0042] Target segments 8-80 nucleobases in length comprising a
stretch of at least eight (8) consecutive nucleobases selected from
within the illustrative preferred target segments are considered to
be suitable for targeting as well.
[0043] Target segments can include DNA or RNA sequences that
comprise at least the 8 consecutive nucleobases from the
5'-terminus of one of the illustrative preferred target segments
(the remaining nucleobases being a consecutive stretch of the same
DNA or RNA beginning immediately upstream of the 5'-terminus of the
target segment and continuing until the DNA or RNA contains about 8
to about 80 nucleobases). Similarly preferred target segments are
represented by DNA or RNA sequences that comprise at least the 8
consecutive nucleobases from the 3'-terminus of one of the
illustrative preferred target segments (the remaining nucleobases
being a consecutive stretch of the same DNA or RNA beginning
immediately downstream of the 3'-terminus of the target segment and
continuing until the DNA or RNA contains about 8 to about 80
nucleobases). It is also understood that preferred antisense target
segments may be represented by DNA or RNA sequences that comprise
at least 8 consecutive nucleobases from an internal portion of the
sequence of an illustrative preferred target segment, and may
extend in either or both directions until the oligonucleotide
contains about 8 to about 80 nucleobases. One having skill in the
art armed with the preferred target segments illustrated herein
will be able, without undue experimentation, to identify further
preferred target segments.
[0044] Once one or more target regions, segments or sites have been
identified, antisense compounds are chosen which are sufficiently
complementary to the target, i.e., hybridize sufficiently well and
with sufficient specificity, to give the desired effect.
[0045] The oligomeric antisense compounds can also be targeted to
regions of a target nucleobase sequence, such as those disclosed
herein. All regions of the target nucleobase sequence to which an
oligomeric antisense compound can be targeted, wherein the regions
are greater than or equal to 8 and less than or equal to 80
nucleobases, are described as follows:
[0046] Let R(n, n+m-1) be a region from a target nucleobase
sequence, where "n" is the 5'-most nucleobase position of the
region, where "n+m-1" is the 3'-most nucleobase position of the
region and where "m" is the length of the region. A set "S(m)", of
regions of length "m" is defined as the regions where n ranges from
1 to L-m+1, where L is the length of the target nucleobase sequence
and L>m. A set, "A", of all regions can be constructed as a
union of the sets of regions for each length from where m is
greater than or equal to 8 and is less than or equal to 80.
[0047] This set of regions can be represented using the following
mathematical notation:
A = m S ( m ) where m .di-elect cons. N 8 .ltoreq. m .ltoreq. 80
##EQU00001## and ##EQU00001.2## S ( m ) = { R n , n + m - 1 n
.di-elect cons. ( 1 , 2 , 3 , , L - m + 1 } } ##EQU00001.3##
[0048] where the mathematical operator | indicates "such that",
[0049] where the mathematical operator .epsilon. indicates "a
member of a set" (e.g. y.epsilon.Z indicates that element y is a
member of set Z),
[0050] where x is a variable,
[0051] where N indicates all natural numbers, defined as positive
integers,
[0052] and where the mathematical operator .orgate. indicates "the
union of sets".
[0053] For example, the set of regions for m equal to 8, 9 and 80
can be constructed in the following manner. The set of regions,
each 8 nucleobases in length, S(m=8), in a target nucleobase
sequence 100 nucleobases in length (L=100), beginning at position 1
(n=1) of the target nucleobase sequence, can be created using the
following expression:
S(8)={R.sub.1,8|n.epsilon.{1,2,3, . . . , 93}}
and describes the set of regions comprising nucleobases 1-8, 2-9,
3-10, 4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19,
13-20, 14-21, 15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28,
22-29, 23-30, 24-31, 25-32, 26-33, 27-34, 28-35, 29-36, 30-37,
31-38, 32-39, 33-40, 34-41, 35-42, 36-43, 37-44, 38-45, 39-46,
40-47, 41-48, 42-49, 43-50, 44-51, 45-52, 46-53, 47-54, 48-55,
49-56, 50-57, 51-58, 52-59, 53-60, 54-61, 55-62, 56-63, 57-64,
58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71, 65-72, 66-73,
67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81, 75-82,
76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91,
85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.
[0054] An additional set for regions 20 nucleobases in length, in a
target sequence 100 nucleobases in length, beginning at position 1
of the target nucleobase sequence, can be described using the
following expression:
S(20)={R.sub.1,20|n.epsilon.{1,2,3, . . . , 81}}
and describes the set of regions comprising nucleobases 1-20, 2-21,
3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31,
13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40,
22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49,
31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58,
40-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67,
49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76,
58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85,
67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94,
76-95, 77-96, 78-97, 79-98, 80-99, 81-100.
[0055] An additional set for regions 80 nucleobases in length, in a
target sequence 100 nucleobases in length, beginning at position 1
of the target nucleobase sequence, can be described using the
following expression:
S(80)={R.sub.1,80n.epsilon.{1,2,3, . . . , 21}}
and describes the set of regions comprising nucleobases 1-80, 2-81,
3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91,
13-92, 14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.
[0056] Thus, in this example, A would include regions 1-8, 2-9,
3-10 . . . 93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82
. . . 21-100.
[0057] The union of these aforementioned example sets and other
sets for lengths from 10 to 19 and 21 to 79 can be described using
the mathematical expression
A = m S ( m ) ##EQU00002##
[0058] where .orgate. represents the union of the sets obtained by
combining all members of all sets.
[0059] The mathematical expressions described herein defines all
possible target regions in a target nucleobase sequence of any
length L, where the region is of length m, and where m is greater
than or equal to 8 and less than or equal to 80 nucleobases and,
and where m is less than L, and where n is less than L-m+1.
D. Screening and Target Validation
[0060] In a further embodiment, the "preferred target segments"
identified herein may be employed in a screen for additional
compounds that modulate the expression of apolipoprotein B.
"Modulators" are those compounds that decrease or increase the
expression of a nucleic acid molecule encoding apolipoprotein B and
which comprise at least an 8-nucleobase portion which is
complementary to a preferred target segment. The screening method
comprises the steps of contacting a preferred target segment of a
nucleic acid molecule encoding apolipoprotein B with one or more
candidate modulators, and selecting for one or more candidate
modulators which decrease or increase the expression of a nucleic
acid molecule encoding apolipoprotein B. Once it is shown that the
candidate modulator or modulators are capable of modulating (e.g.
either decreasing or increasing) the expression of a nucleic acid
molecule encoding apolipoprotein B, the modulator may then be
employed in further investigative studies of the function of
apolipoprotein B, or for use as a research, diagnostic, or
therapeutic agent in accordance with the present invention.
[0061] The preferred target segments of the present invention may
be also be combined with their respective complementary antisense
compounds of the present invention to form stabilized
double-stranded (duplexed) oligonucleotides.
[0062] Such double stranded oligonucleotide moieties have been
shown in the art to modulate target expression and regulate
translation as well as RNA processsing via an antisense mechanism.
Moreover, the double-stranded moieties may be subject to chemical
modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and
Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263,
103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et
al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et
al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,
2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200).
For example, such double-stranded moieties have been shown to
inhibit the target by the classical hybridization of antisense
strand of the duplex to the target, thereby triggering enzymatic
degradation of the target (Tijsterman et al., Science, 2002, 295,
694-697).
[0063] The antisense compounds of the present invention can also be
applied in the areas of drug discovery and target validation. The
present invention comprehends the use of the compounds and
preferred target segments identified herein in drug discovery
efforts to elucidate relationships that exist between
apolipoprotein B and a disease state, phenotype, or condition.
These methods include detecting or modulating apolipoprotein B
comprising contacting a sample, tissue, cell, or organism with the
compounds of the present invention, measuring the nucleic acid or
protein level of apolipoprotein B and/or a related phenotypic or
chemical endpoint at some time after treatment, and optionally
comparing the measured value to a non-treated sample or sample
treated with a further compound of the invention. These methods can
also be performed in parallel or in combination with other
experiments to determine the function of unknown genes for the
process of target validation or to determine the validity of a
particular gene product as a target for treatment or prevention of
a particular disease, condition, or phenotype.
E. Kits, Research Reagents, Diagnostics, and Therapeutics
[0064] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. Furthermore, antisense oligonucleotides, which
are able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes or to distinguish between functions of various
members of a biological pathway.
[0065] For use in kits and diagnostics, the compounds of the
present invention, either alone or in combination with other
compounds or therapeutics, can be used as tools in differential
and/or combinatorial analyses to elucidate expression patterns of a
portion or the entire complement of genes expressed within cells
and tissues.
[0066] As one nonlimiting example, expression patterns within cells
or tissues treated with one or more antisense compounds are
compared to control cells or tissues not treated with antisense
compounds and the patterns produced are analyzed for differential
levels of gene expression as they pertain, for example, to disease
association, signaling pathway, cellular localization, expression
level, size, structure or function of the genes examined. These
analyses can be performed on stimulated or unstimulated cells and
in the presence or absence of other compounds which affect
expression patterns.
[0067] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression) (Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein
arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (To, Comb. Chem. High
Throughput Screen, 2000, 3, 235-41).
[0068] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding apolipoprotein B. For example,
oligonucleotides that are shown to hybridize with such efficiency
and under such conditions as disclosed herein as to be effective
apolipoprotein B inhibitors will also be effective primers or
probes under conditions favoring gene amplification or detection,
respectively. These primers and probes are useful in methods
requiring the specific detection of nucleic acid molecules encoding
apolipoprotein B and in the amplification of said nucleic acid
molecules for detection or for use in further studies of
apolipoprotein B. Hybridization of the antisense oligonucleotides,
particularly the primers and probes, of the invention with a
nucleic acid encoding apolipoprotein B can be detected by means
known in the art. Such means may include conjugation of an enzyme
to the oligonucleotide, radiolabelling of the oligonucleotide or
any other suitable detection means. Kits using such detection means
for detecting the level of apolipoprotein B in a sample may also be
prepared.
[0069] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense compounds have been employed as therapeutic moieties in
the treatment of disease states in animals, including humans.
Antisense oligonucleotide drugs, including ribozymes, have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that
antisense compounds can be useful therapeutic modalities that can
be configured to be useful in treatment regimes for the treatment
of cells, tissues and animals, especially humans.
[0070] For therapeutics, an animal, preferably a human, suspected
of having a disease or disorder which can be treated by modulating
the expression of apolipoprotein B is treated by administering
antisense compounds in accordance with this invention. For example,
in one non-limiting embodiment, the methods comprise the step of
administering to the animal in need of treatment, a therapeutically
effective amount of an apolipoprotein B inhibitor. The
apolipoprotein B inhibitors of the present invention effectively
inhibit the activity of the apolipoprotein B protein or inhibit the
expression of the apolipoprotein B protein. In one embodiment, the
activity or expression of apolipoprotein B in an animal is
inhibited by about 10%. Preferably, the activity or expression of
apolipoprotein B in an animal is inhibited by about 30%. More
preferably, the activity or expression of apolipoprotein B in an
animal is inhibited by 50% or more. Thus, the oligomeric antisense
compounds modulate expression of apolipoprotein B mRNA by at least
10%, by at least 20%, by at least 25%, by at least 30%, by at least
40%, by at least 50%, by at least 60%, by at least 70%, by at least
75%, by at least 80%, by at least 85%, by at least 90%, by at least
95%, by at least 98%, by at least 99%, or by 100%.
[0071] For example, the reduction of the expression of
apolipoprotein B may be measured in serum, adipose tissue, liver or
any other body fluid, tissue or organ of the animal. Preferably,
the cells contained within said fluids, tissues or organs being
analyzed contain a nucleic acid molecule encoding apolipoprotein B
protein and/or the apolipoprotein B protein itself.
[0072] The antisense compounds of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of a
compound to a suitable pharmaceutically acceptable diluent or
carrier. Use of the compounds and methods of the invention may also
be useful prophylactically.
F. Modifications
[0073] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base sometimes referred to as a "nucleobase" or simply
a "base". The two most common classes of such heterocyclic bases
are the purines and the pyrimidines. Nucleotides are nucleosides
that further include a phosphate group covalently linked to the
sugar portion of the nucleoside. For those nucleosides that include
a pentofuranosyl sugar, the phosphate group can be linked to the
2', 3' or 5' hydroxyl moiety of the sugar. In forming
oligonucleotides, the phosphate groups covalently link adjacent
nucleosides to one another to form a linear polymeric compound. In
turn, the respective ends of this linear polymeric compound can be
further joined to form a circular compound, however, linear
compounds are generally preferred. In addition, linear compounds
may have internal nucleobase complementarity and may therefore fold
in a manner as to produce a fully or partially double-stranded
compound. Within oligonucleotides, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
Modified Internucleoside Linkages (Backbones)
[0074] Specific examples of preferred antisense compounds useful in
this invention include oligonucleotides containing modified
backbones or non-natural internucleoside linkages. As defined in
this specification, oligonucleotides having modified backbones
include those that retain a phosphorus atom in the backbone and
those that do not have a phosphorus atom in the backbone. For the
purposes of this specification, and as sometimes referenced in the
art, modified oligonucleotides that do not have a phosphorus atom
in their internucleoside backbone can also be considered to be
oligonucleosides.
[0075] Preferred modified oligonucleotide backbones containing a
phosphorus atom therein include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriaminoalkylphosphotriesters, methyl and other
alkyl phosphonates including 3 `-alkylene phosphonates, 5`-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3 `-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters,
selenophosphates and boranophosphates having normal 3`-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Preferred oligonucleotides having inverted
polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0076] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555;
5,527,899; 5,721,218; 5,672,697 and 5,625,050.
[0077] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
[0078] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608;
5,646,269 and 5,677,439.
Modified Sugar and Internucleoside Linkages-Mimetics
[0079] In other preferred antisense compounds, e.g.,
oligonucleotide mimetics, both the sugar and the internucleoside
linkage (i.e. the backbone), of the nucleotide units are replaced
with novel groups. The nucleobase units are maintained for
hybridization with an appropriate target nucleic acid. One such
compound, an oligonucleotide mimetic that has been shown to have
excellent hybridization properties, is referred to as a peptide
nucleic acid (PNA). In PNA compounds, the sugar-backbone of an
oligonucleotide is replaced with an amide containing backbone, in
particular an aminoethylglycine backbone. The nucleobases are
retained and are bound directly or indirectly to aza nitrogen atoms
of the amide portion of the backbone. Representative United States
patents that teach the preparation of PNA compounds include, but
are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and
5,719,262. Further teaching of PNA compounds can be found in
Nielsen et al., Science, 1991, 254, 1497-1500.
[0080] Preferred embodiments of the invention are oligonucleotides
with phosphorothioate backbones and oligonucleosides with
heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified Sugars
[0081] Modified antisense compounds may also contain one or more
substituted sugar moieties. Preferred are antisense compounds,
preferably antisense oligonucleotides, comprising one of the
following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or
N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the
alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2, also described in
examples hereinbelow.
[0082] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2),
2'-O-allyl(2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F).
The 2'-modification may be in the arabino (up) position or ribo
(down) position. A preferred 2'-arabino modification is 2'-F.
Similar modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Antisense compounds may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920.
[0083] A further preferred modification of the sugar includes
Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is
linked to the 3' or 4' carbon atom of the sugar ring, thereby
forming a bicyclic sugar moiety. The linkage is preferably a
methylene (--CH.sub.2--).sub.n group bridging the 2' oxygen atom
and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation
thereof are described in WO 98/39352 and WO 99/14226.
Natural and Modified Nucleobases
[0084] Antisense compounds may also include nucleobase (often
referred to in the art as heterocyclic base or simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
uracil (U). Modified nucleobases include other synthetic and
natural nucleobases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine
and other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine
and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases
include tricyclic pyrimidines such as phenoxazine cytidine
(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine
cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps
such as a substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the compounds of the
invention. These include 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C. and
are presently preferred base substitutions, even more particularly
when combined with 2'-O-methoxyethyl sugar modifications.
[0085] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653;
5,763,588; 6,005,096; 5,750,692; and 5,681,941.
Conjugates
[0086] Another modification of the antisense compounds of the
invention involves chemically linking to the antisense compound one
or more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. These
moieties or conjugates can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugate groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve uptake,
enhance resistance to degradation, and/or strengthen
sequence-specific hybridization with the target nucleic acid.
Groups that enhance the pharmacokinetic properties, in the context
of this invention, include groups that improve uptake,
distribution, metabolism or excretion of the compounds of the
present invention. Representative conjugate groups are disclosed in
International Patent Application PCT/US92/09196, filed Oct. 23,
1992, and U.S. Pat. No. 6,287,860, the entire disclosures of which
are incorporated herein by reference. Conjugate moieties include
but are not limited to lipid moieties such as a cholesterol moiety,
cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a
thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl
residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a
polyamine or a polyethylene glycol chain, or adamantane acetic
acid, a palmityl moiety, or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety. Antisense compounds of
the invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) which is incorporated herein by reference in its
entirety.
[0087] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941.
Chimeric Compounds
[0088] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an
oligonucleotide.
[0089] The present invention also includes antisense compounds
which are chimeric compounds. "Chimeric" antisense compounds or
"chimeras," in the context of this invention, are antisense
compounds, particularly oligonucleotides, which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of an oligonucleotide
compound. Chimeric antisense oligonucleotides are thus a form of
antisense compound. These oligonucleotides typically contain at
least one region wherein the oligonucleotide is modified so as to
confer upon the oligonucleotide increased resistance to nuclease
degradation, increased cellular uptake, increased stability and/or
increased binding affinity for the target nucleic acid. An
additional region of the oligonucleotide may serve as a substrate
for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way
of example, RNAse H is a cellular endonuclease which cleaves the
RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore,
results in cleavage of the RNA target, thereby greatly enhancing
the efficiency of oligonucleotide-mediated inhibition of gene
expression. The cleavage of RNA:RNA hybrids can, in like fashion,
be accomplished through the actions of endoribonucleases, such as
RNAseL which cleaves both cellular and viral RNA. Cleavage of the
RNA target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known
in the art.
[0090] Chimeric antisense compounds of the invention may be formed
as composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Chimeric antisense compounds can be
of several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers". Such compounds have
also been referred to in the art as hybrids. In a gapmer that is 20
nucleotides in length, a gap or wing can be 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides in length. In
one embodiment, a 20-nucleotide gapmer is comprised of a gap 8
nucleotides in length, flanked on both the 5' and 3' sides by wings
6 nucleotides in length. In another embodiment, a 20-nucleotide
gapmer is comprised of a gap 10 nucleotides in length, flanked on
both the 5' and 3' sides by wings 5 nucleotides in length. In
another embodiment, a 20-nucleotide gapmer is comprised of a gap 12
nucleotides in length flanked on both the 5' and 3' sides by wings
4 nucleotides in length. In a further embodiment, a 20-nucleotide
gapmer is comprised of a gap 14 nucleotides in length flanked on
both the 5' and 3' sides by wings 3 nucleotides in length. In
another embodiment, a 20-nucleotide gapmer is comprised of a gap 16
nucleotides in length flanked on both the 5' and 3' sides by wings
2 nucleotides in length. In a further embodiment, a 20-nucleotide
gapmer is comprised of a gap 18 nucleotides in length flanked on
both the 5' and 3' ends by wings 1 nucleotide in length.
Alternatively, the wings are of different lengths, for example, a
20-nucleotide gapmer may be comprised of a gap 10 nucleotides in
length, flanked by a 6-nucleotide wing on one side (5' or 3') and a
4-nucleotide wing on the other side (5' or 3'). In a hemimer, an
"open end" chimeric antisense compound, 20 nucleotides in length, a
gap segment, located at either the 5' or 3' terminus of the
oligomeric compound, can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18 or 19 nucleotides in length. For example, a
20-nucleotide hemimer can have a gap segment of 10 nucleotides at
the 5' end and a second segment of 10 nucleotides at the 3' end.
Alternatively, a 20-nucleotide hemimer can have a gap segment of 10
nucleotides at the 3' end and a second segment of 10 nucleotides at
the 5' end.
[0091] Representative United States patents that teach the
preparation of such hybrid structures include, but are not limited
to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;
5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;
5,652,356; and 5,700,922.
G. Formulations
[0092] The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor-targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption-assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756.
[0093] 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.
[0094] 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.
[0095] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention. The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration. Pharmaceutical compositions and
formulations for topical administration may include transdermal
patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable. Coated condoms, gloves and the like may
also be useful.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. Emulsions may contain additional components in
addition to the dispersed phases, and the active drug which may be
present as a solution in either the aqueous phase, oily phase or
itself as a separate phase. Microemulsions are included as an
embodiment of the present invention. Emulsions and their uses are
well known in the art and are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety.
[0100] 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.
[0101] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome comprises one or more glycolipids or is
derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. Liposomes and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety.
[0102] The pharmaceutical formulations and compositions of the
present invention may also include surfactants. The use of
surfactants in drug products, formulations and in emulsions is well
known in the art. Surfactants and their uses are further described
in U.S. Pat. No. 6,287,860, which is incorporated herein in its
entirety.
[0103] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides. In addition to aiding the
diffusion of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs. Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants.
Penetration enhancers and their uses are further described in U.S.
Pat. No. 6,287,860, which is incorporated herein in its
entirety.
[0104] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0105] 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).
[0106] For topical or other administration, oligonucleotides of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Preferred fatty acids and esters,
pharmaceutically acceptable salts thereof, and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety. Topical formulations are described in
detail in U.S. patent application Ser. No. 09/315,298 filed on May
20, 1999, which is incorporated herein by reference in its
entirety.
[0107] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Preferred surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Preferred bile
acids/salts and fatty acids and their uses are further described in
U.S. Pat. No. 6,287,860, which is incorporated herein in its
entirety. Also preferred are combinations of penetration enhancers,
for example, fatty acids/salts in combination with bile
acids/salts. A particularly preferred combination is the sodium
salt of lauric acid, capric acid and UDCA. Further penetration
enhancers include polyoxyethylene-9-lauryl ether,
polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention
may be delivered orally, in granular form including sprayed dried
particles, or complexed to form micro or nanoparticles.
Oligonucleotide complexing agents and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein
in its entirety. Oral formulations for oligonucleotides and their
preparation are described in detail in U.S. application Ser. No.
09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20,
1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is
incorporated herein by reference in their entirety.
[0108] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0109] Oligonucleotides may be formulated for delivery in vivo in
an acceptable dosage form, e.g. as parenteral or non-parenteral
formulations. Parenteral formulations include intravenous (IV),
subcutaneous (SC), intraperitoneal (IP), intravitreal and
intramuscular (IM) formulations, as well as formulations for
delivery via pulmonary inhalation, intranasal administration,
topical administration, etc. Non-parenteral formulations include
formulations for delivery via the alimentary canal, e.g. oral
administration, rectal administration, intrajejunal instillation,
etc. Rectal administration includes administration as an enema or a
suppository. Oral administration includes administration as a
capsule, a gel capsule, a pill, an elixir, etc.
[0110] In some embodiments, an oligonucleotide may be administered
to a subject via an oral route of administration. The subject may
be an animal or a human (man). An animal subject may be a mammal,
such as a mouse, a rat, a dog, a guinea pig, a non-human primate, a
cat or a pig. Non-human primates include monkeys and chimpanzees. A
suitable animal subject may be an experimental animal, such as a
mouse, a rat, a dog, a non-human primate, a cat or a pig.
[0111] In some embodiments, the subject may be a human. In certain
embodiments, the subject may be a human patient in need of
therapeutic treatment as discussed in more detail herein. In
certain embodiments, the subject may be in need of modulation of
expression of one or more genes as discussed in more detail herein.
In some particular embodiments, the subject may be in need of
inhibition of expression of one or more genes as discussed in more
detail herein. In particular embodiments, the subject may be in
need of modulation, i.e. inhibition or enhancement, of hepatic
lipase in order to obtain therapeutic indications discussed in more
detail herein.
[0112] In some embodiments, non-parenteral (e.g. oral)
oligonucleotide formulations according to the present invention
result in enhanced bioavailability of the oligonucleotide. In this
context, the term "bioavailability" refers to a measurement of that
portion of an administered drug which reaches the circulatory
system (e.g. blood, especially blood plasma) when a particular mode
of administration is used to deliver the drug. Enhanced
bioavailability refers to a particular mode of administration's
ability to deliver oligonucleotide to the peripheral blood plasma
of a subject relative to another mode of administration. For
example, when a non-parenteral mode of administration (e.g. an oral
mode) is used to introduce the drug into a subject, the
bioavailability for that mode of administration may be compared to
a different mode of administration, e.g. an IV mode of
administration. In some embodiments, the area under a compound's
blood plasma concentration curve (AUC.sub.0) after non-parenteral
(e.g. oral, rectal, intrajejunal) administration may be divided by
the area under the drug's plasma concentration curve after
intravenous (i.v.) administration (AUC.sub.iv) to provide a
dimensionless quotient (relative bioavailability, RB) that
represents fraction of compound absorbed via the non-parenteral
route as compared to the IV route. A composition's bioavailability
is said to be enhanced in comparison to another composition's
bioavailability when the first composition's relative
bioavailability (RB.sub.1) is greater than the second composition's
relative bioavailability (RB.sub.2).
[0113] In general, bioavailability correlates with therapeutic
efficacy when a compound's therapeutic efficacy is related to the
blood concentration achieved, even if the drug's ultimate site of
action is intracellular (van Berge-Henegouwen et al.,
Gastroenterol., 1977, 73, 300). Bioavailability studies have been
used to determine the degree of intestinal absorption of a drug by
measuring the change in peripheral blood levels of the drug after
an oral dose (DiSanto, Chapter 76 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 1451-1458).
[0114] In general, an oral composition's bioavailability is said to
be "enhanced" when its relative bioavailability is greater than the
bioavailability of a composition substantially consisting of pure
oligonucleotide, i.e. oligonucleotide in the absence of a
penetration enhancer.
[0115] Organ bioavailability refers to the concentration of
compound in an organ. Organ bioavailability may be measured in test
subjects by a number of means, such as by whole-body radiography.
Organ bioavailability may be modified, e.g. enhanced, by one or
more modifications to the oligonucleotide, by use of one or more
carrier compounds or excipients, etc. as discussed in more detail
herein. In general, an increase in bioavailability will result in
an increase in organ bioavailability.
[0116] Oral oligonucleotide compositions according to the present
invention may comprise one or more "mucosal penetration enhancers,"
also known as "absorption enhancers" or simply as "penetration
enhancers." Accordingly, some embodiments of the invention comprise
at least one oligonucleotide in combination with at least one
penetration enhancer. In general, a penetration enhancer is a
substance that facilitates the transport of a drug across mucous
membrane(s) associated with the desired mode of administration,
e.g. intestinal epithelial membranes. Accordingly it is desirable
to select one or more penetration enhancers that facilitate the
uptake of an oligonucleotide, without interfering with the activity
of the oligonucleotide, and in a such a manner the oligonucleotide
can be introduced into the body of an animal without unacceptable
side-effects such as toxicity, irritation or allergic response.
[0117] Embodiments of the present invention provide compositions
comprising one or more pharmaceutically acceptable penetration
enhancers, and methods of using such compositions, which result in
the improved bioavailability of oligonucleotides administered via
non-parenteral modes of administration. Heretofore, certain
penetration enhancers have been used to improve the bioavailability
of certain drugs. See Muranishi, Crit. Rev. Ther. Drug Carrier
Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. Drug Carrier
Systems, 1991, 8, 91. It has been found that the uptake and
delivery of oligonucleotides, relatively complex molecules which
are known to be difficult to administer to animals and man, can be
greatly improved even when administered by non-parenteral means
through the use of a number of different classes of penetration
enhancers.
[0118] In some embodiments, compositions for non-parenteral
administration include one or more modifications from
naturally-occurring oligonucleotides (i.e. full-phosphodiester
deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such
modifications may increase binding affinity, nuclease stability,
cell or tissue permeability, tissue distribution, or other
biological or pharmacokinetic property. Modifications may be made
to the base, the linker, or the sugar, in general, as discussed in
more detail herein with regards to oligonucleotide chemistry. In
some embodiments of the invention, compositions for administration
to a subject, and in particular oral compositions for
administration to an animal or human subject, will comprise
modified oligonucleotides having one or more modifications for
enhancing affinity, stability, tissue distribution, or other
biological property.
[0119] Suitable modified linkers include phosphorothioate linkers.
In some embodiments according to the invention, the oligonucleotide
has at least one phosphorothioate linker. Phosphorothioate linkers
provide nuclease stability as well as plasma protein binding
characteristics to the oligonucleotide. Nuclease stability is
useful for increasing the in vivo lifetime of oligonucleotides,
while plasma protein binding decreases the rate of first pass
clearance of oligonucleotide via renal excretion. In some
embodiments according to the present invention, the oligonucleotide
has at least two phosphorothioate linkers. In some embodiments,
wherein the oligonucleotide has exactly n nucleosides, the
oligonucleotide has from one to n-1 phosphorothioate linkages. In
some embodiments, wherein the oligonucleotide has exactly n
nucleosides, the oligonucleotide has n-1 phosphorothioate linkages.
In other embodiments wherein the oligonucleotide has exactly n
nucleoside, and n is even, the oligonucleotide has from 1 to n/2
phosphorothioate linkages, or, when n is odd, from 1 to (n-1)/2
phosphorothioate linkages. In some embodiments, the oligonucleotide
has alternating phosphodiester (PO) and phosphorothioate (PS)
linkages. In other embodiments, the oligonucleotide has at least
one stretch of two or more consecutive PO linkages and at least one
stretch of two or more PS linkages. In other embodiments, the
oligonucleotide has at least two stretches of PO linkages
interrupted by at least on PS linkage.
[0120] In some embodiments, at least one of the nucleosides is
modified on the ribosyl sugar unit by a modification that imparts
nuclease stability, binding affinity or some other beneficial
biological property to the sugar. In some cases, the sugar
modification includes a 2'-modification, e.g. the 2'-OH of the
ribosyl sugar is replaced or substituted. Suitable replacements for
2'-OH include 2'-F and 2'-arabino-F. Suitable substitutions for OH
include 2'-O-alkyl, e.g. 2-O-methyl, and 2'-O-substituted alkyl,
e.g. 2'-O-methoxyethyl, 2'-O-aminopropyl, etc. In some embodiments,
the oligonucleotide contains at least one 2'-modification. In some
embodiments, the oligonucleotide contains at least 2
2'-modifications. In some embodiments, the oligonucleotide has at
least one 2'-modification at each of the termini (i.e. the 3'- and
5'-terminal nucleosides each have the same or different
2'-modifications). In some embodiments, the oligonucleotide has at
least two sequential 2'-modifications at each end of the
oligonucleotide. In some embodiments, oligonucleotides further
comprise at least one deoxynucleoside. In particular embodiments,
oligonucleotides comprise a stretch of deoxynucleosides such that
the stretch is capable of activating RNase (e.g. RNase H) cleavage
of an RNA to which the oligonucleotide is capable of hybridizing.
In some embodiments, a stretch of deoxynucleosides capable of
activating RNase-mediated cleavage of RNA comprises about 6 to
about 16, e.g. about 8 to about 16 consecutive
deoxynucleosides.
[0121] Oral compositions for administration of non-parenteral
oligonucleotide compositions of the present invention may be
formulated in various dosage forms such as, but not limited to,
tablets, capsules, liquid syrups, soft gels, suppositories, and
enemas. The term "alimentary delivery" encompasses e.g. oral,
rectal, endoscopic and sublingual/buccal administration. A common
requirement for these modes of administration is absorption over
some portion or all of the alimentary tract and a need for
efficient mucosal penetration of the nucleic acid(s) so
administered.
[0122] Delivery of a drug via the oral mucosa, as in the case of
buccal and sublingual administration, has several desirable
features, including, in many instances, a more rapid rise in plasma
concentration of the drug than via oral delivery (Harvey, Chapter
35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990, page 711).
[0123] Endoscopy may be used for drug delivery directly to an
interior portion of the alimentary tract. For example, endoscopic
retrograde cystopancreatography (ERCP) takes advantage of extended
gastroscopy and permits selective access to the biliary tract and
the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992,
19(10 Suppl.), 1591). Pharmaceutical compositions, including
liposomal formulations, can be delivered directly into portions of
the alimentary canal, such as, e.g., the duodenum (Somogyi et al.,
Pharm. Res., 1995, 12, 149) or the gastric submucosa (Akamo et al.,
Japanese J. Cancer Res., 1994, 85, 652) via endoscopic means.
Gastric lavage devices (Inoue et al., Artif. Organs, 1997, 21, 28)
and percutaneous endoscopic feeding devices (Pennington et al.,
Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for direct
alimentary delivery of pharmaceutical compositions.
[0124] In some embodiments, oligonucleotide formulations may be
administered through the anus into the rectum or lower intestine.
Rectal suppositories, retention enemas or rectal catheters can be
used for this purpose and may be preferred when patient compliance
might otherwise be difficult to achieve (e.g., in pediatric and
geriatric applications, or when the patient is vomiting or
unconscious). Rectal administration can result in more prompt and
higher blood levels than the oral route. (Harvey, Chapter 35 In:
Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990, page 711). Because about 50% of
the drug that is absorbed from the rectum will bypass the liver,
administration by this route significantly reduces the potential
for first-pass metabolism (Benet et al., Chapter 1 In: Goodman
& Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,
Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).
[0125] One advantageous method of non-parenteral administration
oligonucleotide compositions is oral delivery. Some embodiments
employ various penetration enhancers in order to effect transport
of oligonucleotides and other nucleic acids across mucosal and
epithelial membranes. Penetration enhancers may be classified as
belonging to one of five broad categories--surfactants, fatty
acids, bile salts, chelating agents, and non-chelating
non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, p. 92). Accordingly, some embodiments
comprise oral oligonucleotide compositions comprising at least one
member of the group consisting of surfactants, fatty acids, bile
salts, chelating agents, and non-chelating surfactants. Further
embodiments comprise oral oligonucleotide comprising at least one
fatty acid, e.g. capric or lauric acid, or combinations or salts
thereof. Other embodiments comprise methods of enhancing the oral
bioavailability of an oligonucleotide, the method comprising
co-administering the oligonucleotide and at least one penetration
enhancer.
[0126] Other excipients that may be added to oral oligonucleotide
compositions include surfactants (or "surface-active agents"),
which are chemical entities which, when dissolved in an aqueous
solution, reduce the surface tension of the solution or the
interfacial tension between the aqueous solution and another
liquid, with the result that absorption of oligonucleotides through
the alimentary mucosa and other epithelial membranes is enhanced.
In addition to bile salts and fatty acids, surfactants include, for
example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and
polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, page 92); and
perfluorohemical emulsions, such as FC-43 (Takahashi et al., J.
Pharm. Phamacol., 1988, 40, 252).
[0127] Fatty acids and their derivatives which act as penetration
enhancers and may be used in compositions of the present invention
include, for example, oleic acid, lauric acid, capric acid
(n-decanoic acid), myristic acid, palmitic acid, stearic acid,
linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein
(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic
acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one,
acylcarnitines, acylcholines and mono- and di-glycerides thereof
and/or physiologically acceptable salts thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1; El-Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651).
[0128] In some embodiments, oligonucleotide compositions for oral
delivery comprise at least two discrete phases, which phases may
comprise particles, capsules, gel-capsules, microspheres, etc. Each
phase may contain one or more oligonucleotides, penetration
enhancers, surfactants, bioadhesives, effervescent agents, or other
adjuvant, excipient or diluent. In some embodiments, one phase
comprises at least one oligonucleotide and at lease one penetration
enhancer. In some embodiments, a first phase comprises at least one
oligonucleotide and at least one penetration enhancer, while a
second phase comprises at least one penetration enhancer. In some
embodiments, a first phase comprises at least one oligonucleotide
and at least one penetration enhancer, while a second phase
comprises at least one penetration enhancer and substantially no
oligonucleotide. In some embodiments, at least one phase is
compounded with at least one degradation retardant, such as a
coating or a matrix, which delays release of the contents of that
phase. In some embodiments, a first phase comprises at least one
oligonucleotide, at least one penetration enhancer, while a second
phase comprises at least one penetration enhancer and a
release-retardant. In particular embodiments, an oral
oligonucleotide comprises a first phase comprising particles
containing an oligonucleotide and a penetration enhancer, and a
second phase comprising particles coated with a release-retarding
agent and containing penetration enhancer.
[0129] A variety of bile salts also function as penetration
enhancers to facilitate the uptake and bioavailability of drugs.
The physiological roles of bile include the facilitation of
dispersion and absorption of lipids and fat-soluble vitamins
(Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological
Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill,
New York, N.Y., 1996, pages 934-935). Various natural bile salts,
and their synthetic derivatives, act as penetration enhancers.
Thus, the term "bile salt" includes any of the naturally occurring
components of bile as well as any of their synthetic derivatives.
The bile salts of the invention include, for example, cholic acid
(or its pharmaceutically acceptable sodium salt, sodium cholate),
dehydrocholic acid (sodium dehydrocholate), deoxycholic acid
(sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1; Yamamoto et al., J. Pharm. Exp.
Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79,
579).
[0130] In some embodiments, penetration enhancers useful in some
embodiments of present invention are mixtures of penetration
enhancing compounds. One such penetration enhancer is a mixture of
UDCA (and/or CDCA) with capric and/or lauric acids or salts thereof
e.g. sodium. Such mixtures are useful for enhancing the delivery of
biologically active substances across mucosal membranes, in
particular intestinal mucosa. Other penetration enhancer mixtures
comprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with
5-95% capric and/or lauric acid. Particular penetration enhancers
are mixtures of the sodium salts of UDCA, capric acid and lauric
acid in a ratio of about 1:2:2 respectively. Anther such
penetration enhancer is a mixture of capric and lauric acid (or
salts thereof) in a 0.01:1 to 1:0.01 ratio (mole basis). In
particular embodiments capric acid and lauric acid are present in
molar ratios of e.g. about 0.1:1 to about 1:0.1, in particular
about 0.5:1 to about 1:0.5.
[0131] Other excipients include chelating agents, i.e. compounds
that remove metallic ions from solution by forming complexes
therewith, with the result that absorption of oligonucelotides
through the alimentary and other mucosa is enhanced. With regards
to their use as penetration enhancers in the present invention,
chelating agents have the added advantage of also serving as DNase
inhibitors, as most characterized DNA nucleases require a divalent
metal ion for catalysis and are thus inhibited by chelating agents
(Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents of the
invention include, but are not limited to, disodium
ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,
sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl
derivatives of collagen, laureth-9 and N-amino acyl derivatives of
beta-diketones (enamines)(Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1;
Buur et al., J. Control Rel., 1990, 14, 43).
[0132] As used herein, non-chelating non-surfactant penetration
enhancers may be defined as compounds that demonstrate
insignificant activity as chelating agents or as surfactants but
that nonetheless enhance absorption of oligonucleotides through the
alimentary and other mucosal membranes (Muranishi, Critical Reviews
in Therapeutic Drug Carrier Systems, 1990, 7, 1). This class of
penetration enhancers includes, but is not limited to, unsaturated
cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621).
[0133] Agents that enhance uptake of oligonucleotides at the
cellular level may also be added to the pharmaceutical and other
compositions of the present invention. For example, cationic
lipids, such as lipofectin (Junichi et al, U.S. Pat. No.
5,705,188), cationic glycerol derivatives, and polycationic
molecules, such as polylysine (Lollo et al., PCT Application WO
97/30731), can be used.
[0134] Some oral oligonucleotide compositions also incorporate
carrier compounds in the formulation. As used herein, "carrier
compound" or "carrier" can refer to a nucleic acid, or analog
thereof, which may be inert (i.e., does not possess biological
activity per se) or may be necessary for transport, recognition or
pathway activation or mediation, or is recognized as a nucleic acid
by in vivo processes that reduce the bioavailability of a nucleic
acid having biological activity by, for example, degrading the
biologically active nucleic acid or promoting its removal from
circulation. The coadministration of a nucleic acid and a carrier
compound, typically with an excess of the latter substance, can
result in a substantial reduction of the amount of nucleic acid
recovered in the liver, kidney or other extracirculatory
reservoirs, presumably due to competition between the carrier
compound and the nucleic acid for a common receptor. For example,
the recovery of a partially phosphorothioate oligonucleotide in
hepatic tissue can be reduced when it is coadministered with
polyinosinic acid, dextran sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense
& Nucl. Acid Drug Dev., 1996, 6, 177).
[0135] A "pharmaceutical carrier" or "excipient" may be a
pharmaceutically acceptable solvent, suspending agent or any other
pharmacologically inert vehicle for delivering one or more nucleic
acids to an animal. The excipient may be liquid or solid and is
selected, with the planned manner of administration in mind, so as
to provide for the desired bulk, consistency, etc., when combined
with a nucleic acid and the other components of a given
pharmaceutical composition. Typical pharmaceutical carriers
include, but are not limited to, binding agents (e.g.,
pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl
methylcellulose, etc.); fillers (e.g., lactose and other sugars,
microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl
cellulose, polyacrylates or calcium hydrogen phosphate, etc.);
lubricants (e.g., magnesium stearate, talc, silica, colloidal
silicon dioxide, stearic acid, metallic stearates, hydrogenated
vegetable oils, corn starch, polyethylene glycols, sodium benzoate,
sodium acetate, etc.); disintegrants (e.g., starch, sodium starch
glycolate, EXPLOTAB); and wetting agents (e.g., sodium lauryl
sulphate, etc.).
[0136] Oral oligonucleotide compositions may additionally contain
other adjunct components conventionally found in pharmaceutical
compositions, at their art-established usage levels. Thus, for
example, the compositions may contain additional, compatible,
pharmaceutically-active materials such as, for example,
antipuritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the composition of present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention.
[0137] 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, hexamethyl-melamine, 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.
[0138] In another related embodiment, compositions of the invention
may contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Alternatively, compositions of the invention may contain
two or more antisense compounds targeted to different regions of
the same nucleic acid target. Numerous examples of antisense
compounds are known in the art. Two or more combined compounds may
be used together or sequentially.
H. Dosing
[0139] 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.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides,
and can generally be estimated based on EC.sub.50s found to be
effective in in vitro and in vivo animal models. In general, dosage
is from 0.01 ug to 100 g per kg of body weight, from 0.1 .mu.g to
10 g per kg of body weight, from 1.0 .mu.g to 1 g per kg of body
weight, from 10.0 .mu.g to 100 mg per kg of body weight, from 100
.mu.g to 10 mg per kg of body weight, or from 1 mg to 5 mg per kg
of body weight and may be given once or more daily, weekly, monthly
or yearly, or even once every 2 to 20 years. Persons of ordinary
skill in the art can easily estimate repetition rates for dosing
based on measured residence times and concentrations of the drug in
bodily fluids or tissues. Following successful treatment, it may be
desirable to have the patient undergo maintenance therapy to
prevent the recurrence of the disease state, wherein the
oligonucleotide is administered in maintenance doses, ranging from
0.01 ug to 100 g per kg of body weight, once or more daily, to once
every 20 years.
[0140] The effects of treatments with therapeutic compositions can
be assessed following collection of tissues or fluids from a
patient or subject receiving said treatments. It is known in the
art that a biopsy sample can be procured from certain tissues
without resulting in detrimental effects to a patient or subject.
In certain embodiments, a tissue and its constituent cells
comprise, but are not limited to, blood (e.g., hematopoietic cells,
such as human hematopoietic progenitor cells, human hematopoietic
stem cells, CD34.sup.+ cells CD4.sup.+ cells), lymphocytes and
other blood lineage cells, bone marrow, breast, cervix, colon,
esophagus, lymph node, muscle, peripheral blood, oral mucosa and
skin. In other embodiments, a fluid and its constituent cells
comprise, but are not limited to, blood, urine, semen, synovial
fluid, lymphatic fluid and cerebro-spinal fluid. Tissues or fluids
procured from patients can be evaluated for expression levels of
the target mRNA or protein by techniques known in the art.
Additionally, the mRNA or protein expression levels of other genes
known or suspected to be associated with the specific disease
state, condition or phenotype, or levels of biological markers
associated with the disease state, condition or phenotype, can
similarly be assessed. Target or associated gene mRNA levels can be
measured or evaluated by real-time PCR, Northern blot, in situ
hybridization or DNA array analysis. Target or associated protein
levels or biomarkers can be measured or evaluated by ELISA,
immunoblotting, quantitative protein assays, protein activity
assays (for example, caspase activity assays) immunohistochemistry,
immunocytochemistry or routine clinical analysis.
I. Polymorphisms
[0141] One common allelic genomic sequence for apolipoprotein B is
set forth in SEQ ID NO: 1 and is referred to herein as the
"wild-type" sequence. Novel polymorphic sites have been identified
in this gene at positions 27751, 27735, 27685, 27683, 27679, 27634,
27627 and 27618 of SEQ ID NO: 1 (and corresponding positions 15695,
15711, 15761, 15763, 15767, 15812, 15819 and 15828 of the reverse
complement, SEQ ID NO: 2).
[0142] A polymorphism is a sequence variation in the gene observed
within the population, and can include nucleotide substitutions
(single nucleotide polymorphisms or SNPs), insertions, or
deletions. Polymorphisms may or may not result in detectable
differences in gene expression, protein structure, or protein
function. A polymorphism may alter one or more properties of the
gene or gene products, including DNA or RNA stability, binding of
transcriptional or translation factors to the DNA or RNA,
interactions of the DNA or RNA with other parts of the nuclear or
cytosolic cell machinery, or may confer a change upon the encoded
polypeptide sequence which in turn may alter the polypeptide's
biological activity. Identification of polymorphisms among various
populations is desirable to tailor design of suitable antisense
therapeutics, select antisense therapeutics to administer to a
particular population, and also predict responsiveness to
therapeutics.
[0143] A "polymorphic site" is a position within a genetic locus at
which at least one alternative nucleotide sequence variation (e.g.,
substitution, insertion, or deletion) has been observed in a
population, and includes the position on both complementary strands
at the polymorphic site. The first identified form of the
nucleotide sequence at the polymorphic site is sometimes called the
reference sequence, and the alternative forms are called
alternative or variant alleles (or "allelic variant"). The most
commonly occurring form of the nucleotide sequence at the
polymorphic site is also sometimes called the wild type allele.
Polymorphic sites of the invention are listed in the following
table along with their approximate frequency.
TABLE-US-00001 Approximate frequency of Position in SNP detection
out of 213 Sequence variation SEQ ID NO: 1 samples of diverse
ancestry Substitution of A to G 27751 15% Substitution of C to G
27735 <1% Substitution of T to C 27685 27% Substitution of T to
C 27683 Substitution of T to C 27679 40% Substitution of C to T
27634 <1% Substitution of G to A 27627 2% Substitution of T to C
27618 <1%
[0144] The invention provides a variety of polynucleotides,
including reverse complements, single or double stranded
polynucleotides, RNA, DNA or mimetics thereof, and antisense
compounds, as defined above, that either contain or specifically
hybridize to a polymorphic sequence at one or more of these
polymorphic sites. Such "sequence-specific" polynucleotides can be
used, alone or linked to other moieties, in a variety of areas. For
example, the polynucleotides may be useful as therapeutic products
for inhibiting gene expression, as probes or primers for detecting
the polymorphic sequence as part of genotyping, diagnostic,
pharmacogenomics and/or treatment methods, as part of arrays for
screening, as part of diagnostic or therapeutic kits, or as tools
for producing recombinant protein in host cells or transgenic
organisms.
[0145] Methods of producing polynucleotides are well-known in the
art, including chemical synthesis, cloning, and PCR amplification.
The polymorphic polynucleotide sequences of the invention are
preferably isolated, meaning in a form other than as part of an
intact naturally occurring chromosome. Usually the polynucleotide
sequences will also be purified, meaning at least about 50%, 75%,
80% or 90% pure or substantially free of other polynucleotide
sequences that do not include an apolipoprotein B polynucleotide
sequence or fragment thereof. The polynucleotide sequences can also
be "recombinant", meaning flanked by one or more nucleotides with
which they are not normally associated on a naturally occurring
chromosome.
[0146] As noted elsewhere herein, polynucleotides or
oligonucleotides may include modifications, including but not
limited to modifications to the internucleoside linkages,
modifications to the sugar moieties, and modified nucleobases, so
long as the modified polynucleotides retain the ability to
hybridize specifically to the target polymorphic site.
[0147] Such polynucleotides of the invention may be linked to a
second moiety such as an additional nucleotide sequence for
stabilization purposes or for directing transcription or
translation, a moiety which facilitates linkage to a solid support
(such as a microarray or microparticle), or a label to facilitate
detection of the polynucleotide. Such labels include, without
limitation, a radioactive label, a fluorescent label, a
chemiluminescent label, a paramagnetic label, an enzymatic label,
one member of a high affinity binding partner pair (such as
biotin/avidin) or other labels known in the art. The second moiety
may be attached to any position of the polynucleotide, so long as
the polynucleotide retains its ability to hybridize to the
polymorphic sites described herein. The second moiety may be linked
to the polynucleotide after it has been generated, or may be linked
to a component nucleobase that is then incorporated into the
polynucleotide during synthesis or assembly. Polynucleotides of the
invention can also be attached to the surface of a solid support
through means not involving direct chemical linkage.
[0148] As used herein, "sequence-specific" means that the
polynucleotide, oligonucleotide or antisense compound specifically
hybridizes to one nucleotide sequence at a polymorphic site
compared to another, e.g. preferentially hybridizes more strongly
to the one sequence than to an alternative nucleotide sequence that
has been observed in some individuals at that polymorphic site.
[0149] Sequence-specific polynucleotides when used for sequence
detection must be capable of hybridizing to the polymorphic sites
under conditions of stringency such as those employed in
hybridization-based sequence determination methods, primer
extension-based sequence determination methods, restriction site
analysis, polynucleotide amplification methods, ligase-based
sequencing methods, methods based on enzymatic detection of
mismatches, microarray-based sequence determination methods, and
other sequence determination methods known in the art. In a related
embodiment, the invention also contemplates primer pairs comprising
an oligonucleotide useful for amplification of a polymorphic site
in the gene. Such primer pairs may comprise the polymorphic site or
may surround it. Kits comprising such oligonucleotides and primer
pairs are also contemplated.
[0150] In some embodiments, the invention provides
sequence-specific polynucleotides comprising at least 15 contiguous
nucleotides of SEQ ID NO: 1, or comprising at least 15 continguous
nucleotides of an allelic variant of SEQ ID NO: 1, said
polynucleotide including at least one of:
C at position 27751 of SEQ ID NO: 1; C at position 27735 of SEQ ID
NO: 1; G at position 27685 of SEQ ID NO: 1; G at position 27683 of
SEQ ID NO: 1; G at position 27679 of SEQ ID NO: 1; A at position
27634 of SEQ ID NO: 1; T/U at position 27627 of SEQ ID NO: 1; or G
at position 27618 of SEQ ID NO: 1, wherein G is guanine, C is
cytosine, T is thymine, U is uracil, and A is adenine.
[0151] In related embodiments, the invention further provides
sequence-specific polynucleotides comprising at least 15 contiguous
nucleotides of SEQ ID NO: 2, or comprising at least 15 contiguous
nucleotides of an allelic variant of SEQ ID NO: 2, which is the
reverse complement of SEQ ID NO: 1, said polynucleotide including
at least one of:
G at position 15695 of SEQ ID NO: 2; G at position 15711 of SEQ ID
NO: 2; C at position 15761 of SEQ ID NO: 2; C at position 15763 of
SEQ ID NO: 2; C at position 15767 of SEQ ID NO: 2; T/U at position
15812 of SEQ ID NO: 2; A at position 15819 of SEQ ID NO: 2; or C at
position 15828 of SEQ ID NO: 2.
[0152] Such polynucleotides, including reverse complements, or
single or double stranded polynucleotides, may range in length, for
example, from at least 8, 12, 15, or 20 bases, such as 12-20,
15-30, 15-50, 50-100, 8-80, 8-30, 8-50, 12-50, or 12-30 contiguous
bases, or may correspond to the full length of the encoding
cDNA.
[0153] As part of these above aspects of the invention, the
invention contemplates a sequence-specific oligonucleotide or
antisense compound of no more than 100 nucleobases in length that
hybridize to a portion of SEQ ID NO: 1 including at least one
polymorphic site selected from the group consisting of:
C at position 27751 of SEQ ID NO: 1; C at position 27735 of SEQ ID
NO: 1; G at position 27685 of SEQ ID NO: 1; G at position 27683 of
SEQ ID NO: 1; G at position 27679 of SEQ ID NO: 1; A at position
27634 of SEQ ID NO: 1; T/U at position 27627 of SEQ ID NO: 1; or G
at position 27618 of SEQ ID NO: 1.
[0154] The invention also contemplates a sequence-specific
oligonucleotides or antisense compound of no more than 100
nucleobases in length that hybridizes to a portion of SEQ ID NO: 2,
which is the reverse complement of SEQ ID NO: 1, including at least
one polymorphic site selected from the group consisting of:
G at position 15695 of SEQ ID NO: 2; G at position 15711 of SEQ ID
NO: 2; C at position 15761 of SEQ ID NO: 2; C at position 15763 of
SEQ ID NO: 2; C at position 15767 of SEQ ID NO: 2; T/U at position
15812 of SEQ ID NO: 2; A at position 15819 of SEQ ID NO: 2; or C at
position 15828 of SEQ ID NO: 2.
[0155] The invention further contemplates an oligonucleotide
comprising about 15 to 30 contiguous nucleobases of an allelic
variant of SEQ ID NO: 1, said allelic variant comprising at least
one of:
C at position 27751 of SEQ ID NO: 1; C at position 27735 of SEQ ID
NO: 1; G at position 27685 of SEQ ID NO: 1; G at position 27683 of
SEQ ID NO: 1; G at position 27679 of SEQ ID NO: 1; A at position
27634 of SEQ ID NO: 1; T/U at position 27627 of SEQ ID NO: 1; or G
at position 27618 of SEQ ID NO: 1.
[0156] The invention also contemplates an oligonucleotide
comprising about 15 to 30 contiguous nucleobases of an allelic
variant of SEQ ID NO: 2, said allelic variant comprising at least
one of:
G at position 15695 of SEQ ID NO: 2; G at position 15711 of SEQ ID
NO: 2; C at position 15761 of SEQ ID NO: 2; C at position 15763 of
SEQ ID NO: 2; C at position 15767 of SEQ ID NO: 2; T/U at position
15812 of SEQ ID NO: 2; A at position 15819 of SEQ ID NO: 2; or C at
position 15828 of SEQ ID NO: 2.
[0157] As noted above, such oligonucleotides or antisense compounds
may be single or double stranded, may include reverse complements,
may be RNA, DNA or mimetics, may be chemically modified, and may
range in length, for example, from at least 8, 12, 15, or 20 bases,
such as 12-20, 15-30, 15-50, 50-100, 8-80, 8-30, 8-50, 12-50, or
12-30 contiguous bases. In particular, an oligonucleotide or
antisense compound that specifically hybridizes with a polymorphic
sequence of a polymorphic site identified herein is useful for
antisense therapy as described in other sections herein.
[0158] Where the polymorphism results in a change in the encoded
amino acid sequence, expression vectors, host cells and recombinant
organisms useful for producing the encoded protein are additionally
contemplated. Polypeptides of limited length may also be prepared
using chemical synthesis methods. Expression vectors may include
nucleotide sequences that regulate transcription and/or
translation, which may be inducible or constitutive, and which are
preferably operably linked to coding sequence. Host cells include
any prokaryotic, eukaryotic host cells known in the art, including
bacteria, yeast, insect and mammalian cells. A large variety of
techniques for expressing and purifying recombinant protein in host
cell systems are known in the art. The polynucleotides of the
invention can also be used to generate genetically modified (or
transgenic) non-human animals or site specific gene modifications
in cell lines using techniques known in the art. Such transgenic
animals or cell lines include those in which the polymorphic gene
is deleted or knocked out, those in which an exogenous
polynucleotide comprising the polymorphism is stably inserted and
transmitted to progeny, or those in which an endogenous
polynucleotide comprising the polymorphism is operably linked to an
exogenous regulatory sequence. Homologous recombination techniques
are well known, and may utilize nucleic acid alone or as part of a
suitable vector, such as viral vectors.
[0159] The variant polypeptides encoded by polynucleotides
comprising one or more polymorphisms are also of interest, as are
fragments thereof particularly antigenic epitopes, functional
domains, binding sites, and other regions of interest, and
including fusion proteins thereof. Polypeptides thus expressed are
useful for protein structure analysis, for drug binding studies,
and for screening candidate drugs to treat diseases related to
apolipoprotein B activity. Antibodies specific for the variant
polypeptides that differentiate between variant polypeptides and
wild type polypeptide, including monoclonal antibodies and
humanized or human antibodies, are also contemplated.
[0160] Expression assays can be used to detect differences in
expression of polymorphisms with respect to tissue specificity,
expression level, or expression in response to exposure to various
substrates, and/or timing of expression during development.
Expression assays may be performed in cell-free extracts, or by
transforming cells with a suitable vector. Alterations in
expression may occur in the basal level that is expressed in one or
more cell types, or in the effect that an expression modifier has
on the ability of the gene to be inhibited or induced. Expression
levels of variant alleles are compared by various methods known in
the art.
[0161] Screening can also be performed to determine if the
polymorphisms described herein are genetically linked to other
polymorphisms, to microsatellite markers, or to a phenotypic
variant in apolipoprotein B activity or expression. Two
polymorphisms may be in linkage disequilibrium, i.e. where alleles
show non-random associations between genes even though individual
loci are in Hardy-Weinberg equilibrium. Association of a
polymorphism with a phenotypic trait (risk of a disease, severity
or staging of a disease, or response to a drug) can also be
identified by comparing the frequency of the polymorphism in a
population exhibiting the trait to the frequency in a reference
population; a higher frequency occurrence of the polymorphism in
the population exhibiting the trait indicates that the trait is
associated with the polymorphism. When such an association is
established, the risk of disease, severity or staging of a disease,
or response of an individual to a drug can then be predicted by
determining the patient's genotype with respect to the
polymorphism. Where there is a differential distribution of a
polymorphism by racial background, guidelines for drug
administration can be generally tailored to a particular ethnic
group.
[0162] Identifying the presence or absence of a SNP is useful in
methods of genotyping a human comprising the step of determining
the identity of a nucleotide at a particular polymorphic site, in
either the sense strand or its complement. The genotyping method
may comprise identifying the nucleotide pair that is present at one
or more polymorphic sites described herein. Genotyping compositions
or kits of the invention comprises an oligonucleotide probe or
primer which is designed to specifically hybridize to a target
region containing, or adjacent to, one of these novel polymorphic
sites. A genotyping kit of the invention may further comprise a set
of oligonucleotides designed to genotype other polymorphic
sites.
[0163] Detection of the polymorphism can be performed by DNA or RNA
sequence analysis of any patient sample that contains genetic
material, including biopsied tissue, blood, skin, or other cell
samples. The sample polynucleotide or desired segment thereof can
be amplified or cloned by methods known in the art. The presence or
absence of the polymorphism in question can be determined in a
variety of ways known in the art. For example, the sequence of the
sample polynucleotide may be determined by dideoxy sequencing or
other conventional chemical analytical methods. Hybridization-based
methods include Southern blots or dot blots, detecting a pattern of
hybridization to sets of probes, ligase-based methods, primer
extension-based methods, allele-specific amplification, Taqman, and
other PCR-based methods. Other methods such as single strand
conformational polymorphism (SSCP) analysis, denaturing gradient
gel electrophoresis (DGGE), and heteroduplex analysis in gel
matrices are used to detect conformational changes created by DNA
sequence variation as alterations in electrophoretic mobility. If a
polymorphism creates or destroys a recognition site for a
restriction endonuclease (restriction fragment length polymorphism,
RFLP), polymorphic sequence can be detected by digesting the sample
with that endonuclease, and separating the products by size (e.g.
using gel or capillary electrophoresis) to determine whether the
fragment was digested. Mismatch cleavage detection using enzymes or
chemical cleavage agents followed by detecting product size using
electrophoretic or mass spectrometry methods can also be carried
out. Moreover, in cases where the polymorphism of the invention is
linked to another marker (such as another polymorphism or a
microsatellite marker) then detecting the presence of the marker
serves to detect the presence of the polymorphism.
[0164] In one preferred embodiment, the invention provides a method
of analyzing a patient's polynucleotides for the presence or
absence of a mutation comprising: (a) providing a test sample
comprising polynucleotides or replicas thereof from a biological
sample obtained from the patient; (b) contacting the test sample
with a probe comprising at least 15 contiguous nucleotides of the
nucleotide sequence of SEQ ID NO: 1 or the complement thereof, the
probe comprising at least one of the nucleotides at a polymorphic
site in SEQ ID NO: 1 or the complement thereof; and (c) determining
if the test sample comprises a polynucleotide that specifically
hybridizes to the probe.
[0165] In another preferred embodiment, the invention provides a
method of analyzing a patient's polynucleotides for the presence or
absence of a mutation using PCR comprising: (a) providing a test
sample comprising polynucleotides or replicas thereof from a
biological sample obtained from the patient; (b) contacting the
test sample with at least one primer comprising at least 15
contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1
or its complement, and a polymerase, wherein the primer comprises
at least one of the nucleotides at a polymorphic site in SEQ ID NO:
1 or the complement thereof; and (c) determining if a PCR product
of the appropriate size is amplified.
[0166] Such analysis methods and related kits are useful for
diagnostic, prognostic or pharmacogenomic purposes. Thus, the
invention provides methods of (1) predicting risk of developing a
disease condition (2) diagnosing a condition, and/or (3) predicting
prognosis of a condition comprising: (a) analyzing a patient's
polynucleotides to determine the identity of at least one of the
nucleotides at a polymorphic site in SEQ ID NO: 1, wherein the
presence or absence of the nucleotide correlates with a higher
likelihood of developing said condition. The correlation may be
based on statistically associating either the presence or absence
of a single polymorphism (or multiple polymorphisms) with risk of
developing a disease or condition, or with the diagnosis of a
disease or condition, or with prognosis or staging of a disease or
condition.
[0167] The invention further provides a method for selecting a
treatment for a patient suffering from a disease or condition by
determining whether or not a gene or genes in cells of the patient
contain at least one polymorphism which is correlated to the
effectiveness of the treatment of the disease or condition. The
selection may be the selection of a method or methods which is/are
more or less effective, safer, or toxic than certain other
therapeutic regimens. The selection may involve either choice of a
treatment to use or avoidance of a treatment. For example, a
contra-indicated treatment should be avoided if it will not result
in a therapeutic benefit, or if it will result in an excessive
level of undesirable side effects. Thus, the frequency of the
polymorphism itself may be correlated to the frequency of a
beneficial therapeutic response to a drug or unresponsiveness to
the drug, or it may be correlated to the frequency of an adverse
event resulting from administration of the drug. Even where there
the frequency of the polymorphism does not correspond closely with
the frequency of a beneficial or adverse response, the polymorphism
may still be useful for identifying a patient subset with high
response or toxicity incidence. Preferably, the drug will be
effective in more than 20%, 40% or 60% of individuals with one or
more specific polymorphisms. Alternatively, the drug will be toxic
or create clinically unacceptable side effects in more than 10%,
30%, 50%, 70% or 90% of individuals with one or more specific
polymorphisms.
[0168] The invention thus provides a method of predicting a
beneficial treatment for a patient comprising: (a) analyzing a
patient's polynucleotides to determine the identity of at least one
of the nucleotides at a polymorphic site in SEQ ID NO: 1 wherein
the presence or absence of the nucleotide correlates with a
prediction that the treatment will be beneficial. The method may
further include selecting a suitable dosage amount and/or frequency
of administration.
[0169] Similarly, the invention provides a method of predicting a
contraindicated treatment for a patient comprising: (a) analyzing a
patient's polynucleotides to determine the identity of at least one
of the nucleotides at a polymorphic site in SEQ ID NO: 1, wherein
the presence or absence of the nucleotide correlates with a
prediction that the treatment will not be effective or will have
significantly adverse effects. The correlation may be based on
statistically associating either the presence or absence of a
single polymorphism (or multiple polymorphisms) with a beneficial
effect or contraindicated effect resulting from drug treatment.
[0170] One aspect of the invention specifically provides methods of
treatment with sequence-specific antisense compounds comprising the
step of detecting the presence of a polymorphism of the invention
in the patient's sample prior to treatment with the desired
sequence-specific compound, where detection of the polymorphism
guides selection of the proper sequence-specific compound. For
example, the presence of a polymorphism in a patient's genes may
indicate that treatment with a compound that specifically
hybridizes to the polymorphism may be beneficial. Similarly, the
absence of a polymorphism in the patient's genes may mean that
treatment with a compound that specifically hybridizes to the
polymorphism is contraindicated.
[0171] 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.RTM. accession numbers, and the like recited in
the present application is incorporated herein by reference in its
entirety.
EXAMPLES
Example 1
Synthesis of Nucleoside Phosphoramidites
[0172] The following compounds, including amidites and their
intermediates were prepared as described in U.S. Pat. No. 6,426,220
and published PCT WO 02/36743; 5'-O-Dimethoxytrityl-thymidine
intermediate for 5-methyl dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for
5-methyl-dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcytidine
penultimate intermediate for 5-methyl dC amidite,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N.sup.4-benzoyl-5-methylcy-
tidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(5-methyl dC amidite), 2'-Fluorodeoxyadenosine,
2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine,
2'-O-(2-Methoxyethyl) modified amidites,
2'-O-(2-methoxyethyl)-5-methyluridine intermediate,
5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate
intermediate,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi-
n-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T
amidite),
5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine
intermediate,
5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N.sup.4-benzoyl-5-methyl-cytid-
ine penultimate intermediate,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-benzo-
yl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE 5-Me-C amidite),
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.6-benzo-
yladenosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE A amdite),
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su-
p.4-isobutyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidit-
e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites,
2'-(Dimethylaminooxyethoxy) nucleoside amidites,
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine,
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine,
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine,
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri-
dine,
5'-O-tert-Butyldiphenylsilyl-2'-O--[N,N-dimethylaminooxyethyl]-5-met-
hyluridine, 2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite], 2'-(Aminooxyethoxy)
nucleoside amidites,
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(-
4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphora-
midite], 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside
amidites, 2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl
uridine,
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethyl-aminoethoxy)-ethyl)]-5-methyl
uridine and
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
Example 2
Oligonucleotide and Oligonucleoside Synthesis
[0173] The antisense compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0174] Oligonucleotides: Unsubstituted and substituted
phosphodiester (P.dbd.O) oligonucleotides are synthesized on an
automated DNA synthesizer (Applied Biosystems model 394) using
standard phosphoramidite chemistry with oxidation by iodine.
[0175] Phosphorothioates (P.dbd.S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions:
thiation was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time was increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligonucleotides were recovered by precipitating with >3 volumes
of ethanol from a 1 M NH.sub.4OAc solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270.
[0176] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863.
[0177] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.
[0178] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
[0179] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications PCT/US94/00902 and
PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively).
[0180] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925.
[0181] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243.
[0182] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0183] Oligonucleosides: Methylenemethylimino linked
oligonucleosides, also identified as MMI linked oligonucleosides,
methylenedimethylhydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289, all of which are herein
incorporated by reference.
[0184] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and
5,264,564.
[0185] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618.
Example 3
RNA Synthesis
[0186] In general, RNA synthesis chemistry is based on the
selective incorporation of various protecting groups at strategic
intermediary reactions. Although one of ordinary skill in the art
will understand the use of protecting groups in organic synthesis,
a useful class of protecting groups includes silyl ethers. In
particular bulky silyl ethers are used to protect the 5'-hydroxyl
in combination with an acid-labile orthoester protecting group on
the 2'-hydroxyl. This set of protecting groups is then used with
standard solid-phase synthesis technology. It is important to
lastly remove the acid labile orthoester protecting group after all
other synthetic steps. Moreover, the early use of the silyl
protecting groups during synthesis ensures facile removal when
desired, without undesired deprotection of 2' hydroxyl.
[0187] Following this procedure for the sequential protection of
the 5'-hydroxyl in combination with protection of the 2'-hydroxyl
by protecting groups that are differentially removed and are
differentially chemically labile, RNA oligonucleotides were
synthesized.
[0188] RNA oligonucleotides are synthesized in a stepwise fashion.
Each nucleotide is added sequentially (3'- to 5'-direction) to a
solid support-bound oligonucleotide. The first nucleoside at the
3'-end of the chain is covalently attached to a solid support. The
nucleotide precursor, a ribonucleoside phosphoramidite, and
activator are added, coupling the second base onto the 5'-end of
the first nucleoside. The support is washed and any unreacted
5'-hydroxyl groups are capped with acetic anhydride to yield
5'-acetyl moieties. The linkage is then oxidized to the more stable
and ultimately desired P(V) linkage. At the end of the nucleotide
addition cycle, the 5'-silyl group is cleaved with fluoride. The
cycle is repeated for each subsequent nucleotide.
[0189] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate
(S.sub.2Na.sub.2) in DMF. The deprotection solution is washed from
the solid support-bound oligonucleotide using water. The support is
then treated with 40% methylamine in water for 10 minutes at
55.degree. C. This releases the RNA oligonucleotides into solution,
deprotects the exocyclic amines, and modifies the 2'-groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this
stage.
[0190] The 2'-orthoester groups are the last protecting groups to
be removed. The ethylene glycol monoacetate orthoester protecting
group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is
one example of a useful orthoester protecting group which, has the
following important properties. It is stable to the conditions of
nucleoside phosphoramidite synthesis and oligonucleotide synthesis.
However, after oligonucleotide synthesis the oligonucleotide is
treated with methylamine which not only cleaves the oligonucleotide
from the solid support but also removes the acetyl groups from the
orthoesters. The resulting 2-ethyl-hydroxyl substituents on the
orthoester are less electron withdrawing than the acetylated
precursor. As a result, the modified orthoester becomes more labile
to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is
approximately 10 times faster after the acetyl groups are removed.
Therefore, this orthoester possesses sufficient stability in order
to be compatible with oligonucleotide synthesis and yet, when
subsequently modified, permits deprotection to be carried out under
relatively mild aqueous conditions compatible with the final RNA
oligonucleotide product.
[0191] Additionally, methods of RNA synthesis are well known in the
art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996;
Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821;
Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103,
3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett.,
1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990,
44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25,
4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23,
2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2315-2331).
[0192] RNA antisense compounds (RNA oligonucleotides) of the
present invention can be synthesized by the methods herein or
purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once
synthesized, complementary RNA antisense compounds can then be
annealed by methods known in the art to form double stranded
(duplexed) antisense compounds. For example, duplexes can be formed
by combining 30 .mu.l of each of the complementary strands of RNA
oligonucleotides (50 uM RNA oligonucleotide solution) and 15 .mu.l
of 5.times. annealing buffer (100 mM potassium acetate, 30 mM
HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1
minute at 90.degree. C., then 1 hour at 37.degree. C. The resulting
duplexed antisense compounds can be used in kits, assays, screens,
or other methods to investigate the role of a target nucleic acid,
or for diagnostic or therapeutic purposes.
Example 4
Synthesis of Chimeric Compounds
[0193] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0194] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spectrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry.
[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric
Phosphorothioate Oligonucleotides
[0195] [2'-O-(2-methoxyethyl)]-[2'-deoxy]-[2'-O-(methoxyethyl)]
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
[2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy
Phosphorothioate]-[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0196] [2'-O-(2-methoxyethyl phosphodiester]-[2'-deoxy
phosphorothioate]-[2'-O-(methoxyethyl) phosphodiester] chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidation with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0197] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065.
Example 5
Design and Screening of Duplexed Antisense Compounds Targeting
Apolipoprotein B
[0198] In accordance with the present invention, a series of
nucleic acid duplexes comprising the antisense compounds of the
present invention and their complements can be designed to target
apolipoprotein B. The nucleobase sequence of the antisense strand
of the duplex comprises at least an 8-nucleobase portion of an
oligonucleotide in Table 1. The ends of the strands may be modified
by the addition of one or more natural or modified nucleobases to
form an overhang. The sense strand of the dsRNA is then designed
and synthesized as the complement of the antisense strand and may
also contain modifications or additions to either terminus. For
example, in one embodiment, both strands of the dsRNA duplex would
be complementary over the central nucleobases, each having
overhangs at one or both termini.
[0199] In one embodiment, a duplex comprising an antisense strand
having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 3), can be
prepared with blunt ends (no single stranded overhang) as
shown:
TABLE-US-00002 cgagaggcggacgggaccg Antisense (SEQ ID NO: 3)
||||||||||||||||||| Strand gctctccgcctgccctggc Complement (SEQ ID
NO: 4)
[0200] In another embodiment, both strands of the dsRNA duplex
would be complementary over the central nucleobases, each having
overhangs at one or both termini. For example, a duplex comprising
an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID
NO: 3) and having a two-nucleobase overhang of deoxythymidine (dT)
would have the following structure:
TABLE-US-00003 cgagaggcggacgggaccgTT Antisense (SEQ ID NO: 5)
||||||||||||||||||| Strand TTgctctccgcctgccctggc Complement (SEQ ID
NO: 6)
[0201] Overhangs can range from 2 to 6 nucleobases and these
nucleobases may or may not be complementary to the target nucleic
acid. In another embodiment, the duplexes can have an overhang on
only one terminus.
[0202] The RNA duplex can be unimolecular or bimolecular; i.e, the
two strands can be part of a single molecule or may be separate
molecules.
[0203] RNA strands of the duplex can be synthesized by methods
disclosed herein or purchased from Dharmacon Research Inc.,
(Lafayette, Colo.). Once synthesized, the complementary strands are
annealed. The single strands are aliquoted and diluted to a
concentration of 50 uM. Once diluted, 30 uL of each strand is
combined with 15 uL of a 5.times. solution of annealing buffer. The
final concentration of said buffer is 100 mM potassium acetate, 30
mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume
is 75 uL. This solution is incubated for 1 minute at 90.degree. C.
and then centrifuged for 15 seconds. The tube is allowed to sit for
1 hour at 37.degree. C. at which time the dsRNA duplexes are used
in experimentation. The final concentration of the dsRNA duplex is
20 uM. This solution can be stored frozen (-20.degree. C.) and
freeze-thawed up to 5 times.
[0204] Once prepared, the duplexed compounds are evaluated for
their ability to modulate apolipoprotein B expression. When cells
reach approximately 80% confluency, they are treated with duplexed
compounds of the invention. For cells grown in 96-well plates,
wells are washed once with 200 .mu.L OPTI-MEM.RTM. 1 reduced-serum
medium (Invitrogen Life Technologies, Carlsbad, Calif.) and then
treated with 130 .mu.L of OPTI-MEM.RTM. 1 containing 12 .mu.g/mL
LIPOFECTIN.RTM. (Invitrogen Life Technologies, Carlsbad, Calif.)
and the desired duplex antisense compound (e.g. 200 nM) at a ratio
of 6 .mu.g/mL LIPOFECTIN.RTM. per 100 nM duplex antisense compound.
After approximately 5 hours of treatment, the medium is replaced
with fresh medium. Cells are harvested approximately 16 hours after
treatment, at which time RNA is isolated and target reduction
measured by real-time PCR.
Example 6
Oligonucleotide Isolation
[0205] After cleavage from the controlled pore glass solid support
and deblocking in concentrated ammonium hydroxide at 55.degree. C.
for 12-16 hours, the oligonucleotides or oligonucleosides are
recovered by precipitation out of 1 M NH.sub.4OAc with >3
volumes of ethanol. Synthesized oligonucleotides were analyzed by
electrospray mass spectroscopy (molecular weight determination) and
by capillary gel electrophoresis and judged to be at least 70% full
length material. The relative amounts of phosphorothioate and
phosphodiester linkages obtained in the synthesis was determined by
the ratio of correct molecular weight relative to the -16 amu
product (+/-32 +/-48). For some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7
Oligonucleotide Synthesis
96 Well Plate Format
[0206] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 96-well format.
Phosphodiester internucleotide linkages were afforded by oxidation
with aqueous iodine. Phosphorothioate internucleotide linkages were
generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard
base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were
purchased from commercial vendors (e.g. PE-Applied Biosystems,
Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard
nucleosides are synthesized as per standard or patented methods.
They are utilized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0207] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
Example 8
Oligonucleotide Analysis
96-Well Plate Format
[0208] The concentration of oligonucleotide in each well was
assessed by dilution of samples and UV absorption spectroscopy. The
full-length integrity of the individual products was evaluated by
capillary electrophoresis (CE) in either the 96-well format
(Beckman P/ACE.TM. MDQ) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
Base and backbone composition was confirmed by mass analysis of the
compounds utilizing electrospray-mass spectroscopy. All assay test
plates were diluted from the master plate using single and
multi-channel robotic pipettors. Plates were judged to be
acceptable if at least 85% of the compounds on the plate were at
least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
[0209] The effect of antisense compounds on target nucleic acid
expression can be tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. The following cell types are provided for
illustrative purposes, but other cell types can be routinely used,
provided that the target is expressed in the cell type chosen. This
can be readily determined by methods routine in the art, for
example Northern blot analysis, ribonuclease protection assays, or
RT-PCR.
HepG2 Cells:
[0210] The human hepatoblastoma cell line HepG2 is available from
the American Type Culture Collection (Manassas, Va.). HepG2 cells
are routinely cultured in Eagle's MEM supplemented with 10% fetal
bovine serum, non-essential amino acids, and 1 mM sodium pyruvate
(Invitrogen Life Technologies, Carlsbad, Calif.). Cells are
routinely passaged by trypsinization and dilution when they reach
90% confluence. Cells are seeded into 96-well plates
(Falcon-Primaria #3872, BD Biosciences, Bedford, Mass.) at a
density of approximately 7000 cells/well for use in antisense
oligonucleotide transfection experiments. For Northern blotting or
other analyses, cells may be seeded onto 100 mm or other standard
tissue culture plates and treated similarly, using appropriate
volumes of medium and oligonucleotide.
T-24 Cells:
[0211] The human transitional cell bladder carcinoma cell line T-24
is available from the American Type Culture Collection (ATCC)
(Manassas, Va.). T-24 cells are routinely cultured in complete
McCoy's 5A basal media supplemented with 10% fetal bovine serum,
100 units per mL penicillin, and 100 ug per mL streptomycin (media
and supplements from Invitrogen Life Technologies, Carlsbad,
Calif.). Cells are routinely passaged by trypsinization and
dilution when they reach approximately 90% confluence. Cells are
seeded into 96-well plates (Falcon-Primaria #353872, BD
Biosciences, Bedford, Mass.) at a density of approximately 7000
cells/well for use in antisense oligonucleotide transfection
experiments. For Northern blotting or other analysis, cells may be
seeded onto 100 mm or other standard tissue culture plates and
treated similarly, using appropriate volumes of medium and
oligonucleotide.
A549 Cells:
[0212] The human lung carcinoma cell line A549 is available from
the American Type Culture Collection (ATCC) (Manassas, Va.). A549
cells are routinely cultured in DMEM basal media (Invitrogen
Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine
serum, 100 units per mL penicillin, and 100 ug per mL streptomycin
(media and supplements from Invitrogen Life Technologies, Carlsbad,
Calif.). Cells are routinely passaged by trypsinization and
dilution when they reach approximately 90% confluence. Cells are
seeded into 96-well plates (Falcon-Primaria #353872, BD
Biosciences, Bedford, Mass.) at a density of approximately 7000
cells/well for use in antisense oligonucleotide transfection
experiments. For Northern blotting or other analysis, cells may be
seeded onto 100 mm or other standard tissue culture plates and
treated similarly, using appropriate volumes of medium and
oligonucleotide.
NHDF Cells:
[0213] Human neonatal dermal fibroblast (NHDF) are available from
the Clonetics Corporation (Walkersville, Md.). NHDFs are routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville, Md.) supplemented as recommended by the supplier.
Cells are maintained for up to 10 passages as recommended by the
supplier.
HEK Cells:
[0214] Human embryonic keratinocytes (HEK) are available from the
Clonetics Corporation (Walkersville, Md.). HEKs are routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville, Md.) formulated as recommended by the supplier. Cells
are routinely maintained for up to 10 passages as recommended by
the supplier.
Treatment with Antisense Compounds:
[0215] When cells reached 65-75% confluency, they are treated with
oligonucleotide. Oligonucleotide is mixed with LIPOFECTIN.RTM.
Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM.RTM. 1
reduced serum medium (Invitrogen Life Technologies, Carlsbad,
Calif.) to achieve the desired concentration of oligonucleotide and
a LIPOFECTIN.RTM. concentration of 2.5 or 3 .mu.g/mL per 100 nM
oligonucleotide. This transfection mixture is incubated at room
temperature for approximately 0.5 hours. For cells grown in 96-well
plates, wells are washed once with 100 .mu.L OPTI-MEM.RTM. 1 and
then treated with 130 .mu.L of the transfection mixture. Cells
grown in 24-well plates or other standard tissue culture plates are
treated similarly, using appropriate volumes of medium and
oligonucleotide. Cells are treated and data are obtained in
duplicate or triplicate. After approximately 4-7 hours of treatment
at 37.degree. C., the medium containing the transfection mixture is
replaced with fresh culture medium. Cells are harvested 16-24 hours
after oligonucleotide treatment.
[0216] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is selected from either ISIS 13920
(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 7) which is targeted to human
H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 8) which is
targeted to human Jun-N-terminal kinase-2 (JNK2). ISIS 13920 is a
chimeric oligonucleotide having a 9 nucleotide gap segment composed
of 2'-deoxynucleotides, which is flanked on the 5' side and 3'
sides by 3 nucleotide and 8 nucleotide wing segments, respectively.
ISIS 18078 is a chimeric oligonucleotide having a 5 nucleotide gap
segment composed of 2'-deoxynucleotides, which is flanked on the 5'
and 3' sides by 5 nucleotide and 6 nucleotide wing segments,
respectively. The wings are composed of 2'-O-methoxyethyl
nucleotides. Both compounds have phosphorothioate internucleoside
(backbone) linkages, and cytidines in the wing segments are
5-methylcytidines. For mouse or rat cells the positive control
oligonucleotide is ISIS 15770 (ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 9),
a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in bold),
which is which is targeted to both mouse and rat c-raf. ISIS 15770
is a chimeric oligonucleotide having a 10 nucleotide gap segment
composed of 2'-deoxynucleotides, which is flanked on the 5' side
and 3' sides by 5 nucleotide wing segments. The wings are composed
of 2'-O-methoxyethyl nucleotides. Internucleoside (backbone)
linkages are phosphorothioate and cytidines in the wing segments
are 5-methylcytidines. The concentration of positive control
oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS
13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is
then utilized as the screening concentration for new
oligonucleotides in subsequent experiments for that cell line. If
80% inhibition is not achieved, the lowest concentration of
positive control oligonucleotide that results in 60% inhibition of
c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% inhibition is not achieved, that particular cell line
is deemed as unsuitable for oligonucleotide transfection
experiments. The concentrations of antisense oligonucleotides used
herein are from 50 nM to 300 nM.
Example 10
Analysis of Oligonucleotide Inhibition of Apolipoprotein B
Expression
[0217] Antisense modulation of apolipoprotein B expression can be
assayed in a variety of ways known in the art. For example,
apolipoprotein B mRNA levels can be quantitated by, e.g., Northern
blot analysis, competitive polymerase chain reaction (PCR), or
real-time PCR. Real-time quantitative PCR is presently preferred.
RNA analysis can be performed on total cellular RNA or poly(A)+
mRNA. Methods of RNA isolation are taught in, for example, Ausubel,
F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp.
4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.
Northern blot analysis is routine in the art and is taught in, for
example, Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc.,
1996. Real-time quantitative (PCR) can be conveniently accomplished
using the commercially available ABI PRISM.RTM. 7700 Sequence
Detection System, available from PE-Applied Biosystems, Foster
City, Calif. and used according to manufacturer's instructions.
[0218] Protein levels of apolipoprotein B can be quantitated in a
variety of ways well known in the art, such as immunoprecipitation,
Western blot analysis (immunoblotting), ELISA or
fluorescence-activated cell sorting (FACS). Antibodies directed to
apolipoprotein B can be identified and obtained from a variety of
sources, such as the MSRS catalog of antibodies (Aerie Corporation,
Birmingham, Mich.), or can be prepared via conventional antibody
generation methods. Methods for preparation of polyclonal antisera
are taught in, for example, Ausubel, F. M. et al., Current
Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John
Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies
is taught in, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley
& Sons, Inc., 1997. Immunoprecipitation methods are standard in
the art and can be found at, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 2, pp.
10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot
(immunoblot) analysis is standard in the art and can be found at,
for example, Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc.,
1997. Enzyme-linked immunosorbent assays (ELISA) are standard in
the art and can be found at, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 2, pp.
11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.
Example 11
Poly(A)+ mRNA Isolation
[0219] Poly(A)+ mRNA was isolated according to Miura et al., Clin.
Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA
isolation are taught in, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3,
John Wiley & Sons, Inc., 1993. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C. was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0220] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
Total RNA Isolation
[0221] Total RNA was isolated using an RNEASY.RTM. 96 kit and
buffers purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 100 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 100 .mu.L of 70% ethanol was then added to each well and
the contents mixed by pipetting three times up and down. The
samples were then transferred to the RNEASY.RTM. 96 well plate
attached to a QIAvac manifold fitted with a waste collection tray
and attached to a vacuum source. Vacuum was applied for 15 seconds.
1 mL of Buffer RW1 was added to each well of the RNEASY.RTM. 96
plate and the vacuum again applied for 15 seconds. 1 mL of Buffer
RPE was then added to each well of the RNEASY.RTM. 96 plate and the
vacuum applied for a period of 15 seconds. The Buffer RPE wash was
then repeated and the vacuum was applied for an additional 10
minutes. The plate was then removed from the QIAvac manifold and
blotted dry on paper towels. The plate was then re-attached to the
QIAvac manifold fitted with a collection tube rack containing 1.2
mL collection tubes. RNA was then eluted by pipetting 60 .mu.L
water into each well, incubating 1 minute, and then applying the
vacuum for 30 seconds. The elution step was repeated with an
additional 60 .mu.L water.
[0222] The repetitive pipetting and elution steps may be automated
using a QIAGEN.RTM. Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 13
Real-Time Quantitative PCR Analysis of Apolipoprotein B mRNA
Levels
[0223] Quantitation of apolipoprotein B mRNA levels was determined
by real-time quantitative PCR using the ABI PRISM.RTM. 7700
Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. This is a
closed-tube, non-gel-based, fluorescence detection system which
allows high-throughput quantitation of polymerase chain reaction
(PCR) products in real-time. As opposed to standard PCR, in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., JOE.TM., FAM.TM., or VIC.TM., obtained from
either Operon Technologies Inc., Alameda, Calif. or PE-Applied
Biosystems, Foster City, Calif.) is attached to the 5' end of the
probe and a quencher dye (e.g., TAMRA.TM., obtained from either
Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems,
Foster City, Calif.) is attached to the 3' end of the probe. When
the probe and dyes are intact, reporter dye emission is quenched by
the proximity of the 3' quencher dye. During amplification,
annealing of the probe to the target sequence creates a substrate
that can be cleaved by the 5'-exonuclease activity of Taq
polymerase. During the extension phase of the PCR amplification
cycle, cleavage of the probe by Taq polymerase releases the
reporter dye from the remainder of the probe (and hence from the
quencher moiety) and a sequence-specific fluorescent signal is
generated. With each cycle, additional reporter dye molecules are
cleaved from their respective probes, and the fluorescence
intensity is monitored at regular intervals by laser optics built
into the ABI PRISM.RTM. 7700 Sequence Detection System. In each
assay, a series of parallel reactions containing serial dilutions
of mRNA from untreated control samples generates a standard curve
that is used to quantitate the percent inhibition after antisense
oligonucleotide treatment of test samples.
[0224] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0225] After isolation the RNA is subjected to sequential reverse
transcriptase (RT) reaction and real-time PCR, both of which are
performed in the same well. RT and PCR reagents were obtained from
Invitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR
was carried out in the same by adding 20 .mu.L PCR cocktail
(2.5.times.PCR buffer minus MgCl.sub.2, 6.6 mM MgCl.sub.2, 375
.mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward
primer and reverse primer, 125 nM of probe, 4 Units RNAse
inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse
transcriptase, and 2.5.times.ROX dye) to 96-well plates containing
30 .mu.L total RNA solution (20-200 ng). The RT reaction was
carried out by incubation for 30 minutes at 48.degree. C. Following
a 10 minute incubation at 95.degree. C. to activate the
PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol were
carried out: 95.degree. C. for 15 seconds (denaturation) followed
by 60.degree. C. for 1.5 minutes (annealing/extension).
[0226] Gene target quantities obtained by real time PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RIBOGREEN.RTM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
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
from Molecular Probes. Methods of RNA quantification by
RIBOGREEN.RTM. are taught in Jones, L. J., et al, Analytical
Biochemistry, 1998, 265, 368-374.
[0227] In this assay, 175 .mu.L of RIBOGREEN.RTM. working reagent
(RIBOGREEN.RTM. reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM
EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 480 nm and emission at 520
nm.
[0228] Probes and primers to human apolipoprotein B were designed
to hybridize to a human apolipoprotein B sequence, using published
sequence information (GENBANK.RTM. accession number
NM.sub.--000384.1, incorporated herein as SEQ ID NO: 10). For human
apolipoprotein B the PCR primers were:
[0229] forward primer: TGCTAAAGGCACATATGGCCT (SEQ ID NO: 11)
[0230] reverse primer: CTCAGGTTGGACTCTCCATTGAG (SEQ ID NO: 12) and
the PCR probe was: FAM-CTTGTCAGAGGGATCCTAACACTGGCCG-TAMRA (SEQ ID
NO: 13) where FAM.TM. (PE-Applied Biosystems, Foster City, Calif.)
is the fluorescent reporter dye) and TAMRA.TM. (PE-Applied
Biosystems, Foster City, Calif.) is the quencher dye.
[0231] For human GAPDH the PCR primers were:
[0232] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 14)
[0233] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 15) and the
PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 16)
where JOE.TM. (PE-Applied Biosystems, Foster City, Calif.) is the
fluorescent reporter dye) and TAMRA.TM. (PE-Applied Biosystems,
Foster City, Calif.) is the quencher dye.
[0234] Probes and primers to mouse apolipoprotein B were designed
to hybridize to a mouse apolipoprotein B sequence, using published
sequence information (GENBANK.RTM. accession number M35186,
incorporated herein as SEQ ID NO: 17). For mouse apolipoprotein B
the PCR primers were:
[0235] forward primer: CGTGGGCTCCAGCATTCTA (SEQ ID NO: 18)
[0236] reverse primer: AGTCATTTCTGCCTTTGCGTC (SEQ ID NO: 19) and
the PCR probe was: 5' FAM-CCAATGGTCGGGCACTGCTCAA-TAMRA 3'(SEQ ID
NO: 20) where FAM.TM. (PE-Applied Biosystems, Foster City, Calif.)
is the fluorescent reporter dye) and TAMRA.TM. (PE-Applied
Biosystems, Foster City, Calif.) is the quencher dye. For mouse
GAPDH the PCR primers were:
[0237] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 21)
[0238] reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 22) and the
PCR probe was: 5' JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3' (SEQ ID
NO: 23) where JOE.TM. (PE-Applied Biosystems, Foster City, Calif.)
is the fluorescent reporter dye) and TAMRA.TM. (PE-Applied
Biosystems, Foster City, Calif.) is the quencher dye.
Example 14
Northern Blot Analysis of Apolipoprotein B mRNA Levels
[0239] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.RTM.
(TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared
following manufacturer's recommended protocols. Twenty micrograms
of total RNA was fractionated by electrophoresis through 1.2%
agarose gels containing 1.1% formaldehyde using a MOPS buffer
system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the
gel to HYBOND.RTM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.RTM.
UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then
robed using QUICKHYB.RTM. hybridization solution (Stratagene, La
Jolla, Calif.) using manufacturer's recommendations for stringent
conditions.
[0240] To detect human apolipoprotein B, a human apolipoprotein B
specific probe was prepared by PCR using the forward primer
TGCTAAAGGCACATATGGCCT (SEQ ID NO: 11) and the reverse primer
CTCAGGTTGGACTCTCCATTGAG (SEQ ID NO: 12). To normalize for
variations in loading and transfer efficiency membranes were
stripped and probed for human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
[0241] To detect mouse apolipoprotein B, a human apolipoprotein B
specific probe was prepared by PCR using the forward primer
CGTGGGCTCCAGCATTCTA (SEQ ID NO: 18) and the reverse primer
AGTCATTTCTGCCTTTGCGTC (SEQ ID NO: 19). To normalize for variations
in loading and transfer efficiency membranes were stripped and
probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
RNA (Clontech, Palo Alto, Calif.).
[0242] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.RTM. and IMAGEQUANT.RTM. Software V3.3 (Molecular
Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels
in untreated controls.
Example 15
Western Blot Analysis of Apolipoprotein B Protein Levels
[0243] Western blot analysis (immunoblot analysis) was carried out
using standard methods. Cells were harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a
16% SDS-PAGE gel. Gels were run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to apolipoprotein B was used, with a
radiolabelled or fluorescently labeled secondary antibody directed
against the primary antibody species. Bands were visualized using a
PHOSPHORIMAGER.RTM. (Molecular Dynamics, Sunnyvale Calif.) or the
ECL PLUS.RTM. chemiluminescent detection system (Amersham
Biosciences, Piscataway, N.J.).
Example 16
Antisense Inhibition of Apolipoprotein B Expression
[0244] U.S. Application Publication No. 20040214325 published Oct.
28, 2004 and International Patent Publication WO2004044181
published May 27, 2004, the disclosures and particularly the
examples of which are hereby incorporated by reference in their
entirety, describe the activity in vitro and in vivo of a variety
of different antisense compounds, including dsRNA and chimeric
phosphorothioate oligonucleotides, designed to target different
regions of the human, mouse, rabbit and monkey apolipoprotein B
RNA.
[0245] A number of different compounds demonstrated at least 10%,
at least 30%, and/or at least 50% inhibition of apolipoprotein B
expression. The target regions to which these preferred sequences
are complementary are herein referred to as "preferred target
segments" and are therefore preferred for targeting by compounds of
the present invention. Where sequences are shown to contain thymine
(T) one of skill in the art will appreciate that thymine (T) is
generally replaced by uracil (U) in RNA sequences.
[0246] As these "preferred target segments" have been found by
experimentation to be open to, and accessible for, hybridization
with the antisense compounds of the present invention, one of skill
in the art will recognize or be able to ascertain, using no more
than routine experimentation, further embodiments of the invention
that encompass other compounds that specifically hybridize to these
preferred target segments and consequently inhibit the expression
of apolipoprotein B.
[0247] According to the present invention, antisense compounds
include antisense oligomeric compounds, antisense oligonucleotides,
ribozymes, external guide sequence (EGS) oligonucleotides,
alternate splicers, primers, probes, and other short oligomeric
compounds which hybridize to at least a portion of the target
nucleic acid.
Example 17
Design of Phenotypic Assays for the Use of Apolipoprotein B
Inhibitors
[0248] Once apolipoprotein B inhibitors have been identified by the
methods disclosed herein, the compounds are further investigated in
one or more phenotypic assays, each having measurable endpoints
predictive of efficacy in the treatment of a particular disease
state or condition. Phenotypic assays, kits and reagents for their
use are well known to those skilled in the art and are herein used
to investigate the role and/or association of apolipoprotein B in
health and disease. Representative phenotypic assays, which can be
purchased from any one of several commercial vendors, include those
for determining cell viability, cytotoxicity, proliferation or cell
survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston,
Mass.), protein-based assays including enzymatic assays (Panvera,
LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene
Research Products, San Diego, Calif.), cell regulation, signal
transduction, inflammation, oxidative processes and apoptosis
(Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation
(Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube
formation assays, cytokine and hormone assays and metabolic assays
(Chemicon International Inc., Temecula, Calif.; Amersham
Biosciences, Piscataway, N.J.).
[0249] In one non-limiting example, cells determined to be
appropriate for a particular phenotypic assay (i.e., MCF-7 cells
selected for breast cancer studies; adipocytes for obesity studies)
are treated with apolipoprotein B inhibitors identified from the in
vitro studies as well as control compounds at optimal
concentrations which are determined by the methods described above.
At the end of the treatment period, treated and untreated cells are
analyzed by one or more methods specific for the assay to determine
phenotypic outcomes and endpoints.
[0250] Phenotypic endpoints include changes in cell morphology over
time or treatment dose as well as changes in levels of cellular
components such as proteins, lipids, nucleic acids, hormones,
saccharides or metals. Measurements of cellular status which
include pH, stage of the cell cycle, intake or excretion of
biological indicators by the cell, are also endpoints of
interest.
[0251] Measurement of the expression of one or more of the genes of
the cell after treatment is also used as an indicator of the
efficacy or potency of the apolipoprotein B inhibitors. Hallmark
genes, or those genes suspected to be associated with a specific
disease state, condition, or phenotype, are measured in both
treated and untreated cells.
Example 18
Activity in Animal Models
[0252] U.S. Application Publication No. 20040214325 published Oct.
28, 2004 and International Patent Publication WO2004044181
published May 27, 2004, the disclosures and particularly the
examples of which are hereby incorporated by reference in their
entirety, also describe the in vitro and in vivo biological effects
of antisense inhibition of apolipoprotein B expression in mice and
monkeys.
[0253] Treatment with ISIS 147764, a mouse-specific
oligonucleotide, lowered cholesterol as well as LDL and HDL
lipoproteins and serum glucose in both lean and high fat mice. The
effects demonstrated are, in fact, due to the inhibition of
apolipoprotein B expression as supported by the decrease in mRNA
levels. No significant changes in liver enzyme levels were
observed, indicating that the antisense oligonucleotide was not
toxic to either treatment group.
[0254] Treatment of high fat fed mice with ISIS 147764 decreased
apolipoprotein B protein expression in liver in a dose-dependent
manner, reduced serum cholesterol and triglycerides, lowered levels
of serum HDL, LDL and VLDL lipoproteins, reduced serum glucose
levels, and decreased fat pad weight.
[0255] Treatment of apo E knockout mice with ISIS 147764 lowered
glucose and cholesterol as well as serum HDL, LDL and VLDL
lipoproteins. Further, these decreases correlated with a decrease
in both protein and RNA levels of apolipoprotein B, demonstrating
an antisense mechanism of action. No significant changes in liver
enzyme levels were observed, indicating that the antisense
oligonucleotide was not toxic to either treatment group.
[0256] LDL receptor-deficient mice (LDLr(-/-)mice), a strain that
cannot edit the apolipoprotein B mRNA and therefore synthesize
exclusively apolipoprotein B-100, have markedly elevated LDL
cholesterol and apolipoprotein B-100 levels and develop extensive
atherosclerosis. ISIS 147764 was able to lower cholesterol,
triglycerides, and mRNA levels in a dose-dependent manner in both
male and female LDLr(-/-) mice while the 4-base mismatch ISIS
270906 was not able to do this.
[0257] C57BL/6NTac-TgN(APOB100) transgenic mice have the human
apolipoprotein B gene "knocked-in". These mice express high levels
of human apolipoprotein B100 resulting in mice with elevated serum
levels of LDL cholesterol. Treatment with either of these
oligonucleotides targeted to the human apolipoprotein B which is
expressed in these mice markedly decreased the mRNA levels of the
human apolipoprotein, while the levels of the endogenous mouse
apolipoprotein B were unaffected, indicating that these
oligonucleotides exhibit specificity for the human apolipoprotein
B. Immunoblot analysis of liver protein samples reveals a reduction
in the expression of both forms of human apolipoprotein B,
apolipoprotein B-100 and apolipoprotein B-48. Mouse apolipoprotein
B levels in liver were not significantly changed. LDL-cholesterol
levels were significantly reduced.
[0258] ob/ob mice have a mutation in the leptin gene which results
in obesity and hyperglycemia. Treatment of ob/ob mice receiving a
high fat and cholesterol diet with ISIS 147483 and 147764 were both
able to lower apolipoprotein B mRNA levels, as well as glucose,
cholesterol, and triglyceride levels.
[0259] Toxicity studies in mice revealed no severe toxic effects.
In vitro assays showed that ISIS 301012 does not possess
immunostimulatory activity.
[0260] In Cynomolgus monkeys, antisense inhibition by ISIS 301012
was compared to that of ISIS 326358, which is a perfect match to
the Cynomolgus monkey apolipoprotein B sequence to which ISIS
301012 hybridizes. These data demonstrate that both ISIS 326359 and
ISIS 301012 (despite two mismatches with the Cynomolgus monkey
apolipoprotein B sequence) can inhibit the expression of
apolipoprotein B mRNA in Cynomolgus monkey primary hepatocytes, in
a dose- and time-dependent manner.
TABLE-US-00004 TABLE 1 Effects of antisense inhibition by ISIS
301012 in lean Cynomolgus monkeys Intravenous delivery Subcutaneous
injection 2 mg/kg 4 mg/kg 12 mg/kg 3.5 mg/kg 20 mg/kg
apolipoprotein B expression -45 -76 -96 N.D. -94 % change
normalized to saline antisense oligonucleotide 92 179 550 N.D. 855
concentration .mu.g/g Lipid parameters, % change normalized to
untreated baseline value Saline 2 mg/kg 4 mg/kg 12 mg/kg 3.5 mg/kg
20 mg/kg Total cholesterol +1 -6 -2 -2 +5 -5 LDL-cholesterol +17
+15 +9 +3 -4 -16 HDL-cholesterol -11 -23 -15 -8 +13 +5 LDL/HDL +62
+94 +38 +44 -15 -19 Total cholesterol/HDL +30 +44 +22 +21 -7 -10
Triglyceride +37 +26 +32 +15 +1 -3 LDL Particle concentration +15
+8 +8 -11 -14 -21
[0261] These data show that ISIS 301012 inhibits apolipoprotein B
expression in a dose-dependent manner in a primate species and
concomitantly lowers lipid levels at higher doses of ISIS 301012.
Furthermore, these results demonstrate that antisense
oligonucleotide accumulates in the liver in a dose-dependent
manner.
Example 19
Identification of SNPs
[0262] Polymorphisms were discovered by comparing the
apolipoprotein B genomic sequences of 213 DNA samples. An initial
analysis of 23 DNA samples and a followup analysis of an additional
190 DNA samples was conducted to identify SNPs in the target region
of ISIS 301012 (exon 20, boundary of intron 20). The 190 DNA
samples came from individuals self-identified as belonging to one
of four major population groups: Caucasian (47 individuals),
African descent (48 individuals), Asian (47 individuals), or
Hispanic (48 individuals). All samples were analyzed in replicates
using SEQUENOM's MASSARRAY.RTM. approach including MASSCLEAVE.RTM.
biochemistry.
[0263] Seven previously unknown SNPs with varying frequencies were
discovered in an approximately 541 bp portion of the ISIS 301012
target region, none in the antisense or exon regions. The SNPs and
their positions and frequencies are set forth in Table 2 below.
TABLE-US-00005 TABLE 2 Identification of SNPs Approximate frequency
of Position in SNP detection out of 213 Sequence variation SEQ ID
NO: 1 samples of diverse ancestry Substitution of A to G 27751 15%
Substitution of C to G 27735 <1% Substitution of T to C 27685
27% Substitution of T to C 27683 Substitution of T to C 27679 40%
Substitution of C to T 27634 <1% Substitution of G to A 27627 2%
Substitution of T to C 27618 <1%
[0264] The frequency of the T/C substitution at position 27679 and
the T/C substitution at 27685 was significantly lower in Asian DNA
samples compared to other ethnic groups. See table below.
TABLE-US-00006 Position in African SEQ ID NO: 1 Asian American
Hispanic Caucasian 27685 9.8% 32.4% 42.2% 32.6% 27679 19.7% 45.6%
43.8% 48.6%
[0265] The C/T substitution at position 27634 was only present in
Asian samples. It was detected in one heterozygote Asian sample and
one homozygote Asian sample.
[0266] The distribution of the A/G substitution at position 27751
does not match the Hardy-Weinberg disequilibrium; all samples
carrying this sequence variation are heterozygote.
[0267] The G/A substitution at position 27627 was found only in
four samples of African American origin.
[0268] The T/C substitution at position 27618 was found in one
African American sample.
Sequence CWU 1
1
23143445DNAArtificial Sequenceallelic genomic sequence of
apolipoprotein B 1accaagacag cgctcaggac tggttctcct cgtggctccc
aattcagtcc aggagaagca 60gagattttgt ccccatggtg ggtcatctga agaaggcacc
cctggtcagg gcaggcttct 120cagaccctga ggcgctggcc atggccccac
tgagacacag gaagggccgc gccagagcac 180tgaagacgct tggggaaggg
aacccacctg ggacccagcc cctggtggct gcggctgcat 240cccaggtggg
ccccctcccc gaggctcttc aaggctcaaa gagaagccag tgtagaaaag
300caaacaggtc aggcccggga ggcgcccttt ggaccttttg caatcctggc
gctcttgcag 360cctgggcttc ctataaatgg ggtgcgggcg ccggccgcgc
attcccaccg ggacctgcgg 420ggctgagtgc ccttctcggt tgctgccgct
gaggagcccg cccagccagc cagggccgcg 480aggccgaggc caggccgcag
cccaggagcc gccccaccgc agctggcgat ggacccgccg 540aggcccgcgc
tgctggcgct gctggcgctg cctgcgctgc tgctgctgct gctggcgggc
600gccagggccg gtgagtgcgc ggccgctctg cgggcgcaga gggagcggga
gggagccggc 660ggcacgaggt tggccggggc agcctgggcc taggccagag
ggagggcagc cacagggtcc 720agggcgagtg gggggattgg accagctggc
ggcccctgca ggctcaggat ggggggcgcg 780ggatggaggg gctgaggagg
gggtctccgg agcctgcctc cctcctgaaa ggtgaaacct 840gtgccggtgg
tccccctgtc gggccctagc acccgctggg aagacgtggg aagctcacag
900atttctttct cctgtcttac agaagaggaa atgctggaaa atgtcagcct
ggtctgtcca 960agtaaggcat ctgcgcatgg ggcgtggaag ggcgcccagc
cccgtgcact ctcctacacc 1020cgggtccctg agggcctccc actctacagg
gctgagatgg catcgtggtg tgccttgctc 1080tgaccccagg aagcaagttc
cctgagcctc tgcccacacc caagggatgc caactctctt 1140ctacctggcc
ttctgttctg tcccaaaagt tcagcctggg ggcgggggag ggaagggatt
1200gtctctccgc tggcctgtgc acactttgaa gaaacatcac tgtcctgttt
atcagtgact 1260agtcattgat tcgaagcatg tgagggtgag gaaatactga
ctttaacctt tgtgaagaaa 1320tcgaacctcc acccccttcc tatttacctg
acccctgggg gttaaaggaa ctggcctcca 1380agcgcgaccc tgtgtgctgg
agccgcgggg cggacttctg atggggcagc accgccatct 1440agtggccgtc
tgtcatcact gcagctggac tcaggaccca gatgttcttt ttcttcaatt
1500gttcagaaaa ttcctctcaa ctacagtgga aacctccaga aattcttttc
taggagtttg 1560ttaagttagt tacgcttaat gcttaatgaa ctttgcctta
agtatttggt agtcttagag 1620tcacggaatt acggcgtgtt caagctaaaa
aagcattaga gatagtacta tttgcgtaat 1680gttgtcatct cttaatttgc
cagagggtct ctcatgcaga ttttctgagc cccattactt 1740gacacttgtc
actcccttcc ctgtgcctca gatgagatat tcaagacatg ccagccaatt
1800taaacattag cctcagcaaa aacataatgg agaagtcaaa tctataaagg
aaaattaagt 1860ataaagtcaa ttaaaaaata atttgagttg aattaccatt
tttaattctc tatgccactg 1920cccctctctg cccagaattg gctgtccttg
ggagagctat ttctgctatg tggctgacgt 1980atttctcccc acgttagaag
atgcgacccg attcaagcac ctccggaagt acacatacaa 2040ctatgaggct
gagagttcca gtggagtccc tgggactgct gattcaagaa gtgccaccag
2100gatcaactgc aaggtatgga ggatgcaggc aggagggacc tagagcccac
agctttcccc 2160cagccctgtt ccagcgggcg cccaacacgc gaccttcccg
gagggtgtgt actgagcaaa 2220cgcagaacat cccagaactg ttgtaatctg
atcaaagcac tgggactttg cctctgtttg 2280taagtcagcc acattgctga
gatgtggtct gcccccacca aatttcgcaa gtcagaagta 2340ttttcccgtt
aacttcccag atgcaatagg aatccatgat ctagattagc agcagtgtgg
2400gtctgtagat ttcagcgtga gagaggccca gtaggtgagc tatgggaggc
aggcaactcg 2460gaatcgcact gtgaaatgca gtttttataa tttaagtcaa
acagaatctg ttgctgaaaa 2520atgaatggaa agaagaaaaa aatataaaca
tacagtttgt tctaaaataa aactttgctt 2580attattgaga ctggttgtac
tcatgttaca tacatgtgga gcagatctac aggctgctat 2640tggggtttgg
gtggggaaga gaagtcaagc tgagcagtca ccttttttta gagagtaccg
2700tagctcttgt atgtgctgtc caatatggta gacatgagcc acattgggct
atttaaatgg 2760aatgaaatta aaaattcata ttcgttgtca cattagctgc
atttcaactg ctcaacagcc 2820accctggcta ctggctccca tattgaacag
cacacatgta caacatttct ataaagttat 2880ttgaatagtg ctggataata
agtaggaatc cgttgaaact ccagctatat gcaaagctct 2940aaataggccc
taatagatat aaccagtttt ttgggtgaca ttaaggagac atttgctgtg
3000gaaacgaagg atggccctct tcctgctttc tgtttttctt cttcactttc
actcctagtc 3060tgcagcgctt ctatttaacc acagctcttt ataattaaag
tgagtaactt tagaaccaat 3120aaaaggacat cctccttccc atgcctaggg
gcaaacttaa gaaatgtgtt acccgggagg 3180gggaaaacgt cagcaatagg
actaagtcta ggttggtgca cagagaaccc aggaggcatg 3240ttgataaggc
atgtggtgtt gaggcgcagg cagtggtgtt cccagcacca ttccctttgg
3300tgctctgatt agagattaag ccctgggctt caggggccac ctctcattct
tgatagacaa 3360cctcaatgct ctgctaccct gaattctcag gttgagctgg
aggttcccca gctctgcagc 3420ttcatcctga agaccagcca gtgcaccctg
aaagaggtgt atggcttcaa ccctgagggc 3480aaagccttgc tgaagaaaac
caagaactct gaggagtttg ctgcagccat gtccaggtaa 3540gtcatgttgt
acatgagcac acgcatgtgt gtgtgtccgc tgaggtatga acttgtgtgt
3600ttgcaccagg cacggatgtg actgtaagta tttgtattcc gtatccatcg
tggatcaggg 3660aattactgag ttttcacaat catcaaaaag agagaagcat
tagttaacct tccctagtta 3720ggttccttta attatcattt tcatgtgttt
ctaaaaatct catgctttaa acttcttgag 3780attataaaac tgagatgctt
tgtttaaaca agtgaattct tatttaaaga actagtcaag 3840actagtgctt
ggtggtcttt ggtgtggggt cccagaggca ctggctgctg tggccggcac
3900atggcggggc agggtctgtt caccgcaggg cagaggagca ccaaggcttc
ggtggctccc 3960cctcctaggc tggcattcag ccactgcacg ctgatcggcc
actgcagctg catctctgct 4020gactggtcag ggcccatgtc gcacccattg
taaatatttt caacatcacc cctgcctcat 4080cctcaatcac agtttgtagg
gtcctaggtg tgtatgaata caggcaggat agagttgtta 4140acttggtagc
atcagaaaac tctgtctgta ttagtctgtt ttcatgctgc tgataaagac
4200atacctgaga ctgggcaatt tacaaaagaa aggtttattg gactcacagt
tccacgtggc 4260tggggaggtc tcacaatcat ggcggaaggt gagggacagc
aagtcacatc ttatgtagat 4320ggtggctggc aaagagagct tgtgcagaga
aactcctgtt tttagaacca tcagatctcc 4380cgacacccat ctgcaatcac
gagaacagca cgggaaagac ctgcccccat gattcaatca 4440cctccccccg
ggtccctccc acaacacgtg ggaattatga gagctacgag acgaaatttg
4500ggtggggacg cagagccaaa ccatatcacc atccttgccc atttttcagt
tttgctaaac 4560attagattca gatgccagtc ctttcttgcc aaaataggct
gtgaggcttc tttctttcct 4620atgctttatt ttctccaaga cttaactgta
tatgagggag aggggtatgg tggcaggagg 4680aaagagtggt ttattttttg
gtccttggtc ttctccaaat acagaagaga ctcctgttct 4740tgaaaaggag
ggctttccat gtttgcatct tcatgacttt aactgtcttt tttaaaaatt
4800gacatacaat aattatacat atttattgag aacatagtga tattttgata
catgtaatgt 4860atggtgatca gatcagagta attagcatac ccatcatctc
aaacatttat catttcttcg 4920tgttgggaac tttctgagag agtgtaggct
gtgggagata agtccgtcac cttttcctcc 4980tgatgtaacc agagtggctg
cagccaggtc ctcagaaact cagagagtac ccagtgggaa 5040atccctaaga
ccaaagtcag catgggcttc agccatggcc tgacaccata caaaagaatg
5100actgtccaac aagtgtatga aaataagctc caattcactg gtagtcaaga
aatgcgaatt 5160aatgtaacaa caagatattt atctgctttt acccatcata
ctgcaaaact ggaaaacagt 5220gatagcacct gttgctggca ggccagtgag
gaaaagtgtg ctgtcctgag ctgctggtgg 5280aaacgagagc catcaggcaa
tatctactgt aatttaaaat acttaatacc ctttgacaca 5340gatattttag
tctttgggac tctagcccat gaaaataaaa gcagtaatgt gtgaagatag
5400gcacataagg atgtttgttt tggtattgtt tgtgtggttt aaaaaaaatc
cagaaagaga 5460gagggcaaat gccatcaaat ggggcaatgt gtgaataaat
tatatttagc catggaatgg 5520aatgttctgc atgcagcttt taaaaaaatc
tgttagagct gtaccaagtg actcagaagg 5580atttttgtga agtataatta
agtgagaaaa acaagataaa agtatgcata atacaatgcc 5640acttgtataa
aacaaacaat ggcaaaatct ttgtatgact ctgtttgcac tcacccatgt
5700ttacagagga ttgtatgagt gtgcagaaac aaatggaaca accactcggg
tgtccgtatg 5760gggaggatgg gcaaagagac tgatatgggt ggagaacaga
gcagggctgg atgagccaag 5820caaaaaaagt taaaacacag ctggacctgg
tggctcatgc ctgtagtccc agcactttgg 5880gaggccgagg agggagaatc
acctgaggtc aggagtttga gaccagcctg gccaacatgg 5940tgaaaactgt
ctctactaaa aatacaaaaa ttagctgggt gtgatggcac atgccagtag
6000tcctagctac tccggaggct gaggcaggag aatcacttga tcccaggagg
tggaggttgc 6060agtgagctga ggttgcgcca ttgcactcca gcccgggcga
ccgagcgaga ctccatttca 6120aaaaaagaaa aagaaaaaag aaaaaaagaa
aaaaaaagaa tcaccaaaac ttatgtatat 6180gtgcatactt ttttgaaaat
gtatgtctat gtgtagctat attctatatt tacaaataaa 6240tgatgtcaga
agaacaattg gttaaaaaaa tatgagaaaa gaaacttcag tgccacccag
6300cttacttcca gcaagttgta atggagaagg acatttccgt gaccatcctc
tctctgggac 6360aggtatgagc tcaagctggc cattccagaa gggaagcagg
ttttccttta cccggagaaa 6420gatgaaccta cttacatcct gaacatcaag
aggggcatca tttctgccct cctggttccc 6480ccagagacag aagaagccaa
gcaagtgttg tttctggtga ggatttagaa agctgatagc 6540agtggccctt
gaaactcatc ttcatgtgtt agagaccagt cctaccatat acaaagcaga
6600tcactgagtc agctccatga ctagttacat aggaagccct ggattggcgt
gaaatactgg 6660tgcccgaggt tcctcctgcc ccttaggctc actgacagat
catcccaagc aggcttatca 6720ggttgggtct aattttaaaa cagtcattga
ggagtcctgg ccaccccacc cctgcttttg 6780tttgatgctt cacctgtgtt
tgctgggtta tggtgtacac agtaaatcct gtgtgtattt 6840taaacaccaa
aaataatggg atctgttgct ggtctctttt acgaatttca ggtttcactg
6900tgagacagaa ttcatttcac ctcagtccca tgagcacttt tgtgtgttct
aatttctcta 6960cgacaccata atgggagaag acaccgatgc aacctgcgga
ggcctttctg cagacccacc 7020tttaactggt tttctctctc ccaacttggg
ctggccaggc actagcaaga ccacactctg 7080cataggaaga aaaagaaagt
ccctcccaaa gctagattcc ttctgctttt tctttcacga 7140tccccacccc
atccctccca agtacccaag gatgttgccc gtgttgaata catgtggttg
7200catcttcttc ctccatagga taccgtgtat ggaaactgct ccactcactt
taccgtcaag 7260acgaggaagg gcaatgtggc aacagaaata tccactgaaa
gagacctggg gcagtgtgat 7320cgcttcaagc ccatccgcac aggcatcagc
ccacttgctc tcatcaaagg catggtaagt 7380cccatgtcag cactgtcgtg
cacagcaagg agcatcctct tattaataca attccagaac 7440ttttgagcta
gtgggcacct ttgaggacag cctgccctgg ctgtttttta tacagactag
7500agataggacc ctgagcaggc acgggaaggt ctgcccaggc ttcacggcct
gggatcagtt 7560gagccaaggc ttgagtcagg ctcctccctc ccagcccaga
gctctgtctt tcctcctgtc 7620cttctgtcac tggcaccaaa ctgcctctaa
tctcatcact tgagagtaat gactactcac 7680ctctgagaag gttccgggga
tggatgtagg gcagcaaaac caccttctgt tcttttctgc 7740acaaggactc
cttgtgccag ctccaagcct ctggcctttg aagaagtccc aagacctgtg
7800ttctccccct ctccctcatc ccatgaagtg gagtgactta gagtgctcca
gcttcttgtc 7860cttccacccc cagtaccacc ctgaccaaac atggccccac
tgccaccggc ctggagcacc 7920ctctcctctc tgttaactgg ggccatggag
caccatatta cctgagcctg cctgacccct 7980gcaacatctt ccctgatatg
agccccagcc tgtctcagtg aacatgaata acttgggcaa 8040tcactgtcat
gctgggcgct gttcctggtc attgtcctta gggttgaaaa cagggagtct
8100gatgaccatg agtgccacag tcagaagagg ataatgcact ggcttagggg
tcttttctga 8160gcatctgctg tttgctcaac cccactctgg gcagcaccaa
ggaagggaca gtggcagatg 8220aaccatggac cttcccctca ggatgcttcc
agtctaatgc aggagccagg tcaataaagt 8280atacgtggta tactcaataa
ggtgataagc tgaacagtgc agacaagaag tcctgggcct 8340gaccaggaag
gagaaagaat tattcatgta gctcagcggg caacatttca tggaagatgt
8400ggagcaggaa cccaaaaaat gcaaagaata tgtaaatgaa agagacatgt
aagaatgggc 8460ttttgggcaa agaaaagtta ctgagcaggt gtgtgagggg
ctatgtggtg ggatgggcat 8520gtggaggata caaagtttag acattgtcca
gtgagggtgg aaaaagagga gtctacagct 8580tgactcagct ttggggatgc
cgacttgttg caccccctgg tctaaatgtc aagtacccag 8640ttatcttctt
tctctgagtt tatctagtgg tacaggactc ctgctccctt ctaccttgaa
8700ggtaaatgct tttaacagaa gatacaggga ctgatcaaaa tgctcgtctc
caatctcttt 8760catagacccg ccccttgtca actctgatca gcagcagcca
gtcctgtcag tacacactgg 8820acgctaagag gaagcatgtg gcagaagcca
tctgcaagga gcaacacctc ttcctgcctt 8880tctcctacaa gtaggtcatg
tgatgcaccc ctgatttgtc atttaatggg tcagtgtgaa 8940ctgaacactt
ctcaagtgct ctgttccagg caaacctgtg cctgggaggg aggaatggag
9000agggataaaa tgccgcccct ccctgtcccc ctttttaagc gaacaggcca
tttggcagaa 9060aagtcctagg catgcaaaac aatccaagac caacaaaaga
tatctaagac ccattcttta 9120agggctgtag atccagaaaa cctgaggatc
actgcagggt accctggtta gaaaaggttt 9180catggaagat ttgggatact
gactggaaac ttgtgtatcc aaatccactt tgaaaactga 9240taatcaatga
atatatattg agtaactgcc atattcttgg ctctatgttg tggaagatac
9300gaaagaattt tgagacattg cactagttcc tacctctggc cactccagac
tagtggagag 9360tataaggcac gcatgtcttt ttgatgggag gataactagc
gtgaccagga agaggtggat 9420gttattcatt cagggccaac aatggctgga
tttacccatg ctttgaaaga tgggcaggac 9480ttgggtagat gcagagacag
ggaaaacctt caacatggaa agaatagtat gttctggcca 9540tccgtgacat
ggtgtgcttc cttggttacc aggaataagt atgggatggt agcacaagtg
9600acacagactt tgaaacttga agacacacca aagatcaaca gccgcttctt
tggtgaaggt 9660aagagtttct gtccacatag ttgctggaaa atctactcaa
gatgtgccta tcatggctta 9720gccacttgct gagccctgtt aaatgtctgc
tgactaacaa gtgatacaga cactggtgtt 9780ctggctacct ctagtgagaa
agcaaactca tttcatgatg tcaagttgca atggcataaa 9840ggaaaagaag
ttcccaaagc tacttaggca tttgtaaata gaaaactgga atcctaagtt
9900taacatgaca tatttgatag aactgacatc acccatcctg tgataagatc
cagagctgtc 9960ccagacgagg tggaccaagt gggagagaac cttcagagtc
tggccagata gtaacctcag 10020gagtcagtct ttagaggtag aaggaactct
aacaatctca agtccaaccc ttacccagta 10080ttgtattgta tttatatctg
tccaaattcc ttcttgtaca ttacctcatt gtcctttttg 10140ctcatagcaa
cctgtgatgt caggtggtag agatgtgatt ttatacctat tctacagagg
10200agacagtgac acagagaggc ttagagtttg atgtagtcaa ggccgcagaa
tattagaggg 10260gggaaaataa gtgccaggtt gtaatctaag ccaggactat
tctcattaca ccacatttcc 10320atgatgactt ttacctctct tcctggcata
ggtcacagta ggtggtggag aggatacaaa 10380agtgtctccc ctccccacaa
gctgctggta gacccaatta gaagaaatgg tgataagcac 10440ccatgtgcct
ggtcccagtt gtaaccatgt caacagtagc acctcctcac caattatttc
10500aagctaaggg taacctgatg atagactcag acaagtctgg attccacttt
agctctacct 10560cttagaccct gagagctctt gggaaaccta agttgctcat
ctctgggtca cacttcctca 10620tctctgggtc tcatctcttt gtctcatctc
tgggactcag agctgagatc cagggatgag 10680caatttacat ggcccaaaaa
ctctgtgggt ctcagaagca gggctgaatt tatcattaaa 10740ttgaacaata
atgccacccc acagggatag gatgatgagt cagtgaaaac aagtcaatca
10800cctatggcag agccagatct agcaggcatt gaatacagga tagtttcttt
cccttttccc 10860ctgtgctgat actccacaat ttccagcttc cagtagacaa
agatatggtt gagatgaaga 10920aagctagagt tcctttgaca ctttccatct
tccaggtact aagaagatgg gcctcgcatt 10980tgagagcacc aaatccacat
cacctccaaa gcaggccgaa gctgttttga agactctcca 11040ggaactgaaa
aaactaacca tctctgagca aaatatccag agagctaatc tcttcaataa
11100gctggttact gagctgagag gcctcagtga tgaagcagtc acatctctct
tgccacagct 11160gattgaggtg tccaggtatc taatggttac agctcaactt
tttataaaac tgatggtaac 11220tgactgaact ttcaaacctt ggccaaatgg
agaatctcag ggaccatttg gatatcaatc 11280cagttaatca attagtcaat
cagttcatga ttgctggata gagaactatc agctgctgcg 11340ctgagttcca
tgaaacacac acgcgcatac tgtgttcaag gcagctatgt atttgtgtgt
11400taaaacagaa ggagaatagt tcccacattt tgatgggtaa cttttaattc
ctaggtctat 11460tgcaggtgct ctccagaagc ttataggctg gtggagagag
aactcagacg aaaaatataa 11520tatgatttct ctacccttca aggcactggc
tttaagtgct atgaaggtga gagaagggac 11580tgaggccagg aatgagaccc
agctaatgtt ggccaggcat attctgtgtg ctggccaaag 11640gactgtgata
acagtcttct tgttgctaca gatccacagt cccctcttgg aacttttctc
11700gattgggctt cttctgtggg taatattcct aaggaaagca tcatggttct
gagctccaag 11760ttgggttttg aagttagatt tgaatagtga atgaggtgat
taagggctct cctggcagag 11820gacacaccat gagcaatatt ttatgtgccc
tgaaggtggt ctgtataact ttatccatgt 11880ctttcttctc agccccatca
ctttacaagc cttggttcag tgtggacagc ctcagtgctc 11940cactcacatc
ctccagtggc tgaaacgtgt gcatgccaac ccccttctga tagatgtggt
12000cacctacctg gtggccctga tccccgagcc ctcagcacag cagctgcgag
agatcttcaa 12060catggcgagg gatcagcgca gccgagccac cttgtatgcg
ctgagccacg cggtcaacaa 12120gtgagtttcc acactgtatt tctcctccta
ggagcagagg aacatcttgc acctctgtgc 12180atctctgtat taaaactgaa
cccctccttc cactttcaaa ctctgctcct tactcttgtg 12240ttttttcttg
atcatttttg gggtaatgac ttgaaataag aaatcagcaa acacaaattg
12300aatttttaaa aatattttct ctacattata ttataaaagt ttttgaacat
agcaaagttg 12360acagaatttc acagggaaaa cccctagaaa accagctatc
tcctactatt taagtgttat 12420tatatttgct ttatcacata tacatccatc
cattaattca tcttattttc tgaagcattt 12480caaagtaaat tgcaaacatc
aacacacttt cccctaagta ttacagcttg catattatta 12540acttcagttc
aatattagtt agcagttttt tcctctgaat ttttttgttt gtttgttttg
12600tttttttttg ttgttgttgt ttttttgaga tggtctcact gtgtcaccca
ggctggagtg 12660cagtgatgca gtcacggctc actgaagcct caaattcctg
ggctgaagtg atcctcccac 12720ctcagcctcc tgagtagctg ggaccacagg
tgcatgctac catgccctgg ctaatttttg 12780tattcttggt agatacaggg
tttcaccatg ttgctcaggc tagcaggttt ttcctttgat 12840gaaatttttt
ggctttttct tttttacatt tttatataaa tttatgtgga acaagtgtaa
12900ttttgttaca tgaatagatt gtgcagtagt taagtcaggg ctttcagggt
atccatcacc 12960cagacaacat atagtgtacc cactaagtaa tttctcacca
tccatctccc tccacttcca 13020caccttctga gtctcaattg tctatcattc
cacacactat gtccttgtgt gcacattatt 13080tcactcccac ttataaatga
caacacgcaa tatttgtctt tctgtgactg tcctgtttca 13140cttaagacaa
tgacctccag ttccatccat gttgctgcaa atgacatgat tttattcttt
13200ttatggccga atagtatttt attgcctata catttcacat ttttaatcca
atcgtccatt 13260gatagacact taggttgatt ccatgtcttt gctattgtga
atagtgctgt gataaacata 13320tgggtgcagg tttcctttgg atataatgat
ttcttttcct ttaggtatat acccagtaat 13380gggattgttg gatttattgg
tagttctatt tttagttctt tgagaaatct ctgtattgtt 13440ttccatagtg
gttgtactta tttacaatcc catcaacagt gattaactgt ttccttttct
13500ctgtatcctc accaacaact gttatttttt gtcttttgaa taatggccct
cctgactctt 13560gtaagatgtt atctcattgt ggttttaatt tacatttctc
taatgattag taatgttatg 13620cattttttca tatgcctatt gccatttgta
tgtcttcttt tgaaaaaaat gtctattcat 13680gtcctttgcc tactttttaa
tgggattatt tgggggattt ttttgttgag ttgtttgaat 13740tgcttgtaca
ttccggatat tagtacccca ttggatgaat agtttgcaaa tattttctcc
13800cattctgcag gttaccaccc tgttgattat ttgttttact gtgcagaaac
tttttacttt 13860aattaagttc tatttgtcta ttttttgttt ttgttgtctt
tgcctttgag gtcttattca 13920cgaattcttt gtctaggcca atgtccagag
aagttttccc taggttttct tcttgcattt 13980ttatagtctc aggtcttata
tttaagtctt tgatccatct tgagttgatt tttttatatg 14040gtgacagata
ggagtccagt tttattcttc tgcatatggc aatccatctt tcccagcacc
14100acttattgaa aagggtgtcc tttccctagt gtatgttttt gtcaattttg
tcaaagatcc 14160gttgactgta agtatgtgac tttatttctg ggttcagtat
tctgttccat tgatctatgt 14220gtctattttt atgccagtac catgctgttt
agattactat agccttgttg tataatctga 14280agtcaggtaa tgtgatgcct
ccagctatgt tctttttgct taaaattgct tcagctattc 14340aggctctttt
tggattccat atgaatttta taattatttt ttctaattca caagtttggg
14400ttttaagaca aacctaactg gggttaccaa gtcctgactc tcttctctta
ttctgtagct 14460atcataagac aaaccctaca gggacccagg agctgctgga
cattgctaat tacctgatgg 14520aacagattca agatgactgc actggggatg
aagattacac ctatttgatt ctgcgggtaa 14580tctcagtctt ttatatgaca
tacatcattt cagaagcact tttcctggac accttttact 14640tccctctcct
gcaccctgat gggttcttgt ttcttttctt caatgcaggt cattggaaat
14700atgggccaaa ccatggagca gttaactcca gaactcaagt cttcaatcct
gaaatgtgtc 14760caaagtacaa agccatcact gatgatccag aaagctgcca
tccaggctct gcggaaaatg 14820gagcctaaag acaaggtaaa gtccacaaga
agaggtctga aagtgaaagt ttattaacaa 14880ggatttggaa ggtactaggg
gaatgagact ctagatttca tctactgact ttattctgct 14940gtttctttcc
tttccttcct tccttccttc cttccttcct ccctccctcc ctttcttctt
15000tccttccttc cttccttctt tcgagatgga atctcactct attgcccagg
ctggagtgca 15060gtggcatgat ctcggctcac tgcaacttct gcctcctggg
ttcaagcaat tctctctgcc 15120tcagcctcct gagtaactgg gattacaggc
atgtgccatt acacccagct aatttttgta 15180ttttttagta gagatggagt
tttgccatgt tggccaggct ggtcttgagc tcctgacctc 15240aggtgatccg
cctgcctcag ccttgcaaag tgctgggatt acaggcgtga gccactgcac
15300ctggcctcta ctgttttcta attgcaaatt tcaacaagcc tattgacttg
actgcctagc 15360agtatgtgac gtgagagaaa tacttgactt tgctgctatg
tcaacatgca gaacgtgaga 15420tgtttttgct tcctaccgtc cacctaccag
attgaccatc cctctcatca tggaaaaaca 15480tgcttaattt tcccccaata
agcttaggct aggatagcca acttggcccc ctcttaggtg 15540caaagactcc
agaactttgg aaactaccct atttattagc cccaaactct tactacccct
15600tctcatcttt atcctcacat taaaataact tacgttaaaa caacttgatt
ttcacttagt 15660ggtggatctc caaacaaatc acaacttggc cataatttat
gtgttttaat ggaattgaat 15720tcaacaggca ttccacaggc tttttctggg
aacccttact tgatagtgct ctaggaaaca 15780ctggcaagaa gattcaatac
cagcatttga agaacgatta cagagaaatt agacctgtgc 15840ttaagaaaga
gctagcagac aatgccagtg tttgccaggc atgttctgtg ttctgaccac
15900aggacagtga taaccatctc ctcttttgac tgcaggacca ggaggttctt
cttcagactt 15960tccttgatga tgcttctccg ggagataagc gactggctgc
ctatcttatg ttgatgagga 16020gtccttcaca ggcagatatt aacaaaattg
tccaaattct accatgggaa cagaatgagc 16080aagtgaagaa ctttgtggct
tcccatattg ccaatatctt gaactcagaa gaattggata 16140tccaagagta
agtaagagct attcacccca tataccactg agggccctga gctggaattc
16200caaccctagg ttttggcata gccactgtct gcccttgctt ctgaaacaaa
cacttgtgca 16260aatgtgtagc agatctagac ccaaagactt agggtcaatg
aaatcaagac attttggtag 16320tgattggaaa tccatattta cttggggtgc
aagagtcaaa ggataataac atggtgtgtc 16380agctcaaaat atacttcttc
ttatctagtc tgaaaaagtt agtgaaagaa gctctgaaag 16440aatctcaact
tccaactgtc atggacttca gaaaattctc tcggaactat caactctaca
16500aatctgtttc tcttccatca cttgacccag cctcagccaa aatagaaggg
aatcttatat 16560ttgatccaaa taactacctt cctaaagaaa gcatgctgaa
aactaccctc actgcctttg 16620gatttgcttc agctgacctc atcgaggtaa
gtgtgaagag tttgaggttc tctagcccat 16680tttgtacagc atcataaaca
gagagtccct gggagccagg agctacccag aggaaaacta 16740agaaccacca
ggcacttcct accatgattc tgaggctttc ttctttccct ccttccccgc
16800cttcctctct ccccgctagg ggtcacctga agcatgactt cttaacatta
atagaaatgc 16860aggcctggcg aggtggctca ctcctgtaat cccagcactt
tgggaggccg aggcgggtgg 16920atcatgaggt caggatatcg acaccatcct
ggctaacacg gtgaaagccc atctctacta 16980aaaatacaaa aaattagccg
ggcgtggtgg caggcacctg tagtcccagc tacttgggag 17040gatgaggcag
gagaatggcg tgaacccagg aggctgagct tgcagtgagc cgagagattg
17100cgccactgcg ctccagcctg ggcgacagag caagactcca tctcaaaaaa
aaaaaaaaaa 17160aaaaaaattg aaatgcaaat gtctcgtctt taagtcccaa
agccaaggaa gcatatgtgc 17220tgcctagtca gatctgcttc aaatctcaaa
tcactcccaa ctctgaatcc tttgttgaat 17280tatttgtcct atctgaacct
tagctgcctc ttctagaaaa aagcaagtaa taaggtcaag 17340attctagtga
gattttaata aagcagctcc tgtgaaatgc taaggtcagc tcctggcctg
17400tggtattcaa atacttgttt agataaatgg acatcaagag tggggactac
taggctggca 17460tacaacaaag aaacctgatg ccattttctt gtctgatttt
ctttctcaga ttggcttgga 17520aggaaaaggc tttgagccaa cattggaagc
tctttttggg aagcaaggat ttttcccaga 17580cagtgtcaac aaagctttgt
actgggttaa tggtcaagtt cctgatggtg tctctaaggt 17640cttagtggac
cactttggct ataccaaaga tgataaacat gagcaggtgt gtatttgtga
17700agtatcttct taaggaaagc tttgggtctc aatgcaaaaa caattctttt
ctaagcatgg 17760aagtcctcaa aatactatct aactgaaggg ataactatgg
tttttatcaa ccagacctgc 17820tggggtaagg gccagtatcc tctgcagtta
aagatctcct gaattcagtg tgcccagaaa 17880ccagactcac aataagtact
ctaggataac aagagtatga actctgggct gggtgtggtg 17940gttcatgcct
gtaatcccag cactttggga ggccaaggtg ggcagatcac aaggtcagga
18000atttgagacc agcctggcca acatactgaa accccgtctc tactaaaaat
acaaaaaaac 18060tagctgggca tggtagtggg tgcctgtaat cctagctact
cgggaggctg agacaggaga 18120attgcttgaa cccgggaggt ggaggttgca
gtgagccgag atcacgccgt tacactccag 18180cccgggtgac agtgtgagac
tgtatcttaa aaaaaaaaaa agtatgaact ctgggcatag 18240atttaattct
aacttccctg tcttgaagct gtgcgcactt ggggaagttg gttgatatta
18300tgtgtatctg tttctgtctg tatcccagac tactaataac agtccaaacc
tcacaaggtt 18360atttaaagac aatgaaataa ggcatctaaa atgccaagca
cagtgcctga tgctggcatt 18420ggttgttcaa taagcagaca ctattacgag
ttctaaatta atattttcat tattattaac 18480tgctgtcttt ggctctcact
cccatcagtg cactagcaaa tgagaccaaa cttccacttt 18540gaagctagca
atgagccccc atttaaggag ggaaataggt tgtatgatct ggagcttatt
18600cttgaatttt ttgctaccca aagtgtggtc tggtcagaaa tacagcttct
catgcttcac 18660ccacaatcta ctgaatcaga agcgcatttt agcaagacct
catgtgactt gtatgcacat 18720tcaactttgc agagcaaggc agtaatttac
ccctccaggc tcactgttga gcacgagctc 18780catcttctaa tttcctgacc
cccacttgag gccgaggatc tttgatctgc tttgagtctg 18840tcagtttcac
attttttttt tcccaatgcc tgggcatcca tctctgagat tcttcttctc
18900tctgagaaga acttgtctag gatcaagtgt ttttcaaact tctggtgaat
ttatataaca 18960gctacatttt cttaagaaac accttgtagt cttcactggt
caaagaagag aaggctaagc 19020agggaacggg tgggggatag aggatcttct
aatcttgagg atcctggcat actggagaat 19080agggacccct cctctcatcc
caccacatct tactatgtct acagattttt taattaagaa 19140tagctttagg
agtgccacta tccctgacaa gaccttagtt ctttaatctc tgcttagagg
19200aattagcctg gacttcagtg tctccctgtt cctcacctgg agcatttttt
aggcccatcc 19260tggctgcatc agacaggtcc cacattggga actgaaaggt
gtttgacatt gctgacatct 19320cactggccat tttattacta aactctcagg
atatggtaaa tggaataatg ctcagtgttg 19380agaagctgat taaagatttg
aaatccaaag aagtcccgga agccagagcc tacctccgca 19440tcttgggaga
ggagcttggt tttgccagtc tccatgacct ccagctcctg ggaaagctgc
19500ttctgatggg tgcccgcact ctgcagggga tcccccagat ggtaagtcag
caggccccac 19560tgggggccca tgagaccaga cgttggtttt tttttagatc
gcccagactc ccttacgatc 19620ccagctgcac aagcccgaaa agatgcttgt
actttcttca gagatggagg tttgccttga 19680atttcactga agatgactct
tggatcacat ggaaatgtta acatttagaa attaagctat 19740tcataatgtt
agctgtattt ttaagagcat taatttattc atctggaaaa caatgttcgg
19800tataccttcc tctacctttg ctgaaggtcc ttttattttt atttttattt
ttttaatttt 19860ttgagatgga gtcttgctcc caggctggag tgcagtgata
caatctcggc tcactgcaac 19920tctgccttcc gggttcaagc aattctcctg
cctcagcctc ccaagtagct gggactgtgg 19980acgtgcacca gcatgcccgg
ctaatttgtg tatctttagt agagacaagc ctgttgacaa 20040ccatgtcagg
ctggtttcga actcctgacc tcaagtgatc ctccagcctg ggcctcccac
20100agtgctggaa taacaggtgt gagccactgc acctgacctg aaggtccttt
taagattgaa 20160atgatacaat gattataaaa gaaagtattt ggcaaactat
aattcactat ctaaatatgc 20220tataattttt attattaatt cataaaagga
aatatataaa tgtactccta tggcttgatt 20280aaaaaaatgt tgactttaag
aaaacaggtc tcaagctatt ttattgaaat attatttaaa 20340aaataaaacc
caatgcaaat tgatatgtac atcatctcaa taggcctttg gtttcaaaaa
20400attgatttta tcataatata atacatttca agtacacctt cacttacagt
cagactccag 20460aacaccagaa ttaagccatg gcatatatga tacttaaagt
ccataaagct ctgaggccca 20520gcaatattct taagagcctt ctgagtccac
ttgaaaatga catgatatct atctagtgaa 20580atttcttata tcctgattca
ctgaaaacgg taaaaacatc agtttgatct ttatttatca 20640aactattcag
ctcatcaaaa tatgctagtc cttcctttcc agataaagag gaattactct
20700ccaatgtatg ggaggttgta attaacaaaa ccgactttaa aaagacttac
ttttatttgc 20760tctcccttgt tgggtctaca gattggagag gtcatcagga
agggctcaaa gaatgacttt 20820tttcttcact acatcttcat ggagaatgcc
tttgaactcc ccactggagc tggattacag 20880ttgcaaatat cttcatctgg
agtcattgct cccggagcca aggctggagt aaaactggaa 20940gtagccaacg
taagattctg tttgcctttt gatttcttag gttattactt tcttccaggg
21000tgcatttctt gttaaaacat atttaaaaat gtgtttccac ttcaagacaa
aatgcttcat 21060cattgtaatc acctcattat ttttttatga aaaacttcaa
gcttccacca gaatgcacta 21120cctcactagc tccagtagtg gtatggccat
aagacaagaa ctcagttctc tcaacaaatg 21180agtattccta tcatcttttt
aatctggttt tgcctcacgt taactcaggt gctttctagt 21240tctgggtagt
atactccaac tctagagaac tgagaactcg ctttccttct tccaaacaaa
21300tcccagtaat gtttccaaag gtctgagtta tccaggaaat ctttgcccgg
aggtgagaaa 21360gggtggttga tctgactgac aggggactga agtatttaat
gaatctgaat aggttgtttt 21420ctgacttata gatgcaggct gaactggtgg
caaaaccctc cgtgtctgtg gagtttgtga 21480caaatatggg catcatcatt
ccggacttcg ctaggagtgg ggtccagatg aacaccaact 21540tcttccacga
gtcgggtctg gaggctcatg ttgccctaaa agctgggaag ctgaagttta
21600tcattccttc cccaaagaga ccagtcaagc tgctcagtgg agggtaattc
tttcagccaa 21660gtctgcctag ccagtttgaa agagagaaca gagaatgtac
ctgcagaatt ttgccaggct 21720aaacagttga ttgagatcat tcaggtcctg
aggaagcagg agaggagtag aaaggaaaga 21780ttccgggtta cctattttaa
ttctagccta gacttactac ataactacat aattaccttt 21840cttctacttt
tcacatttta ctaaactgtc ctttatcttt ctgctttgag acttattaag
21900acctactgct taattagttt ttattaagtt gtgatttttt gttatctatt
tgttttgaga 21960atgaagaaac aatagctctg gagagatcat ctttggaaaa
ttaatatttt cccccccaaa 22020aaatacctaa gaacatattg atttgaggta
gctaggtagg taaagcatga aactcctaac 22080ctcgtgataa tggaatacag
cctcttttgg agagttccat tttaagtggc accctcaacc 22140attgatttgc
cttagttttc atattttaga cacattcatg tgttcattca aaaataatat
22200ttaattggcc agccacggtg gttcatgcct gtaatcctag cactttggga
gccccaggtg 22260gatggatcgc ttgagccctg gtgtttggat accagcctgg
gcaacatggc aaaaccccat 22320ctctacaaaa aaaattaaat aaataacaaa
attagccagt cgtggtggca catgcctgta 22380gctccagcta ctcagaaggc
tgagatggga ggatcaactg agcccaagag ttcaagcctt 22440cagtgaacca
tgcttgcacc actgcactcc agcctgggag acagagcaag atcctgtctc
22500acaaaaaaca aaaaatagta tatttaattg cctaatatat accacgtatg
ttgagtgaga 22560cacacaaggt ccctgacctt tgaacgctta cattttataa
gggagacaca caattaagca 22620agcagtaatc atagagtaag ggctaagtta
tagaaagtat tagagtacca tgaaatttta 22680tatcatgtag cctgtgctag
tcagggaatg cattctgaag caagtgtact tgacctgata 22740actgaggact
gtgtcagagt catttaggca aaggagaaag gagtgagtgt tccaggcaaa
22800aggaaaagca tgtaatggcc tgaaggtaaa ggaatatggt tcaaggaact
ggaagaagtg 22860cagaatggta aggggctcag agatgatggg gagaggtagg
caggggagag agcatgccca 22920gctgcgaaag ccatcctaag gagtttggac
tcttttgaag gcacaggagt tgaaaagggg 22980agcagaaata agataggggt
gatgttttag aagaaatact ctgactctag tgtggaagat 23040gggtgagaag
gaggcacagc tggacacgaa gagaccattg gacatctctt acgatcctat
23100gtggctaaga gctgataatg gcctgcagtg gagaaaagcc aggtatagaa
aggagtgagc 23160agattctaca actttctaag aggcagaatc ataagtactg
ggtgattaac tgggtatggg 23220gacaaggcaa aagaaagaag aaaagaggaa
ggaggcgccc ttcattttaa taagaactac 23280agtgggagag cttctggttt
caaggaaagt gacaaattca gttttggatg tgctgtattt 23340gatgtcctcc
tatgaaacaa ccagtttaga aatctagctg tcaaatagac ctatggatct
23400gagcccagta aagaggcttg ggctccacat atggatttgg gaatcattag
tatacagagg 23460ttgttgtggt taaacagcaa ctggtataga gtgagacatg
agagatgagg acagaaatat 23520ggagaagaca aacatataaa ggaagaaggg
gaataaccag caatgagtta gaagaagtga 23580ccagagaagc agaaggagaa
ccaaagccat aaaaggtcac agaagccaaa gagcagccac 23640aggggagatc
accccatggg taggcgaaag ctggcattag gactccagca catcagcaaa
23700gcttggtctt gtggcacccc caacttggag aaacaatact tggaggaaaa
tgtgctattt 23760caaagaaagc atccttagaa aaaaccaggc caatgttgaa
ctttcttaca tgtactaagt 23820ttttaagtac acacttggaa ggaaggtgcc
atcatctctt cagatgtgag aggctccagc 23880gtcttagtct ggtcatgagt
gcgcaactct atggaaggct tctgggaggt caaggaagat 23940gaaacctaaa
tatgcccatt ggatgtagga gcaaggaggg cattagagac attgatgaaa
24000gcattttcag gagatggagt gagcagtcag agcacattgg gaggaagtag
agactgcaaa 24060ggcagacaac tcttgatggt gaggaagatg agaaagcaag
aaaagaaaga aaggagcata 24120ggggaggggc acaggggaag agacttgagc
gtgcttaatg caggtggaag gaagcaggta 24180gagagtagga gatttcatat
gaaagagaca gtttctcttg ccctgcattg taggaaggaa 24240ggggcacact
gaagttcagc cccagtgatc agctatttaa catctctgag cctctgcttc
24300tgtaaaatga gaaccataag cctactgttg tggggattac aggtaacaga
tggaaagaac 24360tcagccagaa gcttcagagt cactctcatg gcttgtcatg
ttgatgttct ttctaatatt 24420atttgtttct cagtaaatta aatagttaga
gataggtgtg gactgaggga agacaggagg 24480ataagggggt atttgcaccc
tgagaatttg tgatgtccat tttgattcat gacttggcaa 24540taactcaggt
atttttgttc ttcaccagca acacattaca tttggtctct accaccaaaa
24600cggaggtgat cccacctctc attgagaaca ggcagtcctg gtcagtttgc
aagcaagtct 24660ttcctggcct gaattactgc acctcaggcg cttactccaa
cgccagctcc acagactccg 24720cctcctacta tccgctgacc ggggacacca
ggttagagat gctcagtgcc tgacccagca 24780ttttctcacc ttccacatca
tggccaccta gcatggcaca ggaaaaaata ctctgtgttg 24840taagaccctg
tcactagcct tctgggtttg caccatcttt gggtatttaa agcagggtcc
24900tctggccaac acattgggtg tcaccttttg cttccttgtg catgggatgg
gatcacagca 24960cagatcccaa tttgctccta attcagtgtc catgtttctg
agcctccaga cccatcgcta 25020tgagcttcct ggagcccacc aatgtgcttg
aagccttcac cgtacttagg tggctccctg 25080tcttcagccc ccaagttcca
gtgcttgttc tcagctttgc tgaaacaacc agccaactcc 25140tgctctgctt
gtccaaagtc ttgggaatcc tggtgtctgc ccttgccttg ggttcttgta
25200ggactgaggg atcaaaaaga tcatcttagt taagggcaag agacaatgtt
aaaataagga 25260ccatattttt gttgcatttg aggctgaatt gttttgggaa
cataatcacc atccttgaaa 25320gctctaacat tatgcactgt cttcattgta
atgtctttag attagagctg gaactgaggc 25380ctacaggaga gattgagcag
tattctgtca gcgcaaccta tgagctccag agagaggaca 25440gagccttggt
ggataccctg aagtttgtaa ctcaagcaga aggtgagtat tcaaaacaca
25500gctgcctcat ctctgctcgc agtctcaggt tcagaattca tgaggagaag
acatgtaatt 25560taacctattt aacaaatagg ttaactgagt acccactaag
cggcaggcct attctaagac 25620ctgggttaac tgagtaccca ataagcggca
ggcctattct aagacctggg gctagaacag 25680tgaacaatgg agtctctgcc
ttcatggaag ttacagtgaa caaccaaaca agttaatatt 25740tggaatatca
gataagtact gaggaggaaa acagagcgta gactggtcta tggagggcta
25800ggagtaggag ggaggaagaa gggcagggaa agcagtgcat ttggaataat
aagggaaagt 25860ctccctggta aagtgagcat aaggagacct atcagaaata
agaggagaag ccgtgtggta 25920agactgttaa caggcagagg gaccagcaag
tgcaaaggcc ctgaggctga cacactacta 25980ccatgtttca aggaaaggaa
ggaagacagt atggctggag cagaaagacc agggagaaaa 26040gaggtagaag
atgaggacag agagatatgg agaggtgaag gaaggataat ctcataggcc
26100atggtaagaa ctttggcttt ttctatgaat taaacgaaag ccattgggga
gtcctcatga 26160tttgatttat gtttatgttg agaaaagact atgggcagac
aagggcagag aaactaatat 26220gtaggttatc acaataatcc aggcaggaat
cagtgttgtt ttggatcagg gcaatggcag 26280aagagatatg agaaggggat
ggattctggc catattttga agattaggct gacaagattt 26340gctgatacag
tggatgttga gtgtaagagg aaaaggggaa tgaagacaaa cctaaggttt
26400ttggcccggg caactgaaaa atggaacttc catttattga gatggaaagg
gctactggag 26460gagcaggttt tagggaatgg gagaaattta ggtgttcact
ttggaaaaaa aattatatag 26520ggatagcgag gagcaggttt tagggaatgg
ggcacattta ggtgttcact ttggaaaaat 26580ttttatatag ggatagcata
tcacagaatt aaactaggaa gaaaatccca tgatagaaag 26640cactggagga
gcagggcacg ctggggaaat agtgtttggt aaacattgtt ttacgaagga
26700tataaaatgg accagcctat ggattgaagg acgcccggga atcttgttac
aaagaaaggg 26760ggagttgggg agatggagcc cagggcaagg gcagcaagga
accaggacag gcatcttggg 26820tagaaagtaa tatagagatg tcgtgtcttc
ctggcccaga agggctgcga gcctttgctg 26880ttccacaaac aagctaagtg
ctccccattt cagggccttt gcattcctga ccttctgcct 26940ggaatgtgct
cctcccagaa ctcagcgtgg ctccaacctc ttttcattct ggtctctgcc
27000cacatgtgcc cttatcagag agaatttctc tgaccaccaa gtatgaaata
acacttcttc 27060tatccctttc ttttatcctt gtatccagtt ttactcttct
tcataacatt cattaccatc 27120tgacatgagc aagttacttg tttattgcct
gtacacctcc cccactagaa ggtaagcccc 27180atgaaagcaa ggattcccca
gtaccaagag cagtgcccag cacacaatag gctcataaca 27240ggcaatccat
aaagacttgc atacatgaac acaactgagt ttaaaattat cagtaaatga
27300gacccattaa aaaattttaa tgagaaaaaa aaaattcagt aaaatcctga
actgtgtttt 27360tgtttaagca cattgattcc ttggagtttc tctacctttt
cctctctttc cttccaaaac 27420atagcttctt tatttattta tttatttatt
tgtttgttta tttatttatt tatttattta 27480tttatttttt gagatggagt
ctcgctcttt tgcccaggct gcagtgcagt ggtgccatct 27540cggctcactg
caagccccgc ctcccgggtt catgccattc tcctgcctca gcctcctgag
27600tagctgggac tacaggcacc caccaacgcg cccggctaat tttttgtatt
tttagtagag 27660acggggtttc accatgttag ccagaatggt cttgatctcc
tgacctcatg atctgcccgc 27720cttggcctcc caaagtgctg ggattacagg
tgtgagccac cgcacccggc ccaaaacata 27780gcttcttacc acacatctct
tgattctctt atacactcgt ccaggtgcga agcagactga 27840ggctaccatg
acattcaaat ataatcggca gagtatgacc ttgtccagtg aagtccaaat
27900tccggatttt gatgttgacc tcggaacaat cctcagagtt aatgatgaat
ctactgaggg 27960caaaacgtct tacagactca ccctggacat tcagaacaag
aaaattactg aggtcgccct 28020catgggccac ctaaggtaaa gaaggccgag
ggtcatctga cctgcactgc aggcctgggt 28080ggttcttttc attattcctc
ttccacttca tacctgacca agccatgttc tcccctagtc 28140tacaatcaga
gtggcagaga gagccctcaa caattttttt tttttttgag atggagtctc
28200actctgtcac caggctggag tgcagtggca caatctcggc tcactgcaac
ctccgcctcc 28260cgagttcaag tgattctcct gcttaagcct cccaaggagc
tggaactata ggtgcatgcc 28320accacaccca gctaattttt atatttttag
tagagacagg gtttcaccat attgaccagg 28380atggtctcga tctcctgacc
tcgtgatcca cctgccttgg cctcccaaag tgctgggatt 28440acaggtgtaa
gccactgcac ccggccaagc tctcaacatt ttaaccctct gcgcatgtcc
28500agttggattt tcctaccatt tatcaggcac ttactattca tgtatcaagc
acagtgctgg 28560gtgctttaaa gaaattatct cggtcctcac aataaactgc
gaggtcactg tgagttttcc 28620tgtttcatgg ataaggaaat ggtagctcag
aggggttaaa tcatttggtc aaaatcacag 28680agctagtaaa tagcagagca
ggattcaaac agttttcaaa aaacttctct ttctcctaaa 28740cctgtttgca
aagtccttaa tttgtgctga atgttggctt tagaagttga tgagtttgat
28800ctgtggctgt ttctctgaac catccttgta tctggttttg atcaccacaa
atggaacttc 28860tgtttaatcc tgcatatctc cattgaaagg acaaaatcat
tggtgccaac tgattttctt 28920taccatagtt gtgacacaaa ggaagaaaga
aaaatcaagg gtgttatttc cataccccgt 28980ttgcaagcag aagccagaag
tgagatcctc gcccactggt cgcctgccaa actgcttctc 29040caaatggact
catctgctac agcttatggc tccacagttt ccaagagggt ggcatggcat
29100tatggtatgt gtctcttccc ctgtgtgagc acttccaaag taatgcaggt
gttgagacct 29160gtggttacag gctgaactag taccattcac aactatttcc
tacgtatttt cagatgaaga 29220gaagattgaa tttgaatgga acacaggcac
caatgtagat accaaaaaaa tgacttccaa 29280tttccctgtg gatctctccg
attatcctaa gagcttgcat atgtatgcta atagactcct 29340ggatcacaga
gtccctcaaa cagacatgac tttccggcac gtgggttcca aattaatagt
29400tgtaagtatg agtctgccag tcaataaata catggatata agtgctaatt
acatcctcaa 29460ctctgagcta ggtgcaggaa ggtttccaaa gatgtataag
gcatgcttcc ttccccccag 29520ggaattcttg gggagaaaaa aaaactttca
caagtgtgta gttacccagt tacacaaagc 29580tgaatgtgat acatatcaaa
gagatgctac taagtagaac agttctttgc ctagtggtat 29640caaaggaagc
ttcaggacac cagctaggag gctgactatg ttagacattc cttttataaa
29700tatggacagt gatcagtgac tggcaacgaa gattcataat tttctgttat
ttatttttaa 29760ctttcagtgc attgtccagc ttaataatta acttgtcaaa
tcggtatttt tgcctaatgt 29820tcattgctct ttgaggctca tccaagccca
ttaccttaaa aatctcctgt cattttgtag 29880gcaatgagct catggcttca
gaaggcatct gggagtcttc cttataccca gactttgcaa 29940gaccacctca
atagcctgaa ggagttcaac ctccagaaca tgggattgcc agacttccac
30000atcccagaaa acctcttctt aaaaaggtaa aagaagaaag cagcaaggct
tcttgaacca
30060tgcaaagtaa atgaaagatt ttacatagca tgatttagac atttttttaa
atttttaaag 30120gaaataattt aagcatttta aggagattaa taactatagc
acaaacactg tggcatcttt 30180gcattagtaa acatgagaac accaaccctg
tcaggaagaa tctaagaaag tcattagagg 30240attctggtac tttcacccta
agatatttta ttcagtacaa cctgttataa gcaaattctc 30300cctctgactg
tgaagaattc agaatggcta gaggcgttat tgactacagg cttgctgtta
30360agctagagag agtcagaaca gccattgagc actaaatgga ggcagcattc
tgagaaaata 30420ctttaaccca ggcttactga cttccatacc tatgttcttt
ccacaaatca agttgtctca 30480attcagttta gcaaatttgt atcaagtatc
ccctatgtgc aaaatgctag actaggtaca 30540gtgagaagat agaaactggg
taaggtatag ccttttcttt caagaagata ccatggagac 30600atcaacaaat
gagaaataat taattatata agcaaaatta tgacatgctc tttgagaaag
30660gtgcaaggga ctatgtaact gtaagaatga gacaaattgg ctatgactta
ggtgggatgg 30720taatgataag gagtggccct tagaagagct ttgtcaggat
ttgagtgttt gacaggtgga 30780ggtaaaagca aaggggtcca ggcataggag
tagcacaaag aaaagtgcag agtggctttg 30840ggaatggggc aagtacaata
ttgttgtgaa ggtcagaggc agagaacttt gaatgactga 30900tgtctgactg
tggggatgtt atctttgttg ttcatttcag cgatggccgg gtcaaatata
30960ccttgaacaa gaacagtttg aaaattgaga ttcctttgcc ttttggtggc
aaatcctcca 31020gagatctaaa gatgttagag actgttagga caccagccct
ccacttcaag tctgtgggat 31080tccatctgcc atctcgagag ttccaagtcc
ctacttttac cattcccaag ttgtatcaac 31140tgcaagtgcc tctcctgggt
gttctagacc tctccacgaa tgtctacagc aacttgtaca 31200actggtccgc
ctcctacagt ggtggcaaca ccagcacaga ccatttcagc cttcgggctc
31260gttaccacat gaaggctgac tctgtggttg acctgctttc ctacaatgtg
caaggtgagc 31320tatgctcagg taaagggtgc accgggctag ttcatggcag
gctctaagag gagagcctcc 31380tccagggagg aaaggacttt ggctttctag
cagataatct tccttgctac ttggaagtct 31440tttattttat tcaacaaata
gaaatattta ttaaacatat cacgtgtatt aaatattcta 31500gtaggcagta
acagaaagta gacagataag ccagcaatta taattcagtg tgagaggtgc
31560tatgataaag tgtagtatat aagtataagg tagagtggaa gcactcaaca
agggaaccta 31620aacaaagcct gtggtggtca ggcaaggctt cctggaggaa
tgccttttgc tatcagattt 31680tatctttgca ttacagatgg aggagtctat
tgcacaattg gcccagaaaa atggggcttt 31740attattgaaa gactttcaac
atagagattg ctctggaaat gtactgctta atttaaccaa 31800tgtcttttca
tttttatgtt aggatctgga gaaacaacat atgaccacaa gaatacgttc
31860acactatcat atgatgggtc tctacgccac aaatttctag attcgaatat
caaattcagt 31920catgtagaaa aacttggaaa caacccagtc tcaaaaggtt
tactaatatt cgatgcatct 31980agttcctggg gaccacagat gtctgcttca
gttcatttgg actccaaaaa gaaacagcat 32040ttgtttgtca aagaagtcaa
gattgatggg cagttcagag tctcttcgtt ctatgctaaa 32100ggcacatatg
gcctgtcttg tcagagggat cctaacactg gccggctcaa tggagagtcc
32160aacctgaggt ttaactcctc ctacctccaa ggcaccaacc agataacagg
aagatatgaa 32220gatggaaccc tctccctcac ctccacctct gatctgcaaa
gtggcatcat taaaaatact 32280gcttccctaa agtatgagaa ctacgagctg
actttaaaat ctgacaccaa tgggaagtat 32340aagaactttg ccacttctaa
caagatggat atgaccttct ctaagcaaaa tgcactgctg 32400cgttctgaat
atcaggctga ttacgagtca ttgaggttct tcagcctgct ttctggatca
32460ctaaattccc atggtcttga gttaaatgct gacatcttag gcactgacaa
aattaatagt 32520ggtgctcaca aggcgacact aaggattggc caagatggaa
tatctaccag tgcaacgacc 32580aacttgaagt gtagtctcct ggtgctggag
aatgagctga atgcagagct tggcctctct 32640ggggcatcta tgaaattaac
aacaaatggc cgcttcaggg aacacaatgc aaaattcagt 32700ctggatggga
aagccgccct cacagagcta tcactgggaa gtgcttatca ggccatgatt
32760ctgggtgtcg acagcaaaaa cattttcaac ttcaaggtca gtcaagaagg
acttaagctc 32820tcaaatgaca tgatgggctc atatgctgaa atgaaatttg
accacacaaa cagtctgaac 32880attgcaggct tatcactgga cttctcttca
aaacttgaca acatttacag ctctgacaag 32940ttttataagc aaactgttaa
tttacagcta cagccctatt ctctggtaac tactttaaac 33000agtgacctga
aatacaatgc tctggatctc accaacaatg ggaaactacg gctagaaccc
33060ctgaagctgc atgtggctgg taacctaaaa ggagcctacc aaaataatga
aataaaacac 33120atctatgcca tctcttctgc tgccttatca gcaagctata
aagcagacac tgttgctaag 33180gttcagggtg tggagtttag ccatcggctc
aacacagaca tcgctgggct ggcttcagcc 33240attgacatga gcacaaacta
taattcagac tcactgcatt tcagcaatgt cttccgttct 33300gtaatggccc
cgtttaccat gaccatcgat gcacatacaa atggcaatgg gaaactcgct
33360ctctggggag aacatactgg gcagctgtat agcaaattcc tgttgaaagc
agaacctctg 33420gcatttactt tctctcatga ttacaaaggc tccacaagtc
atcatctcgt gtctaggaaa 33480agcatcagtg cagctcttga acacaaagtc
agtgccctgc ttactccagc tgagcagaca 33540ggcacctgga aactcaagac
ccaatttaac aacaatgaat acagccagga cttggatgct 33600tacaacacta
aagataaaat tggcgtggag cttactggac gaactctggc tgacctaact
33660ctactagact ccccaattaa agtgccactt ttactcagtg agcccatcaa
tatcattgat 33720gctttagaga tgagagatgc cgttgagaag ccccaagaat
ttacaattgt tgcttttgta 33780aagtatgata aaaaccaaga tgttcactcc
attaacctcc cattttttga gaccttgcaa 33840gaatattttg agaggaatcg
acaaaccatt atagttgtac tggaaaacgt acagagaaac 33900ctgaagcaca
tcaatattga tcaatttgta agaaaataca gagcagccct gggaaaactc
33960ccacagcaag ctaatgatta tctgaattca ttcaattggg agagacaagt
ttcacatgcc 34020aaggagaaac tgactgctct cacaaaaaag tatagaatta
cagaaaatga tatacaaatt 34080gcattagatg atgccaaaat caactttaat
gaaaaactat ctcaactgca gacatatatg 34140atacaatttg atcagtatat
taaagatagt tatgatttac atgatttgaa aatagctatt 34200gctaatatta
ttgatgaaat cattgaaaaa ttaaaaagtc ttgatgagca ctatcatatc
34260cgtgtaaatt tagtaaaaac aatccatgat ctacatttgt ttattgaaaa
tattgatttt 34320aacaaaagtg gaagtagtac tgcatcctgg attcaaaatg
tggatactaa gtaccaaatc 34380agaatccaga tacaagaaaa actgcagcag
cttaagagac acatacagaa tatagacatc 34440cagcacctag ctggaaagtt
aaaacaacac attgaggcta ttgatgttag agtgctttta 34500gatcaattgg
gaactacaat ttcatttgaa agaataaatg acattcttga gcatgtcaaa
34560cactttgtta taaatcttat tggggatttt gaagtagctg agaaaatcaa
tgccttcaga 34620gccaaagtcc atgagttaat cgagaggtat gaagtagacc
aacaaatcca ggttttaatg 34680gataaattag tagagttggc ccaccaatac
aagttgaagg agactattca gaagctaagc 34740aatgtcctac aacaagttaa
gataaaagat tactttgaga aattggttgg atttattgat 34800gatgctgtca
agaagcttaa tgaattatct tttaaaacat tcattgaaga tgttaacaaa
34860ttccttgaca tgttgataaa gaaattaaag tcatttgatt accaccagtt
tgtagatgaa 34920accaatgaca aaatccgtga ggtgactcag agactcaatg
gtgaaattca ggctctggaa 34980ctaccacaaa aagctgaagc attaaaactg
tttttagagg aaaccaaggc cacagttgca 35040gtgtatctgg aaagcctaca
ggacaccaaa ataaccttaa tcatcaattg gttacaggag 35100gctttaagtt
cagcatcttt ggctcacatg aaggccaaat tccgagagac cctagaagat
35160acacgagacc gaatgtatca aatggacatt cagcaggaac ttcaacgata
cctgtctctg 35220gtaggccagg tttatagcac acttgtcacc tacatttctg
attggtggac tcttgctgct 35280aagaacctta ctgactttgc agagcaatat
tctatccaag attgggctaa acgtatgaaa 35340gcattggtag agcaagggtt
cactgttcct gaaatcaaga ccatccttgg gaccatgcct 35400gcctttgaag
tcagtcttca ggctcttcag aaagctacct tccagacacc tgattttata
35460gtccccctaa cagatttgag gattccatca gttcagataa acttcaaaga
cttaaaaaat 35520ataaaaatcc catccaggtt ttccacacca gaatttacca
tccttaacac cttccacatt 35580ccttccttta caattgactt tgtagaaatg
aaagtaaaga tcatcagaac cattgaccag 35640atgctgaaca gtgagctgca
gtggcccgtt ccagatatat atctcaggga tctgaaggtg 35700gaggacattc
ctctagcgag aatcaccctg ccagacttcc gtttaccaga aatcgcaatt
35760ccagaattca taatcccaac tctcaacctt aatgattttc aagttcctga
ccttcacata 35820ccagaattcc agcttcccca catctcacac acaattgaag
tacctacttt tggcaagcta 35880tacagtattc tgaaaatcca atctcctctt
ttcacattag atgcaaatgc tgacataggg 35940aatggaacca cctcagcaaa
cgaagcaggt atcgcagctt ccatcactgc caaaggagag 36000tccaaattag
aagttctcaa ttttgatttt caagcaaatg cacaactctc aaaccctaag
36060attaatccgc tggctctgaa ggagtcagtg aagttctcca gcaagtacct
gagaacggag 36120catgggagtg aaatgctgtt ttttggaaat gctattgagg
gaaaatcaaa cacagtggca 36180agtttacaca cagaaaaaaa tacactggag
cttagtaatg gagtgattgt caagataaac 36240aatcagctta ccctggatag
caacactaaa tacttccaca aattgaacat ccccaaactg 36300gacttctcta
gtcaggctga cctgcgcaac gagatcaaga cactgttgaa agctggccac
36360atagcatgga cttcttctgg aaaagggtca tggaaatggg cctgccccag
attctcagat 36420gagggaacac atgaatcaca aattagtttc accatagaag
gacccctcac ttcctttgga 36480ctgtccaata agatcaatag caaacaccta
agagtaaacc aaaacttggt ttatgaatct 36540ggctccctca acttttctaa
acttgaaatt caatcacaag tcgattccca gcatgtgggc 36600cacagtgttc
taactgctaa aggcatggca ctgtttggag aagggaaggc agagtttact
36660gggaggcatg atgctcattt aaatggaaag gttattggaa ctttgaaaaa
ttctcttttc 36720ttttcagccc agccatttga gatcacggca tccacaaaca
atgaagggaa tttgaaagtt 36780cgttttccat taaggttaac agggaagata
gacttcctga ataactatgc actgtttctg 36840agtcccagtg cccagcaagc
aagttggcaa gtaagtgcta ggttcaatca gtataagtac 36900aaccaaaatt
tctctgctgg aaacaacgag aacattatgg aggcccatgt aggaataaat
36960ggagaagcaa atctggattt cttaaacatt cctttaacaa ttcctgaaat
gcgtctacct 37020tacacaataa tcacaactcc tccactgaaa gatttctctc
tatgggaaaa aacaggcttg 37080aaggaattct tgaaaacgac aaagcaatca
tttgatttaa gtgtaaaagc tcagtataag 37140aaaaacaaac acaggcattc
catcacaaat cctttggctg tgctttgtga gtttatcagt 37200cagagcatca
aatcctttga caggcatttt gaaaaaaaca gaaacaatgc attagatttt
37260gtcaccaaat cctataatga aacaaaaatt aagtttgata agtacaaagc
tgaaaaatct 37320cacgacgagc tccccaggac ctttcaaatt cctggataca
ctgttccagt tgtcaatgtt 37380gaagtgtctc cattcaccat agagatgtcg
gcattcggct atgtgttccc aaaagcagtc 37440agcatgccta gtttctccat
cctaggttct gacgtccgtg tgccttcata cacattaatc 37500ctgccatcat
tagagctgcc agtccttcat gtccctagaa atctcaagct ttctcttcca
37560gatttcaagg aattgtgtac cataagccat atttttattc ctgccatggg
caatattacc 37620tatgatttct cctttaaatc aagtgtcatc acactgaata
ccaatgctga actttttaac 37680cagtcagata ttgttgctca tctcctttct
tcatcttcat ctgtcattga tgcactgcag 37740tacaaattag agggcaccac
aagattgaca agaaaaaggg gattgaagtt agccacagct 37800ctgtctctga
gcaacaaatt tgtggagggt agtcataaca gtactgtgag cttaaccacg
37860aaaaatatgg aagtgtcagt ggcaacaacc acaaaagccc aaattccaat
tttgagaatg 37920aatttcaagc aagaacttaa tggaaatacc aagtcaaaac
ctactgtctc ttcctccatg 37980gaatttaagt atgatttcaa ttcttcaatg
ctgtactcta ccgctaaagg agcagttgac 38040cacaagctta gcttggaaag
cctcacctct tacttttcca ttgagtcatc taccaaagga 38100gatgtcaagg
gttcggttct ttctcgggaa tattcaggaa ctattgctag tgaggccaac
38160acttacttga attccaagag cacacggtct tcagtgaagc tgcagggcac
ttccaaaatt 38220gatgatatct ggaaccttga agtaaaagaa aattttgctg
gagaagccac actccaacgc 38280atatattccc tctgggagca cagtacgaaa
aaccacttac agctagaggg cctctttttc 38340accaacggag aacatacaag
caaagccacc ctggaactct ctccatggca aatgtcagct 38400cttgttcagg
tccatgcaag tcagcccagt tccttccatg atttccctga ccttggccag
38460gaagtggccc tgaatgctaa cactaagaac cagaagatca gatggaaaaa
tgaagtccgg 38520attcattctg ggtctttcca gagccaggtc gagctttcca
atgaccaaga aaaggcacac 38580cttgacattg caggatcctt agaaggacac
ctaaggttcc tcaaaaatat catcctacca 38640gtctatgaca agagcttatg
ggatttccta aagctggatg taaccaccag cattggtagg 38700agacagcatc
ttcgtgtttc aactgccttt gtgtacacca aaaaccccaa tggctattca
38760ttctccatcc ctgtaaaagt tttggctgat aaattcatta ttcctgggct
gaaactaaat 38820gatctaaatt cagttcttgt catgcctacg ttccatgtcc
catttacaga tcttcaggtt 38880ccatcgtgca aacttgactt cagagaaata
caaatctata agaagctgag aacttcatca 38940tttgccctca acctaccaac
actccccgag gtaaaattcc ctgaagttga tgtgttaaca 39000aaatattctc
aaccagaaga ctccttgatt cccttttttg agataaccgt gcctgaatct
39060cagttaactg tgtcccagtt cacgcttcca aaaagtgttt cagatggcat
tgctgctttg 39120gatctaaatg cagtagccaa caagatcgca gactttgagt
tgcccaccat catcgtgcct 39180gagcagacca ttgagattcc ctccattaag
ttctctgtac ctgctggaat tgtcattcct 39240tcctttcaag cactgactgc
acgctttgag gtagactctc ccgtgtataa tgccacttgg 39300agtgccagtt
tgaaaaacaa agcagattat gttgaaacag tcctggattc cacatgcagc
39360tcaaccgtac agttcctaga atatgaacta aatggtaaga aatatcctgc
ctcctctcct 39420agatactgta tattttcaat gagagttatg agtaaataat
tatgtattta gttgtgagta 39480gatgtacaat tactcaatgt cacaaaattt
taagtaagaa aagagataca tgtataccct 39540acacgtaaaa accaaactgt
agaaaatcta gtgtcattca agacaaacag ctttaaagaa 39600aatggatttt
tctgtaatta ttttaggact aacaatgtct tttaactatt tattttaaaa
39660taagtgtgag ctgtacattg catattttaa acacaagtga aatatctggt
taggatagaa 39720ttctcccagt tttcacaatg aaaacatcaa cgtcctactg
ttatgaatct aataaaatac 39780aaaatctctc ctatacagtt ttgggaacac
acaaaatcga agatggtacg ttagcctcta 39840agactaaagg aacatttgca
caccgtgact tcagtgcaga atatgaagaa gatggcaaat 39900atgaaggact
tcagtatgga gcttttattg aattgaaacc ttataccttt tgaaaactca
39960ttgtgatttt cttcatctcc ataccccttt cgtgatagct catctgtttt
tctgctttca 40020gggaatggga aggaaaagcg cacctcaata tcaaaagccc
agcgttcacc gatctccatc 40080tgcgctacca gaaagacaag aaaggcatct
ccacctcagc agcctcccca gccgtaggca 40140ccgtgggcat ggatatggat
gaagatgacg acttttctaa atggaacttc tactacagcc 40200ctcaggtaaa
taccacctaa tgagtgacac gcccccaaga gcgagtggag aattggggca
40260gatacattta attcaggacc aaatattcag agattcccca aactaggtga
aagacaggcg 40320gtaagcaact tcttctctga ggaaatattc tctagaaagt
attacaatga gtccttgatt 40380gattttaatg tttagatgca cacatgacat
cccatcagca ctattattta ttaattctgg 40440gcaaatccag gaagatgagg
gttatacctc atcatctaaa tcataggcaa gctcagccat 40500aggcagggta
tatttttcag agaggactgg tttctgtagt atttaaaact ttaaaattct
40560tccccacaat agaattgcta gatgagatac atcaaattcc tctcatgtca
tttacaagct 40620ctgccagggc caaatcaagg gtgacattac cagaggagaa
gaccaaacat ggttctatga 40680ctgttactaa aagtttgtca tgggcttgga
gaatgcgtac tgatgttggg attctgggtc 40740tctgcagggt gggctccaac
ttgccttttt tgctatttct tcttttccta tctgtcattt 40800cctgactctt
cttctctctc ctcttctttc tcttcccccc actcctcttc cagttttcag
40860tcctaggaag gctttaattt taagtgtcac aatgtaaatg acaaacagca
agcgtttttg 40920ttaaatcctt tctggggcat gtgataaaga gaaattaaca
acagtagact tatttaacca 40980taaaacaaac acatgaactg acatatgaaa
gataaatccc tttcagtata tgaaagattc 41040tctgatcttt atttttaact
gctaatgaag ttttagtgta ctatattgtg taattggagt 41100aattgaaaac
atgttatttt tttttttctc tctgtttagt cctctccaga taaaaaactc
41160accatattca aaactgagtt gagggtccgg gaatctgatg aggaaactca
gatcaaagtt 41220aattgggaag aagaggcagc ttctggcttg ctaacctctc
tgaaagacaa cgtgcccaag 41280gccacagggg tcctttatga ttatgtcaac
aagtaccact gggaacacac agggctcacc 41340ctgagagaag tgtcttcaaa
gctgagaaga aatctgcaga acaatgctga gtgggtttat 41400caaggggcca
ttaggcaaat tgatgatatc gacgtgaggt tccagaaagc agccagtggc
41460accactggga cctaccaaga gtggaaggac aaggcccaga atctgtacca
ggaactgttg 41520actcaggaag gccaagccag tttccaggga ctcaaggata
acgtgtttga tggcttggta 41580cgagttactc aagaattcca tatgaaagtc
aagcatctga ttgactcact cattgatttt 41640ctgaacttcc ccagattcca
gtttccgggg aaacctggga tatacactag ggaggaactt 41700tgcactatgt
tcataaggga ggtagggacg gtactgtccc aggtatattc gaaagtccat
41760aatggttcag aaatactgtt ttcctatttc caagacctag tgattacact
tcctttcgag 41820ttaaggaaac ataaactaat agatgtaatc tcgatgtata
gggaactgtt gaaagattta 41880tcaaaagaag cccaagaggt atttaaagcc
attcagtctc tcaagaccac agaggtgcta 41940cgtaatcttc aggacctttt
acaattcatt ttccaactaa tagaagataa cattaaacag 42000ctgaaagaga
tgaaatttac ttatcttatt aattatatcc aagatgagat caacacaatc
42060ttcagtgatt atatcccata tgtttttaaa ttgttgaaag aaaacctatg
ccttaatctt 42120cataagttca atgaatttat tcaaaacgag cttcaggaag
cttctcaaga gttacagcag 42180atccatcaat acattatggc ccttcgtgaa
gaatattttg atccaagtat agttggctgg 42240acagtgaaat attatgaact
tgaagaaaag atagtcagtc tgatcaagaa cctgttagtt 42300gctcttaagg
acttccattc tgaatatatt gtcagtgcct ctaactttac ttcccaactc
42360tcaagtcaag ttgagcaatt tctgcacaga aatattcagg aatatcttag
catccttacc 42420gatccagatg gaaaagggaa agagaagatt gcagagcttt
ctgccactgc tcaggaaata 42480attaaaagcc aggccattgc gacgaagaaa
ataatttctg attaccacca gcagtttaga 42540tataaactgc aagatttttc
agaccaactc tctgattact atgaaaaatt tattgctgaa 42600tccaaaagat
tgattgacct gtccattcaa aactaccaca catttctgat atacatcacg
42660gagttactga aaaagctgca atcaaccaca gtcatgaacc cctacatgaa
gcttgctcca 42720ggagaactta ctatcatcct ctaatttttt aaaagaaatc
ttcatttatt cttcttttcc 42780aattgaactt tcacatagca cagaaaaaat
tcaaactgcc tatattgata aaaccataca 42840gtgagccagc cttgcagtag
gcagtagact ataagcagaa gcacatatga actggacctg 42900caccaaagct
ggcaccaggg ctcggaaggt ctctgaactc agaaggatgg cattttttgc
42960aagttaaaga aaatcaggat ctgagttatt ttgctaaact tgggggagga
ggaacaaata 43020aatggagtct ttattgtgta tcataccact gaatgtggct
catttgtatt gaaagacagt 43080gaaacgaggg cattgataaa atgttctggc
acagcaaaac ctctagaaca catagtgtga 43140tttaagtaac agaataaaaa
tggaaacgga gaaattatgg agggaaatat tttgcaaaaa 43200tatttaaaaa
gatgaggtaa ttgtgttttt ataattaaat attttataat taaaatattt
43260ataattaaaa tatttataat taaatatttt ataattaaaa tatttataat
taaatatttt 43320ataattaaag tatttataat taaatatttt ataattaaaa
tatttataat taaatatttt 43380ataattaaaa tatttataat taaatatttt
ataattaaaa tatttataat taaatatttt 43440ataat
43445243445DNAArtificial Sequencereverse complement sequence of SEQ
ID NO. 1 2attataaaat atttaattat aaatatttta attataaaat atttaattat
aaatatttta 60attataaaat atttaattat aaatatttta attataaaat atttaattat
aaatacttta 120attataaaat atttaattat aaatatttta attataaaat
atttaattat aaatatttta 180attataaata ttttaattat aaaatattta
attataaaaa cacaattacc tcatcttttt 240aaatattttt gcaaaatatt
tccctccata atttctccgt ttccattttt attctgttac 300ttaaatcaca
ctatgtgttc tagaggtttt gctgtgccag aacattttat caatgccctc
360gtttcactgt ctttcaatac aaatgagcca cattcagtgg tatgatacac
aataaagact 420ccatttattt gttcctcctc ccccaagttt agcaaaataa
ctcagatcct gattttcttt 480aacttgcaaa aaatgccatc cttctgagtt
cagagacctt ccgagccctg gtgccagctt 540tggtgcaggt ccagttcata
tgtgcttctg cttatagtct actgcctact gcaaggctgg 600ctcactgtat
ggttttatca atataggcag tttgaatttt ttctgtgcta tgtgaaagtt
660caattggaaa agaagaataa atgaagattt cttttaaaaa attagaggat
gatagtaagt 720tctcctggag caagcttcat gtaggggttc atgactgtgg
ttgattgcag ctttttcagt 780aactccgtga tgtatatcag aaatgtgtgg
tagttttgaa tggacaggtc aatcaatctt 840ttggattcag caataaattt
ttcatagtaa tcagagagtt ggtctgaaaa atcttgcagt 900ttatatctaa
actgctggtg gtaatcagaa attattttct tcgtcgcaat ggcctggctt
960ttaattattt cctgagcagt ggcagaaagc tctgcaatct tctctttccc
ttttccatct 1020ggatcggtaa ggatgctaag atattcctga atatttctgt
gcagaaattg ctcaacttga 1080cttgagagtt gggaagtaaa gttagaggca
ctgacaatat attcagaatg gaagtcctta 1140agagcaacta acaggttctt
gatcagactg actatctttt cttcaagttc ataatatttc 1200actgtccagc
caactatact tggatcaaaa tattcttcac gaagggccat aatgtattga
1260tggatctgct gtaactcttg agaagcttcc tgaagctcgt tttgaataaa
ttcattgaac 1320ttatgaagat taaggcatag gttttctttc aacaatttaa
aaacatatgg gatataatca 1380ctgaagattg tgttgatctc atcttggata
taattaataa gataagtaaa tttcatctct 1440ttcagctgtt taatgttatc
ttctattagt tggaaaatga attgtaaaag gtcctgaaga 1500ttacgtagca
cctctgtggt cttgagagac tgaatggctt taaatacctc ttgggcttct
1560tttgataaat ctttcaacag ttccctatac atcgagatta catctattag
tttatgtttc 1620cttaactcga aaggaagtgt aatcactagg tcttggaaat
aggaaaacag tatttctgaa 1680ccattatgga ctttcgaata tacctgggac
agtaccgtcc ctacctccct tatgaacata 1740gtgcaaagtt cctccctagt
gtatatccca ggtttccccg gaaactggaa tctggggaag 1800ttcagaaaat
caatgagtga gtcaatcaga tgcttgactt tcatatggaa ttcttgagta
1860actcgtacca agccatcaaa cacgttatcc ttgagtccct ggaaactggc
ttggccttcc 1920tgagtcaaca gttcctggta cagattctgg gccttgtcct
tccactcttg gtaggtccca 1980gtggtgccac tggctgcttt ctggaacctc
acgtcgatat catcaatttg cctaatggcc 2040ccttgataaa cccactcagc
attgttctgc agatttcttc tcagctttga agacacttct 2100ctcagggtga
gccctgtgtg ttcccagtgg tacttgttga cataatcata aaggacccct
2160gtggccttgg gcacgttgtc tttcagagag gttagcaagc cagaagctgc
ctcttcttcc 2220caattaactt tgatctgagt ttcctcatca gattcccgga
ccctcaactc agttttgaat 2280atggtgagtt ttttatctgg agaggactaa
acagagagaa aaaaaaaaat aacatgtttt 2340caattactcc aattacacaa
tatagtacac taaaacttca ttagcagtta aaaataaaga 2400tcagagaatc
tttcatatac tgaaagggat ttatctttca tatgtcagtt catgtgtttg
2460ttttatggtt aaataagtct actgttgtta atttctcttt atcacatgcc
ccagaaagga 2520tttaacaaaa acgcttgctg tttgtcattt acattgtgac
acttaaaatt aaagccttcc 2580taggactgaa aactggaaga ggagtggggg
gaagagaaag aagaggagag agaagaagag 2640tcaggaaatg acagatagga
aaagaagaaa tagcaaaaaa ggcaagttgg agcccaccct 2700gcagagaccc
agaatcccaa catcagtacg cattctccaa gcccatgaca aacttttagt
2760aacagtcata gaaccatgtt tggtcttctc ctctggtaat gtcacccttg
atttggccct 2820ggcagagctt gtaaatgaca tgagaggaat ttgatgtatc
tcatctagca attctattgt 2880ggggaagaat tttaaagttt taaatactac
agaaaccagt cctctctgaa aaatataccc 2940tgcctatggc tgagcttgcc
tatgatttag atgatgaggt ataaccctca tcttcctgga 3000tttgcccaga
attaataaat aatagtgctg atgggatgtc atgtgtgcat ctaaacatta
3060aaatcaatca aggactcatt gtaatacttt ctagagaata tttcctcaga
gaagaagttg 3120cttaccgcct gtctttcacc tagtttgggg aatctctgaa
tatttggtcc tgaattaaat 3180gtatctgccc caattctcca ctcgctcttg
ggggcgtgtc actcattagg tggtatttac 3240ctgagggctg tagtagaagt
tccatttaga aaagtcgtca tcttcatcca tatccatgcc 3300cacggtgcct
acggctgggg aggctgctga ggtggagatg cctttcttgt ctttctggta
3360gcgcagatgg agatcggtga acgctgggct tttgatattg aggtgcgctt
ttccttccca 3420ttccctgaaa gcagaaaaac agatgagcta tcacgaaagg
ggtatggaga tgaagaaaat 3480cacaatgagt tttcaaaagg tataaggttt
caattcaata aaagctccat actgaagtcc 3540ttcatatttg ccatcttctt
catattctgc actgaagtca cggtgtgcaa atgttccttt 3600agtcttagag
gctaacgtac catcttcgat tttgtgtgtt cccaaaactg tataggagag
3660attttgtatt ttattagatt cataacagta ggacgttgat gttttcattg
tgaaaactgg 3720gagaattcta tcctaaccag atatttcact tgtgtttaaa
atatgcaatg tacagctcac 3780acttatttta aaataaatag ttaaaagaca
ttgttagtcc taaaataatt acagaaaaat 3840ccattttctt taaagctgtt
tgtcttgaat gacactagat tttctacagt ttggttttta 3900cgtgtagggt
atacatgtat ctcttttctt acttaaaatt ttgtgacatt gagtaattgt
3960acatctactc acaactaaat acataattat ttactcataa ctctcattga
aaatatacag 4020tatctaggag aggaggcagg atatttctta ccatttagtt
catattctag gaactgtacg 4080gttgagctgc atgtggaatc caggactgtt
tcaacataat ctgctttgtt tttcaaactg 4140gcactccaag tggcattata
cacgggagag tctacctcaa agcgtgcagt cagtgcttga 4200aaggaaggaa
tgacaattcc agcaggtaca gagaacttaa tggagggaat ctcaatggtc
4260tgctcaggca cgatgatggt gggcaactca aagtctgcga tcttgttggc
tactgcattt 4320agatccaaag cagcaatgcc atctgaaaca ctttttggaa
gcgtgaactg ggacacagtt 4380aactgagatt caggcacggt tatctcaaaa
aagggaatca aggagtcttc tggttgagaa 4440tattttgtta acacatcaac
ttcagggaat tttacctcgg ggagtgttgg taggttgagg 4500gcaaatgatg
aagttctcag cttcttatag atttgtattt ctctgaagtc aagtttgcac
4560gatggaacct gaagatctgt aaatgggaca tggaacgtag gcatgacaag
aactgaattt 4620agatcattta gtttcagccc aggaataatg aatttatcag
ccaaaacttt tacagggatg 4680gagaatgaat agccattggg gtttttggtg
tacacaaagg cagttgaaac acgaagatgc 4740tgtctcctac caatgctggt
ggttacatcc agctttagga aatcccataa gctcttgtca 4800tagactggta
ggatgatatt tttgaggaac cttaggtgtc cttctaagga tcctgcaatg
4860tcaaggtgtg ccttttcttg gtcattggaa agctcgacct ggctctggaa
agacccagaa 4920tgaatccgga cttcattttt ccatctgatc ttctggttct
tagtgttagc attcagggcc 4980acttcctggc caaggtcagg gaaatcatgg
aaggaactgg gctgacttgc atggacctga 5040acaagagctg acatttgcca
tggagagagt tccagggtgg ctttgcttgt atgttctccg 5100ttggtgaaaa
agaggccctc tagctgtaag tggtttttcg tactgtgctc ccagagggaa
5160tatatgcgtt ggagtgtggc ttctccagca aaattttctt ttacttcaag
gttccagata 5220tcatcaattt tggaagtgcc ctgcagcttc actgaagacc
gtgtgctctt ggaattcaag 5280taagtgttgg cctcactagc aatagttcct
gaatattccc gagaaagaac cgaacccttg 5340acatctcctt tggtagatga
ctcaatggaa aagtaagagg tgaggctttc caagctaagc 5400ttgtggtcaa
ctgctccttt agcggtagag tacagcattg aagaattgaa atcatactta
5460aattccatgg aggaagagac agtaggtttt gacttggtat ttccattaag
ttcttgcttg 5520aaattcattc tcaaaattgg aatttgggct tttgtggttg
ttgccactga cacttccata 5580tttttcgtgg ttaagctcac agtactgtta
tgactaccct ccacaaattt gttgctcaga 5640gacagagctg tggctaactt
caatcccctt tttcttgtca atcttgtggt gccctctaat 5700ttgtactgca
gtgcatcaat gacagatgaa gatgaagaaa ggagatgagc aacaatatct
5760gactggttaa aaagttcagc attggtattc agtgtgatga cacttgattt
aaaggagaaa 5820tcataggtaa tattgcccat ggcaggaata aaaatatggc
ttatggtaca caattccttg 5880aaatctggaa gagaaagctt gagatttcta
gggacatgaa ggactggcag ctctaatgat 5940ggcaggatta atgtgtatga
aggcacacgg acgtcagaac ctaggatgga gaaactaggc 6000atgctgactg
cttttgggaa cacatagccg aatgccgaca tctctatggt gaatggagac
6060acttcaacat tgacaactgg aacagtgtat ccaggaattt gaaaggtcct
ggggagctcg 6120tcgtgagatt tttcagcttt gtacttatca aacttaattt
ttgtttcatt ataggatttg 6180gtgacaaaat ctaatgcatt gtttctgttt
ttttcaaaat gcctgtcaaa ggatttgatg 6240ctctgactga taaactcaca
aagcacagcc aaaggatttg tgatggaatg cctgtgtttg 6300tttttcttat
actgagcttt tacacttaaa tcaaatgatt gctttgtcgt tttcaagaat
6360tccttcaagc ctgttttttc ccatagagag aaatctttca gtggaggagt
tgtgattatt 6420gtgtaaggta gacgcatttc aggaattgtt aaaggaatgt
ttaagaaatc cagatttgct 6480tctccattta ttcctacatg ggcctccata
atgttctcgt tgtttccagc agagaaattt 6540tggttgtact tatactgatt
gaacctagca cttacttgcc aacttgcttg ctgggcactg 6600ggactcagaa
acagtgcata gttattcagg aagtctatct tccctgttaa ccttaatgga
6660aaacgaactt tcaaattccc ttcattgttt gtggatgccg tgatctcaaa
tggctgggct 6720gaaaagaaaa gagaattttt caaagttcca ataacctttc
catttaaatg agcatcatgc 6780ctcccagtaa actctgcctt cccttctcca
aacagtgcca tgcctttagc agttagaaca 6840ctgtggccca catgctggga
atcgacttgt gattgaattt caagtttaga aaagttgagg 6900gagccagatt
cataaaccaa gttttggttt actcttaggt gtttgctatt gatcttattg
6960gacagtccaa aggaagtgag gggtccttct atggtgaaac taatttgtga
ttcatgtgtt 7020ccctcatctg agaatctggg gcaggcccat ttccatgacc
cttttccaga agaagtccat 7080gctatgtggc cagctttcaa cagtgtcttg
atctcgttgc gcaggtcagc ctgactagag 7140aagtccagtt tggggatgtt
caatttgtgg aagtatttag tgttgctatc cagggtaagc 7200tgattgttta
tcttgacaat cactccatta ctaagctcca gtgtattttt ttctgtgtgt
7260aaacttgcca ctgtgtttga ttttccctca atagcatttc caaaaaacag
catttcactc 7320ccatgctccg ttctcaggta cttgctggag aacttcactg
actccttcag agccagcgga 7380ttaatcttag ggtttgagag ttgtgcattt
gcttgaaaat caaaattgag aacttctaat 7440ttggactctc ctttggcagt
gatggaagct gcgatacctg cttcgtttgc tgaggtggtt 7500ccattcccta
tgtcagcatt tgcatctaat gtgaaaagag gagattggat tttcagaata
7560ctgtatagct tgccaaaagt aggtacttca attgtgtgtg agatgtgggg
aagctggaat 7620tctggtatgt gaaggtcagg aacttgaaaa tcattaaggt
tgagagttgg gattatgaat 7680tctggaattg cgatttctgg taaacggaag
tctggcaggg tgattctcgc tagaggaatg 7740tcctccacct tcagatccct
gagatatata tctggaacgg gccactgcag ctcactgttc 7800agcatctggt
caatggttct gatgatcttt actttcattt ctacaaagtc aattgtaaag
7860gaaggaatgt ggaaggtgtt aaggatggta aattctggtg tggaaaacct
ggatgggatt 7920tttatatttt ttaagtcttt gaagtttatc tgaactgatg
gaatcctcaa atctgttagg 7980gggactataa aatcaggtgt ctggaaggta
gctttctgaa gagcctgaag actgacttca 8040aaggcaggca tggtcccaag
gatggtcttg atttcaggaa cagtgaaccc ttgctctacc 8100aatgctttca
tacgtttagc ccaatcttgg atagaatatt gctctgcaaa gtcagtaagg
8160ttcttagcag caagagtcca ccaatcagaa atgtaggtga caagtgtgct
ataaacctgg 8220cctaccagag acaggtatcg ttgaagttcc tgctgaatgt
ccatttgata cattcggtct 8280cgtgtatctt ctagggtctc tcggaatttg
gccttcatgt gagccaaaga tgctgaactt 8340aaagcctcct gtaaccaatt
gatgattaag gttattttgg tgtcctgtag gctttccaga 8400tacactgcaa
ctgtggcctt ggtttcctct aaaaacagtt ttaatgcttc agctttttgt
8460ggtagttcca gagcctgaat ttcaccattg agtctctgag tcacctcacg
gattttgtca 8520ttggtttcat ctacaaactg gtggtaatca aatgacttta
atttctttat caacatgtca 8580aggaatttgt taacatcttc aatgaatgtt
ttaaaagata attcattaag cttcttgaca 8640gcatcatcaa taaatccaac
caatttctca aagtaatctt ttatcttaac ttgttgtagg 8700acattgctta
gcttctgaat agtctccttc aacttgtatt ggtgggccaa ctctactaat
8760ttatccatta aaacctggat ttgttggtct acttcatacc tctcgattaa
ctcatggact 8820ttggctctga aggcattgat tttctcagct acttcaaaat
ccccaataag atttataaca 8880aagtgtttga catgctcaag aatgtcattt
attctttcaa atgaaattgt agttcccaat 8940tgatctaaaa gcactctaac
atcaatagcc tcaatgtgtt gttttaactt tccagctagg 9000tgctggatgt
ctatattctg tatgtgtctc ttaagctgct gcagtttttc ttgtatctgg
9060attctgattt ggtacttagt atccacattt tgaatccagg atgcagtact
acttccactt 9120ttgttaaaat caatattttc aataaacaaa tgtagatcat
ggattgtttt tactaaattt 9180acacggatat gatagtgctc atcaagactt
tttaattttt caatgatttc atcaataata 9240ttagcaatag ctattttcaa
atcatgtaaa tcataactat ctttaatata ctgatcaaat 9300tgtatcatat
atgtctgcag ttgagatagt ttttcattaa agttgatttt ggcatcatct
9360aatgcaattt gtatatcatt ttctgtaatt ctatactttt ttgtgagagc
agtcagtttc 9420tccttggcat gtgaaacttg tctctcccaa ttgaatgaat
tcagataatc attagcttgc 9480tgtgggagtt ttcccagggc tgctctgtat
tttcttacaa attgatcaat attgatgtgc 9540ttcaggtttc tctgtacgtt
ttccagtaca actataatgg tttgtcgatt cctctcaaaa 9600tattcttgca
aggtctcaaa aaatgggagg ttaatggagt gaacatcttg gtttttatca
9660tactttacaa aagcaacaat tgtaaattct tggggcttct caacggcatc
tctcatctct 9720aaagcatcaa tgatattgat gggctcactg agtaaaagtg
gcactttaat tggggagtct 9780agtagagtta ggtcagccag agttcgtcca
gtaagctcca cgccaatttt atctttagtg 9840ttgtaagcat ccaagtcctg
gctgtattca ttgttgttaa attgggtctt gagtttccag 9900gtgcctgtct
gctcagctgg agtaagcagg gcactgactt tgtgttcaag agctgcactg
9960atgcttttcc tagacacgag atgatgactt gtggagcctt tgtaatcatg
agagaaagta 10020aatgccagag gttctgcttt caacaggaat ttgctataca
gctgcccagt atgttctccc 10080cagagagcga gtttcccatt gccatttgta
tgtgcatcga tggtcatggt aaacggggcc 10140attacagaac ggaagacatt
gctgaaatgc agtgagtctg aattatagtt tgtgctcatg 10200tcaatggctg
aagccagccc agcgatgtct gtgttgagcc gatggctaaa ctccacaccc
10260tgaaccttag caacagtgtc tgctttatag cttgctgata aggcagcaga
agagatggca 10320tagatgtgtt ttatttcatt attttggtag gctcctttta
ggttaccagc cacatgcagc 10380ttcaggggtt ctagccgtag tttcccattg
ttggtgagat ccagagcatt gtatttcagg 10440tcactgttta aagtagttac
cagagaatag ggctgtagct gtaaattaac agtttgctta 10500taaaacttgt
cagagctgta aatgttgtca agttttgaag agaagtccag tgataagcct
10560gcaatgttca gactgtttgt gtggtcaaat ttcatttcag catatgagcc
catcatgtca 10620tttgagagct taagtccttc ttgactgacc ttgaagttga
aaatgttttt gctgtcgaca 10680cccagaatca tggcctgata agcacttccc
agtgatagct ctgtgagggc ggctttccca 10740tccagactga attttgcatt
gtgttccctg aagcggccat ttgttgttaa tttcatagat 10800gccccagaga
ggccaagctc tgcattcagc tcattctcca gcaccaggag actacacttc
10860aagttggtcg ttgcactggt agatattcca tcttggccaa tccttagtgt
cgccttgtga 10920gcaccactat taattttgtc agtgcctaag atgtcagcat
ttaactcaag accatgggaa 10980tttagtgatc cagaaagcag gctgaagaac
ctcaatgact cgtaatcagc ctgatattca 11040gaacgcagca gtgcattttg
cttagagaag gtcatatcca tcttgttaga agtggcaaag 11100ttcttatact
tcccattggt gtcagatttt aaagtcagct cgtagttctc atactttagg
11160gaagcagtat ttttaatgat gccactttgc agatcagagg tggaggtgag
ggagagggtt 11220ccatcttcat atcttcctgt tatctggttg gtgccttgga
ggtaggagga gttaaacctc 11280aggttggact ctccattgag ccggccagtg
ttaggatccc tctgacaaga caggccatat 11340gtgcctttag catagaacga
agagactctg aactgcccat caatcttgac ttctttgaca 11400aacaaatgct
gtttcttttt ggagtccaaa tgaactgaag cagacatctg tggtccccag
11460gaactagatg catcgaatat tagtaaacct tttgagactg ggttgtttcc
aagtttttct 11520acatgactga atttgatatt cgaatctaga aatttgtggc
gtagagaccc atcatatgat 11580agtgtgaacg tattcttgtg gtcatatgtt
gtttctccag atcctaacat aaaaatgaaa 11640agacattggt taaattaagc
agtacatttc cagagcaatc tctatgttga aagtctttca 11700ataataaagc
cccatttttc tgggccaatt gtgcaataga ctcctccatc tgtaatgcaa
11760agataaaatc tgatagcaaa aggcattcct ccaggaagcc ttgcctgacc
accacaggct 11820ttgtttaggt tcccttgttg agtgcttcca ctctacctta
tacttatata ctacacttta 11880tcatagcacc tctcacactg aattataatt
gctggcttat ctgtctactt tctgttactg 11940cctactagaa tatttaatac
acgtgatatg tttaataaat atttctattt gttgaataaa 12000ataaaagact
tccaagtagc aaggaagatt atctgctaga aagccaaagt cctttcctcc
12060ctggaggagg ctctcctctt agagcctgcc atgaactagc ccggtgcacc
ctttacctga 12120gcatagctca ccttgcacat tgtaggaaag caggtcaacc
acagagtcag ccttcatgtg 12180gtaacgagcc cgaaggctga aatggtctgt
gctggtgttg ccaccactgt aggaggcgga 12240ccagttgtac aagttgctgt
agacattcgt ggagaggtct agaacaccca ggagaggcac 12300ttgcagttga
tacaacttgg gaatggtaaa agtagggact tggaactctc gagatggcag
12360atggaatccc acagacttga agtggagggc tggtgtccta acagtctcta
acatctttag 12420atctctggag gatttgccac caaaaggcaa aggaatctca
attttcaaac tgttcttgtt 12480caaggtatat ttgacccggc catcgctgaa
atgaacaaca aagataacat ccccacagtc 12540agacatcagt cattcaaagt
tctctgcctc tgaccttcac aacaatattg tacttgcccc 12600attcccaaag
ccactctgca cttttctttg tgctactcct atgcctggac ccctttgctt
12660ttacctccac ctgtcaaaca ctcaaatcct gacaaagctc ttctaagggc
cactccttat 12720cattaccatc ccacctaagt catagccaat ttgtctcatt
cttacagtta catagtccct 12780tgcacctttc tcaaagagca tgtcataatt
ttgcttatat aattaattat ttctcatttg 12840ttgatgtctc catggtatct
tcttgaaaga aaaggctata ccttacccag tttctatctt 12900ctcactgtac
ctagtctagc attttgcaca taggggatac ttgatacaaa tttgctaaac
12960tgaattgaga caacttgatt tgtggaaaga acataggtat ggaagtcagt
aagcctgggt 13020taaagtattt tctcagaatg ctgcctccat ttagtgctca
atggctgttc tgactctctc 13080tagcttaaca gcaagcctgt agtcaataac
gcctctagcc attctgaatt cttcacagtc 13140agagggagaa tttgcttata
acaggttgta ctgaataaaa tatcttaggg tgaaagtacc 13200agaatcctct
aatgactttc ttagattctt cctgacaggg ttggtgttct catgtttact
13260aatgcaaaga tgccacagtg tttgtgctat agttattaat ctccttaaaa
tgcttaaatt 13320atttccttta aaaatttaaa aaaatgtcta aatcatgcta
tgtaaaatct ttcatttact 13380ttgcatggtt caagaagcct tgctgctttc
ttcttttacc tttttaagaa gaggttttct 13440gggatgtgga agtctggcaa
tcccatgttc tggaggttga actccttcag gctattgagg 13500tggtcttgca
aagtctgggt ataaggaaga ctcccagatg ccttctgaag ccatgagctc
13560attgcctaca aaatgacagg agatttttaa ggtaatgggc ttggatgagc
ctcaaagagc 13620aatgaacatt aggcaaaaat accgatttga caagttaatt
attaagctgg acaatgcact 13680gaaagttaaa aataaataac agaaaattat
gaatcttcgt tgccagtcac tgatcactgt 13740ccatatttat aaaaggaatg
tctaacatag tcagcctcct agctggtgtc ctgaagcttc 13800ctttgatacc
actaggcaaa gaactgttct acttagtagc atctctttga tatgtatcac
13860attcagcttt gtgtaactgg gtaactacac acttgtgaaa gttttttttt
ctccccaaga 13920attccctggg gggaaggaag catgccttat acatctttgg
aaaccttcct gcacctagct 13980cagagttgag gatgtaatta gcacttatat
ccatgtattt attgactggc agactcatac 14040ttacaactat taatttggaa
cccacgtgcc ggaaagtcat gtctgtttga gggactctgt 14100gatccaggag
tctattagca tacatatgca agctcttagg ataatcggag agatccacag
14160ggaaattgga agtcattttt ttggtatcta cattggtgcc tgtgttccat
tcaaattcaa 14220tcttctcttc atctgaaaat acgtaggaaa tagttgtgaa
tggtactagt tcagcctgta 14280accacaggtc tcaacacctg cattactttg
gaagtgctca cacaggggaa gagacacata 14340ccataatgcc atgccaccct
cttggaaact gtggagccat aagctgtagc agatgagtcc 14400atttggagaa
gcagtttggc aggcgaccag tgggcgagga tctcacttct ggcttctgct
14460tgcaaacggg gtatggaaat aacacccttg atttttcttt cttcctttgt
gtcacaacta 14520tggtaaagaa aatcagttgg caccaatgat tttgtccttt
caatggagat atgcaggatt 14580aaacagaagt tccatttgtg gtgatcaaaa
ccagatacaa ggatggttca gagaaacagc 14640cacagatcaa actcatcaac
ttctaaagcc aacattcagc acaaattaag gactttgcaa 14700acaggtttag
gagaaagaga agttttttga aaactgtttg aatcctgctc tgctatttac
14760tagctctgtg attttgacca aatgatttaa cccctctgag ctaccatttc
cttatccatg 14820aaacaggaaa actcacagtg acctcgcagt ttattgtgag
gaccgagata atttctttaa 14880agcacccagc actgtgcttg atacatgaat
agtaagtgcc tgataaatgg taggaaaatc 14940caactggaca tgcgcagagg
gttaaaatgt tgagagcttg gccgggtgca gtggcttaca 15000cctgtaatcc
cagcactttg ggaggccaag gcaggtggat cacgaggtca ggagatcgag
15060accatcctgg tcaatatggt gaaaccctgt ctctactaaa aatataaaaa
ttagctgggt 15120gtggtggcat gcacctatag ttccagctcc ttgggaggct
taagcaggag aatcacttga 15180actcgggagg cggaggttgc agtgagccga
gattgtgcca ctgcactcca gcctggtgac 15240agagtgagac tccatctcaa
aaaaaaaaaa aattgttgag ggctctctct gccactctga 15300ttgtagacta
ggggagaaca tggcttggtc aggtatgaag tggaagagga ataatgaaaa
15360gaaccaccca ggcctgcagt gcaggtcaga tgaccctcgg ccttctttac
cttaggtggc 15420ccatgagggc gacctcagta attttcttgt tctgaatgtc
cagggtgagt ctgtaagacg 15480ttttgccctc agtagattca tcattaactc
tgaggattgt tccgaggtca acatcaaaat 15540ccggaatttg gacttcactg
gacaaggtca tactctgccg attatatttg aatgtcatgg 15600tagcctcagt
ctgcttcgca cctggacgag tgtataagag aatcaagaga tgtgtggtaa
15660gaagctatgt tttgggccgg gtgcggtggc tcacacctgt aatcccagca
ctttgggagg 15720ccaaggcggg cagatcatga ggtcaggaga tcaagaccat
tctggctaac atggtgaaac 15780cccgtctcta ctaaaaatac aaaaaattag
ccgggcgcgt tggtgggtgc ctgtagtccc 15840agctactcag gaggctgagg
caggagaatg gcatgaaccc gggaggcggg gcttgcagtg 15900agccgagatg
gcaccactgc actgcagcct gggcaaaaga gcgagactcc atctcaaaaa
15960ataaataaat aaataaataa ataaataaac aaacaaataa ataaataaat
aaataaagaa 16020gctatgtttt ggaaggaaag agaggaaaag gtagagaaac
tccaaggaat caatgtgctt 16080aaacaaaaac acagttcagg attttactga
attttttttt tctcattaaa attttttaat 16140gggtctcatt tactgataat
tttaaactca gttgtgttca tgtatgcaag tctttatgga 16200ttgcctgtta
tgagcctatt gtgtgctggg cactgctctt ggtactgggg aatccttgct
16260ttcatggggc ttaccttcta gtgggggagg tgtacaggca ataaacaagt
aacttgctca 16320tgtcagatgg taatgaatgt tatgaagaag agtaaaactg
gatacaagga taaaagaaag 16380ggatagaaga agtgttattt catacttggt
ggtcagagaa attctctctg ataagggcac 16440atgtgggcag agaccagaat
gaaaagaggt tggagccacg ctgagttctg ggaggagcac 16500attccaggca
gaaggtcagg aatgcaaagg ccctgaaatg gggagcactt agcttgtttg
16560tggaacagca aaggctcgca gcccttctgg gccaggaaga cacgacatct
ctatattact
16620ttctacccaa gatgcctgtc ctggttcctt gctgcccttg ccctgggctc
catctcccca 16680actccccctt tctttgtaac aagattcccg ggcgtccttc
aatccatagg ctggtccatt 16740ttatatcctt cgtaaaacaa tgtttaccaa
acactatttc cccagcgtgc cctgctcctc 16800cagtgctttc tatcatggga
ttttcttcct agtttaattc tgtgatatgc tatccctata 16860taaaaatttt
tccaaagtga acacctaaat gtgccccatt ccctaaaacc tgctcctcgc
16920tatccctata taattttttt tccaaagtga acacctaaat ttctcccatt
ccctaaaacc 16980tgctcctcca gtagcccttt ccatctcaat aaatggaagt
tccatttttc agttgcccgg 17040gccaaaaacc ttaggtttgt cttcattccc
cttttcctct tacactcaac atccactgta 17100tcagcaaatc ttgtcagcct
aatcttcaaa atatggccag aatccatccc cttctcatat 17160ctcttctgcc
attgccctga tccaaaacaa cactgattcc tgcctggatt attgtgataa
17220cctacatatt agtttctctg cccttgtctg cccatagtct tttctcaaca
taaacataaa 17280tcaaatcatg aggactcccc aatggctttc gtttaattca
tagaaaaagc caaagttctt 17340accatggcct atgagattat ccttccttca
cctctccata tctctctgtc ctcatcttct 17400acctcttttc tccctggtct
ttctgctcca gccatactgt cttccttcct ttccttgaaa 17460catggtagta
gtgtgtcagc ctcagggcct ttgcacttgc tggtccctct gcctgttaac
17520agtcttacca cacggcttct cctcttattt ctgataggtc tccttatgct
cactttacca 17580gggagacttt cccttattat tccaaatgca ctgctttccc
tgcccttctt cctccctcct 17640actcctagcc ctccatagac cagtctacgc
tctgttttcc tcctcagtac ttatctgata 17700ttccaaatat taacttgttt
ggttgttcac tgtaacttcc atgaaggcag agactccatt 17760gttcactgtt
ctagccccag gtcttagaat aggcctgccg cttattgggt actcagttaa
17820cccaggtctt agaataggcc tgccgcttag tgggtactca gttaacctat
ttgttaaata 17880ggttaaatta catgtcttct cctcatgaat tctgaacctg
agactgcgag cagagatgag 17940gcagctgtgt tttgaatact caccttctgc
ttgagttaca aacttcaggg tatccaccaa 18000ggctctgtcc tctctctgga
gctcataggt tgcgctgaca gaatactgct caatctctcc 18060tgtaggcctc
agttccagct ctaatctaaa gacattacaa tgaagacagt gcataatgtt
18120agagctttca aggatggtga ttatgttccc aaaacaattc agcctcaaat
gcaacaaaaa 18180tatggtcctt attttaacat tgtctcttgc ccttaactaa
gatgatcttt ttgatccctc 18240agtcctacaa gaacccaagg caagggcaga
caccaggatt cccaagactt tggacaagca 18300gagcaggagt tggctggttg
tttcagcaaa gctgagaaca agcactggaa cttgggggct 18360gaagacaggg
agccacctaa gtacggtgaa ggcttcaagc acattggtgg gctccaggaa
18420gctcatagcg atgggtctgg aggctcagaa acatggacac tgaattagga
gcaaattggg 18480atctgtgctg tgatcccatc ccatgcacaa ggaagcaaaa
ggtgacaccc aatgtgttgg 18540ccagaggacc ctgctttaaa tacccaaaga
tggtgcaaac ccagaaggct agtgacaggg 18600tcttacaaca cagagtattt
tttcctgtgc catgctaggt ggccatgatg tggaaggtga 18660gaaaatgctg
ggtcaggcac tgagcatctc taacctggtg tccccggtca gcggatagta
18720ggaggcggag tctgtggagc tggcgttgga gtaagcgcct gaggtgcagt
aattcaggcc 18780aggaaagact tgcttgcaaa ctgaccagga ctgcctgttc
tcaatgagag gtgggatcac 18840ctccgttttg gtggtagaga ccaaatgtaa
tgtgttgctg gtgaagaaca aaaatacctg 18900agttattgcc aagtcatgaa
tcaaaatgga catcacaaat tctcagggtg caaatacccc 18960cttatcctcc
tgtcttccct cagtccacac ctatctctaa ctatttaatt tactgagaaa
19020caaataatat tagaaagaac atcaacatga caagccatga gagtgactct
gaagcttctg 19080gctgagttct ttccatctgt tacctgtaat ccccacaaca
gtaggcttat ggttctcatt 19140ttacagaagc agaggctcag agatgttaaa
tagctgatca ctggggctga acttcagtgt 19200gccccttcct tcctacaatg
cagggcaaga gaaactgtct ctttcatatg aaatctccta 19260ctctctacct
gcttccttcc acctgcatta agcacgctca agtctcttcc cctgtgcccc
19320tcccctatgc tcctttcttt cttttcttgc tttctcatct tcctcaccat
caagagttgt 19380ctgcctttgc agtctctact tcctcccaat gtgctctgac
tgctcactcc atctcctgaa 19440aatgctttca tcaatgtctc taatgccctc
cttgctccta catccaatgg gcatatttag 19500gtttcatctt ccttgacctc
ccagaagcct tccatagagt tgcgcactca tgaccagact 19560aagacgctgg
agcctctcac atctgaagag atgatggcac cttccttcca agtgtgtact
19620taaaaactta gtacatgtaa gaaagttcaa cattggcctg gttttttcta
aggatgcttt 19680ctttgaaata gcacattttc ctccaagtat tgtttctcca
agttgggggt gccacaagac 19740caagctttgc tgatgtgctg gagtcctaat
gccagctttc gcctacccat ggggtgatct 19800cccctgtggc tgctctttgg
cttctgtgac cttttatggc tttggttctc cttctgcttc 19860tctggtcact
tcttctaact cattgctggt tattcccctt cttcctttat atgtttgtct
19920tctccatatt tctgtcctca tctctcatgt ctcactctat accagttgct
gtttaaccac 19980aacaacctct gtatactaat gattcccaaa tccatatgtg
gagcccaagc ctctttactg 20040ggctcagatc cataggtcta tttgacagct
agatttctaa actggttgtt tcataggagg 20100acatcaaata cagcacatcc
aaaactgaat ttgtcacttt ccttgaaacc agaagctctc 20160ccactgtagt
tcttattaaa atgaagggcg cctccttcct cttttcttct ttcttttgcc
20220ttgtccccat acccagttaa tcacccagta cttatgattc tgcctcttag
aaagttgtag 20280aatctgctca ctcctttcta tacctggctt ttctccactg
caggccatta tcagctctta 20340gccacatagg atcgtaagag atgtccaatg
gtctcttcgt gtccagctgt gcctccttct 20400cacccatctt ccacactaga
gtcagagtat ttcttctaaa acatcacccc tatcttattt 20460ctgctcccct
tttcaactcc tgtgccttca aaagagtcca aactccttag gatggctttc
20520gcagctgggc atgctctctc ccctgcctac ctctccccat catctctgag
ccccttacca 20580ttctgcactt cttccagttc cttgaaccat attcctttac
cttcaggcca ttacatgctt 20640ttccttttgc ctggaacact cactcctttc
tcctttgcct aaatgactct gacacagtcc 20700tcagttatca ggtcaagtac
acttgcttca gaatgcattc cctgactagc acaggctaca 20760tgatataaaa
tttcatggta ctctaatact ttctataact tagcccttac tctatgatta
20820ctgcttgctt aattgtgtgt ctcccttata aaatgtaagc gttcaaaggt
cagggacctt 20880gtgtgtctca ctcaacatac gtggtatata ttaggcaatt
aaatatacta ttttttgttt 20940tttgtgagac aggatcttgc tctgtctccc
aggctggagt gcagtggtgc aagcatggtt 21000cactgaaggc ttgaactctt
gggctcagtt gatcctccca tctcagcctt ctgagtagct 21060ggagctacag
gcatgtgcca ccacgactgg ctaattttgt tatttattta attttttttg
21120tagagatggg gttttgccat gttgcccagg ctggtatcca aacaccaggg
ctcaagcgat 21180ccatccacct ggggctccca aagtgctagg attacaggca
tgaaccaccg tggctggcca 21240attaaatatt atttttgaat gaacacatga
atgtgtctaa aatatgaaaa ctaaggcaaa 21300tcaatggttg agggtgccac
ttaaaatgga actctccaaa agaggctgta ttccattatc 21360acgaggttag
gagtttcatg ctttacctac ctagctacct caaatcaata tgttcttagg
21420tattttttgg gggggaaaat attaattttc caaagatgat ctctccagag
ctattgtttc 21480ttcattctca aaacaaatag ataacaaaaa atcacaactt
aataaaaact aattaagcag 21540taggtcttaa taagtctcaa agcagaaaga
taaaggacag tttagtaaaa tgtgaaaagt 21600agaagaaagg taattatgta
gttatgtagt aagtctaggc tagaattaaa ataggtaacc 21660cggaatcttt
cctttctact cctctcctgc ttcctcagga cctgaatgat ctcaatcaac
21720tgtttagcct ggcaaaattc tgcaggtaca ttctctgttc tctctttcaa
actggctagg 21780cagacttggc tgaaagaatt accctccact gagcagcttg
actggtctct ttggggaagg 21840aatgataaac ttcagcttcc cagcttttag
ggcaacatga gcctccagac ccgactcgtg 21900gaagaagttg gtgttcatct
ggaccccact cctagcgaag tccggaatga tgatgcccat 21960atttgtcaca
aactccacag acacggaggg ttttgccacc agttcagcct gcatctataa
22020gtcagaaaac aacctattca gattcattaa atacttcagt cccctgtcag
tcagatcaac 22080caccctttct cacctccggg caaagatttc ctggataact
cagacctttg gaaacattac 22140tgggatttgt ttggaagaag gaaagcgagt
tctcagttct ctagagttgg agtatactac 22200ccagaactag aaagcacctg
agttaacgtg aggcaaaacc agattaaaaa gatgatagga 22260atactcattt
gttgagagaa ctgagttctt gtcttatggc cataccacta ctggagctag
22320tgaggtagtg cattctggtg gaagcttgaa gtttttcata aaaaaataat
gaggtgatta 22380caatgatgaa gcattttgtc ttgaagtgga aacacatttt
taaatatgtt ttaacaagaa 22440atgcaccctg gaagaaagta ataacctaag
aaatcaaaag gcaaacagaa tcttacgttg 22500gctacttcca gttttactcc
agccttggct ccgggagcaa tgactccaga tgaagatatt 22560tgcaactgta
atccagctcc agtggggagt tcaaaggcat tctccatgaa gatgtagtga
22620agaaaaaagt cattctttga gcccttcctg atgacctctc caatctgtag
acccaacaag 22680ggagagcaaa taaaagtaag tctttttaaa gtcggttttg
ttaattacaa cctcccatac 22740attggagagt aattcctctt tatctggaaa
ggaaggacta gcatattttg atgagctgaa 22800tagtttgata aataaagatc
aaactgatgt ttttaccgtt ttcagtgaat caggatataa 22860gaaatttcac
tagatagata tcatgtcatt ttcaagtgga ctcagaaggc tcttaagaat
22920attgctgggc ctcagagctt tatggacttt aagtatcata tatgccatgg
cttaattctg 22980gtgttctgga gtctgactgt aagtgaaggt gtacttgaaa
tgtattatat tatgataaaa 23040tcaatttttt gaaaccaaag gcctattgag
atgatgtaca tatcaatttg cattgggttt 23100tattttttaa ataatatttc
aataaaatag cttgagacct gttttcttaa agtcaacatt 23160tttttaatca
agccatagga gtacatttat atatttcctt ttatgaatta ataataaaaa
23220ttatagcata tttagatagt gaattatagt ttgccaaata ctttctttta
taatcattgt 23280atcatttcaa tcttaaaagg accttcaggt caggtgcagt
ggctcacacc tgttattcca 23340gcactgtggg aggcccaggc tggaggatca
cttgaggtca ggagttcgaa accagcctga 23400catggttgtc aacaggcttg
tctctactaa agatacacaa attagccggg catgctggtg 23460cacgtccaca
gtcccagcta cttgggaggc tgaggcagga gaattgcttg aacccggaag
23520gcagagttgc agtgagccga gattgtatca ctgcactcca gcctgggagc
aagactccat 23580ctcaaaaaat taaaaaaata aaaataaaaa taaaaggacc
ttcagcaaag gtagaggaag 23640gtataccgaa cattgttttc cagatgaata
aattaatgct cttaaaaata cagctaacat 23700tatgaatagc ttaatttcta
aatgttaaca tttccatgtg atccaagagt catcttcagt 23760gaaattcaag
gcaaacctcc atctctgaag aaagtacaag catcttttcg ggcttgtgca
23820gctgggatcg taagggagtc tgggcgatct aaaaaaaaac caacgtctgg
tctcatgggc 23880ccccagtggg gcctgctgac ttaccatctg ggggatcccc
tgcagagtgc gggcacccat 23940cagaagcagc tttcccagga gctggaggtc
atggagactg gcaaaaccaa gctcctctcc 24000caagatgcgg aggtaggctc
tggcttccgg gacttctttg gatttcaaat ctttaatcag 24060cttctcaaca
ctgagcatta ttccatttac catatcctga gagtttagta ataaaatggc
24120cagtgagatg tcagcaatgt caaacacctt tcagttccca atgtgggacc
tgtctgatgc 24180agccaggatg ggcctaaaaa atgctccagg tgaggaacag
ggagacactg aagtccaggc 24240taattcctct aagcagagat taaagaacta
aggtcttgtc agggatagtg gcactcctaa 24300agctattctt aattaaaaaa
tctgtagaca tagtaagatg tggtgggatg agaggagggg 24360tccctattct
ccagtatgcc aggatcctca agattagaag atcctctatc ccccacccgt
24420tccctgctta gccttctctt ctttgaccag tgaagactac aaggtgtttc
ttaagaaaat 24480gtagctgtta tataaattca ccagaagttt gaaaaacact
tgatcctaga caagttcttc 24540tcagagagaa gaagaatctc agagatggat
gcccaggcat tgggaaaaaa aaaatgtgaa 24600actgacagac tcaaagcaga
tcaaagatcc tcggcctcaa gtgggggtca ggaaattaga 24660agatggagct
cgtgctcaac agtgagcctg gaggggtaaa ttactgcctt gctctgcaaa
24720gttgaatgtg catacaagtc acatgaggtc ttgctaaaat gcgcttctga
ttcagtagat 24780tgtgggtgaa gcatgagaag ctgtatttct gaccagacca
cactttgggt agcaaaaaat 24840tcaagaataa gctccagatc atacaaccta
tttccctcct taaatggggg ctcattgcta 24900gcttcaaagt ggaagtttgg
tctcatttgc tagtgcactg atgggagtga gagccaaaga 24960cagcagttaa
taataatgaa aatattaatt tagaactcgt aatagtgtct gcttattgaa
25020caaccaatgc cagcatcagg cactgtgctt ggcattttag atgccttatt
tcattgtctt 25080taaataacct tgtgaggttt ggactgttat tagtagtctg
ggatacagac agaaacagat 25140acacataata tcaaccaact tccccaagtg
cgcacagctt caagacaggg aagttagaat 25200taaatctatg cccagagttc
atactttttt tttttttaag atacagtctc acactgtcac 25260ccgggctgga
gtgtaacggc gtgatctcgg ctcactgcaa cctccacctc ccgggttcaa
25320gcaattctcc tgtctcagcc tcccgagtag ctaggattac aggcacccac
taccatgccc 25380agctagtttt tttgtatttt tagtagagac ggggtttcag
tatgttggcc aggctggtct 25440caaattcctg accttgtgat ctgcccacct
tggcctccca aagtgctggg attacaggca 25500tgaaccacca cacccagccc
agagttcata ctcttgttat cctagagtac ttattgtgag 25560tctggtttct
gggcacactg aattcaggag atctttaact gcagaggata ctggccctta
25620ccccagcagg tctggttgat aaaaaccata gttatccctt cagttagata
gtattttgag 25680gacttccatg cttagaaaag aattgttttt gcattgagac
ccaaagcttt ccttaagaag 25740atacttcaca aatacacacc tgctcatgtt
tatcatcttt ggtatagcca aagtggtcca 25800ctaagacctt agagacacca
tcaggaactt gaccattaac ccagtacaaa gctttgttga 25860cactgtctgg
gaaaaatcct tgcttcccaa aaagagcttc caatgttggc tcaaagcctt
25920ttccttccaa gccaatctga gaaagaaaat cagacaagaa aatggcatca
ggtttctttg 25980ttgtatgcca gcctagtagt ccccactctt gatgtccatt
tatctaaaca agtatttgaa 26040taccacaggc caggagctga ccttagcatt
tcacaggagc tgctttatta aaatctcact 26100agaatcttga ccttattact
tgcttttttc tagaagaggc agctaaggtt cagataggac 26160aaataattca
acaaaggatt cagagttggg agtgatttga gatttgaagc agatctgact
26220aggcagcaca tatgcttcct tggctttggg acttaaagac gagacatttg
catttcaatt 26280tttttttttt tttttttttt tgagatggag tcttgctctg
tcgcccaggc tggagcgcag 26340tggcgcaatc tctcggctca ctgcaagctc
agcctcctgg gttcacgcca ttctcctgcc 26400tcatcctccc aagtagctgg
gactacaggt gcctgccacc acgcccggct aattttttgt 26460atttttagta
gagatgggct ttcaccgtgt tagccaggat ggtgtcgata tcctgacctc
26520atgatccacc cgcctcggcc tcccaaagtg ctgggattac aggagtgagc
cacctcgcca 26580ggcctgcatt tctattaatg ttaagaagtc atgcttcagg
tgacccctag cggggagaga 26640ggaaggcggg gaaggaggga aagaagaaag
cctcagaatc atggtaggaa gtgcctggtg 26700gttcttagtt ttcctctggg
tagctcctgg ctcccaggga ctctctgttt atgatgctgt 26760acaaaatggg
ctagagaacc tcaaactctt cacacttacc tcgatgaggt cagctgaagc
26820aaatccaaag gcagtgaggg tagttttcag catgctttct ttaggaaggt
agttatttgg 26880atcaaatata agattccctt ctattttggc tgaggctggg
tcaagtgatg gaagagaaac 26940agatttgtag agttgatagt tccgagagaa
ttttctgaag tccatgacag ttggaagttg 27000agattctttc agagcttctt
tcactaactt tttcagacta gataagaaga agtatatttt 27060gagctgacac
accatgttat tatcctttga ctcttgcacc ccaagtaaat atggatttcc
27120aatcactacc aaaatgtctt gatttcattg accctaagtc tttgggtcta
gatctgctac 27180acatttgcac aagtgtttgt ttcagaagca agggcagaca
gtggctatgc caaaacctag 27240ggttggaatt ccagctcagg gccctcagtg
gtatatgggg tgaatagctc ttacttactc 27300ttggatatcc aattcttctg
agttcaagat attggcaata tgggaagcca caaagttctt 27360cacttgctca
ttctgttccc atggtagaat ttggacaatt ttgttaatat ctgcctgtga
27420aggactcctc atcaacataa gataggcagc cagtcgctta tctcccggag
aagcatcatc 27480aaggaaagtc tgaagaagaa cctcctggtc ctgcagtcaa
aagaggagat ggttatcact 27540gtcctgtggt cagaacacag aacatgcctg
gcaaacactg gcattgtctg ctagctcttt 27600cttaagcaca ggtctaattt
ctctgtaatc gttcttcaaa tgctggtatt gaatcttctt 27660gccagtgttt
cctagagcac tatcaagtaa gggttcccag aaaaagcctg tggaatgcct
27720gttgaattca attccattaa aacacataaa ttatggccaa gttgtgattt
gtttggagat 27780ccaccactaa gtgaaaatca agttgtttta acgtaagtta
ttttaatgtg aggataaaga 27840tgagaagggg tagtaagagt ttggggctaa
taaatagggt agtttccaaa gttctggagt 27900ctttgcacct aagagggggc
caagttggct atcctagcct aagcttattg ggggaaaatt 27960aagcatgttt
ttccatgatg agagggatgg tcaatctggt aggtggacgg taggaagcaa
28020aaacatctca cgttctgcat gttgacatag cagcaaagtc aagtatttct
ctcacgtcac 28080atactgctag gcagtcaagt caataggctt gttgaaattt
gcaattagaa aacagtagag 28140gccaggtgca gtggctcacg cctgtaatcc
cagcactttg caaggctgag gcaggcggat 28200cacctgaggt caggagctca
agaccagcct ggccaacatg gcaaaactcc atctctacta 28260aaaaatacaa
aaattagctg ggtgtaatgg cacatgcctg taatcccagt tactcaggag
28320gctgaggcag agagaattgc ttgaacccag gaggcagaag ttgcagtgag
ccgagatcat 28380gccactgcac tccagcctgg gcaatagagt gagattccat
ctcgaaagaa ggaaggaagg 28440aaggaaagaa gaaagggagg gagggaggaa
ggaaggaagg aaggaaggaa ggaaaggaaa 28500gaaacagcag aataaagtca
gtagatgaaa tctagagtct cattccccta gtaccttcca 28560aatccttgtt
aataaacttt cactttcaga cctcttcttg tggactttac cttgtcttta
28620ggctccattt tccgcagagc ctggatggca gctttctgga tcatcagtga
tggctttgta 28680ctttggacac atttcaggat tgaagacttg agttctggag
ttaactgctc catggtttgg 28740cccatatttc caatgacctg cattgaagaa
aagaaacaag aacccatcag ggtgcaggag 28800agggaagtaa aaggtgtcca
ggaaaagtgc ttctgaaatg atgtatgtca tataaaagac 28860tgagattacc
cgcagaatca aataggtgta atcttcatcc ccagtgcagt catcttgaat
28920ctgttccatc aggtaattag caatgtccag cagctcctgg gtccctgtag
ggtttgtctt 28980atgatagcta cagaataaga gaagagagtc aggacttggt
aaccccagtt aggtttgtct 29040taaaacccaa acttgtgaat tagaaaaaat
aattataaaa ttcatatgga atccaaaaag 29100agcctgaata gctgaagcaa
ttttaagcaa aaagaacata gctggaggca tcacattacc 29160tgacttcaga
ttatacaaca aggctatagt aatctaaaca gcatggtact ggcataaaaa
29220tagacacata gatcaatgga acagaatact gaacccagaa ataaagtcac
atacttacag 29280tcaacggatc tttgacaaaa ttgacaaaaa catacactag
ggaaaggaca cccttttcaa 29340taagtggtgc tgggaaagat ggattgccat
atgcagaaga ataaaactgg actcctatct 29400gtcaccatat aaaaaaatca
actcaagatg gatcaaagac ttaaatataa gacctgagac 29460tataaaaatg
caagaagaaa acctagggaa aacttctctg gacattggcc tagacaaaga
29520attcgtgaat aagacctcaa aggcaaagac aacaaaaaca aaaaatagac
aaatagaact 29580taattaaagt aaaaagtttc tgcacagtaa aacaaataat
caacagggtg gtaacctgca 29640gaatgggaga aaatatttgc aaactattca
tccaatgggg tactaatatc cggaatgtac 29700aagcaattca aacaactcaa
caaaaaaatc ccccaaataa tcccattaaa aagtaggcaa 29760aggacatgaa
tagacatttt tttcaaaaga agacatacaa atggcaatag gcatatgaaa
29820aaatgcataa cattactaat cattagagaa atgtaaatta aaaccacaat
gagataacat 29880cttacaagag tcaggagggc cattattcaa aagacaaaaa
ataacagttg ttggtgagga 29940tacagagaaa aggaaacagt taatcactgt
tgatgggatt gtaaataagt acaaccacta 30000tggaaaacaa tacagagatt
tctcaaagaa ctaaaaatag aactaccaat aaatccaaca 30060atcccattac
tgggtatata cctaaaggaa aagaaatcat tatatccaaa ggaaacctgc
30120acccatatgt ttatcacagc actattcaca atagcaaaga catggaatca
acctaagtgt 30180ctatcaatgg acgattggat taaaaatgtg aaatgtatag
gcaataaaat actattcggc 30240cataaaaaga ataaaatcat gtcatttgca
gcaacatgga tggaactgga ggtcattgtc 30300ttaagtgaaa caggacagtc
acagaaagac aaatattgcg tgttgtcatt tataagtggg 30360agtgaaataa
tgtgcacaca aggacatagt gtgtggaatg atagacaatt gagactcaga
30420aggtgtggaa gtggagggag atggatggtg agaaattact tagtgggtac
actatatgtt 30480gtctgggtga tggataccct gaaagccctg acttaactac
tgcacaatct attcatgtaa 30540caaaattaca cttgttccac ataaatttat
ataaaaatgt aaaaaagaaa aagccaaaaa 30600atttcatcaa aggaaaaacc
tgctagcctg agcaacatgg tgaaaccctg tatctaccaa 30660gaatacaaaa
attagccagg gcatggtagc atgcacctgt ggtcccagct actcaggagg
30720ctgaggtggg aggatcactt cagcccagga atttgaggct tcagtgagcc
gtgactgcat 30780cactgcactc cagcctgggt gacacagtga gaccatctca
aaaaaacaac aacaacaaaa 30840aaaaacaaaa caaacaaaca aaaaaattca
gaggaaaaaa ctgctaacta atattgaact 30900gaagttaata atatgcaagc
tgtaatactt aggggaaagt gtgttgatgt ttgcaattta 30960ctttgaaatg
cttcagaaaa taagatgaat taatggatgg atgtatatgt gataaagcaa
31020atataataac acttaaatag taggagatag ctggttttct aggggttttc
cctgtgaaat 31080tctgtcaact ttgctatgtt caaaaacttt tataatataa
tgtagagaaa atatttttaa 31140aaattcaatt tgtgtttgct gatttcttat
ttcaagtcat taccccaaaa atgatcaaga 31200aaaaacacaa gagtaaggag
cagagtttga aagtggaagg aggggttcag ttttaataca 31260gagatgcaca
gaggtgcaag atgttcctct gctcctagga ggagaaatac agtgtggaaa
31320ctcacttgtt gaccgcgtgg ctcagcgcat acaaggtggc tcggctgcgc
tgatccctcg 31380ccatgttgaa gatctctcgc agctgctgtg ctgagggctc
ggggatcagg gccaccaggt 31440aggtgaccac atctatcaga agggggttgg
catgcacacg tttcagccac tggaggatgt 31500gagtggagca ctgaggctgt
ccacactgaa ccaaggcttg taaagtgatg gggctgagaa 31560gaaagacatg
gataaagtta tacagaccac cttcagggca cataaaatat tgctcatggt
31620gtgtcctctg ccaggagagc ccttaatcac ctcattcact attcaaatct
aacttcaaaa
31680cccaacttgg agctcagaac catgatgctt tccttaggaa tattacccac
agaagaagcc 31740caatcgagaa aagttccaag aggggactgt ggatctgtag
caacaagaag actgttatca 31800cagtcctttg gccagcacac agaatatgcc
tggccaacat tagctgggtc tcattcctgg 31860cctcagtccc ttctctcacc
ttcatagcac ttaaagccag tgccttgaag ggtagagaaa 31920tcatattata
tttttcgtct gagttctctc tccaccagcc tataagcttc tggagagcac
31980ctgcaataga cctaggaatt aaaagttacc catcaaaatg tgggaactat
tctccttctg 32040ttttaacaca caaatacata gctgccttga acacagtatg
cgcgtgtgtg tttcatggaa 32100ctcagcgcag cagctgatag ttctctatcc
agcaatcatg aactgattga ctaattgatt 32160aactggattg atatccaaat
ggtccctgag attctccatt tggccaaggt ttgaaagttc 32220agtcagttac
catcagtttt ataaaaagtt gagctgtaac cattagatac ctggacacct
32280caatcagctg tggcaagaga gatgtgactg cttcatcact gaggcctctc
agctcagtaa 32340ccagcttatt gaagagatta gctctctgga tattttgctc
agagatggtt agttttttca 32400gttcctggag agtcttcaaa acagcttcgg
cctgctttgg aggtgatgtg gatttggtgc 32460tctcaaatgc gaggcccatc
ttcttagtac ctggaagatg gaaagtgtca aaggaactct 32520agctttcttc
atctcaacca tatctttgtc tactggaagc tggaaattgt ggagtatcag
32580cacaggggaa aagggaaaga aactatcctg tattcaatgc ctgctagatc
tggctctgcc 32640ataggtgatt gacttgtttt cactgactca tcatcctatc
cctgtggggt ggcattattg 32700ttcaatttaa tgataaattc agccctgctt
ctgagaccca cagagttttt gggccatgta 32760aattgctcat ccctggatct
cagctctgag tcccagagat gagacaaaga gatgagaccc 32820agagatgagg
aagtgtgacc cagagatgag caacttaggt ttcccaagag ctctcagggt
32880ctaagaggta gagctaaagt ggaatccaga cttgtctgag tctatcatca
ggttaccctt 32940agcttgaaat aattggtgag gaggtgctac tgttgacatg
gttacaactg ggaccaggca 33000catgggtgct tatcaccatt tcttctaatt
gggtctacca gcagcttgtg gggaggggag 33060acacttttgt atcctctcca
ccacctactg tgacctatgc caggaagaga ggtaaaagtc 33120atcatggaaa
tgtggtgtaa tgagaatagt cctggcttag attacaacct ggcacttatt
33180ttcccccctc taatattctg cggccttgac tacatcaaac tctaagcctc
tctgtgtcac 33240tgtctcctct gtagaatagg tataaaatca catctctacc
acctgacatc acaggttgct 33300atgagcaaaa aggacaatga ggtaatgtac
aagaaggaat ttggacagat ataaatacaa 33360tacaatactg ggtaagggtt
ggacttgaga ttgttagagt tccttctacc tctaaagact 33420gactcctgag
gttactatct ggccagactc tgaaggttct ctcccacttg gtccacctcg
33480tctgggacag ctctggatct tatcacagga tgggtgatgt cagttctatc
aaatatgtca 33540tgttaaactt aggattccag ttttctattt acaaatgcct
aagtagcttt gggaacttct 33600tttcctttat gccattgcaa cttgacatca
tgaaatgagt ttgctttctc actagaggta 33660gccagaacac cagtgtctgt
atcacttgtt agtcagcaga catttaacag ggctcagcaa 33720gtggctaagc
catgataggc acatcttgag tagattttcc agcaactatg tggacagaaa
33780ctcttacctt caccaaagaa gcggctgttg atctttggtg tgtcttcaag
tttcaaagtc 33840tgtgtcactt gtgctaccat cccatactta ttcctggtaa
ccaaggaagc acaccatgtc 33900acggatggcc agaacatact attctttcca
tgttgaaggt tttccctgtc tctgcatcta 33960cccaagtcct gcccatcttt
caaagcatgg gtaaatccag ccattgttgg ccctgaatga 34020ataacatcca
cctcttcctg gtcacgctag ttatcctccc atcaaaaaga catgcgtgcc
34080ttatactctc cactagtctg gagtggccag aggtaggaac tagtgcaatg
tctcaaaatt 34140ctttcgtatc ttccacaaca tagagccaag aatatggcag
ttactcaata tatattcatt 34200gattatcagt tttcaaagtg gatttggata
cacaagtttc cagtcagtat cccaaatctt 34260ccatgaaacc ttttctaacc
agggtaccct gcagtgatcc tcaggttttc tggatctaca 34320gcccttaaag
aatgggtctt agatatcttt tgttggtctt ggattgtttt gcatgcctag
34380gacttttctg ccaaatggcc tgttcgctta aaaaggggga cagggagggg
cggcatttta 34440tccctctcca ttcctccctc ccaggcacag gtttgcctgg
aacagagcac ttgagaagtg 34500ttcagttcac actgacccat taaatgacaa
atcaggggtg catcacatga cctacttgta 34560ggagaaaggc aggaagaggt
gttgctcctt gcagatggct tctgccacat gcttcctctt 34620agcgtccagt
gtgtactgac aggactggct gctgctgatc agagttgaca aggggcgggt
34680ctatgaaaga gattggagac gagcattttg atcagtccct gtatcttctg
ttaaaagcat 34740ttaccttcaa ggtagaaggg agcaggagtc ctgtaccact
agataaactc agagaaagaa 34800gataactggg tacttgacat ttagaccagg
gggtgcaaca agtcggcatc cccaaagctg 34860agtcaagctg tagactcctc
tttttccacc ctcactggac aatgtctaaa ctttgtatcc 34920tccacatgcc
catcccacca catagcccct cacacacctg ctcagtaact tttctttgcc
34980caaaagccca ttcttacatg tctctttcat ttacatattc tttgcatttt
ttgggttcct 35040gctccacatc ttccatgaaa tgttgcccgc tgagctacat
gaataattct ttctccttcc 35100tggtcaggcc caggacttct tgtctgcact
gttcagctta tcaccttatt gagtatacca 35160cgtatacttt attgacctgg
ctcctgcatt agactggaag catcctgagg ggaaggtcca 35220tggttcatct
gccactgtcc cttccttggt gctgcccaga gtggggttga gcaaacagca
35280gatgctcaga aaagacccct aagccagtgc attatcctct tctgactgtg
gcactcatgg 35340tcatcagact ccctgttttc aaccctaagg acaatgacca
ggaacagcgc ccagcatgac 35400agtgattgcc caagttattc atgttcactg
agacaggctg gggctcatat cagggaagat 35460gttgcagggg tcaggcaggc
tcaggtaata tggtgctcca tggccccagt taacagagag 35520gagagggtgc
tccaggccgg tggcagtggg gccatgtttg gtcagggtgg tactgggggt
35580ggaaggacaa gaagctggag cactctaagt cactccactt catgggatga
gggagagggg 35640gagaacacag gtcttgggac ttcttcaaag gccagaggct
tggagctggc acaaggagtc 35700cttgtgcaga aaagaacaga aggtggtttt
gctgccctac atccatcccc ggaaccttct 35760cagaggtgag tagtcattac
tctcaagtga tgagattaga ggcagtttgg tgccagtgac 35820agaaggacag
gaggaaagac agagctctgg gctgggaggg aggagcctga ctcaagcctt
35880ggctcaactg atcccaggcc gtgaagcctg ggcagacctt cccgtgcctg
ctcagggtcc 35940tatctctagt ctgtataaaa aacagccagg gcaggctgtc
ctcaaaggtg cccactagct 36000caaaagttct ggaattgtat taataagagg
atgctccttg ctgtgcacga cagtgctgac 36060atgggactta ccatgccttt
gatgagagca agtgggctga tgcctgtgcg gatgggcttg 36120aagcgatcac
actgccccag gtctctttca gtggatattt ctgttgccac attgcccttc
36180ctcgtcttga cggtaaagtg agtggagcag tttccataca cggtatccta
tggaggaaga 36240agatgcaacc acatgtattc aacacgggca acatccttgg
gtacttggga gggatggggt 36300ggggatcgtg aaagaaaaag cagaaggaat
ctagctttgg gagggacttt ctttttcttc 36360ctatgcagag tgtggtcttg
ctagtgcctg gccagcccaa gttgggagag agaaaaccag 36420ttaaaggtgg
gtctgcagaa aggcctccgc aggttgcatc ggtgtcttct cccattatgg
36480tgtcgtagag aaattagaac acacaaaagt gctcatggga ctgaggtgaa
atgaattctg 36540tctcacagtg aaacctgaaa ttcgtaaaag agaccagcaa
cagatcccat tatttttggt 36600gtttaaaata cacacaggat ttactgtgta
caccataacc cagcaaacac aggtgaagca 36660tcaaacaaaa gcaggggtgg
ggtggccagg actcctcaat gactgtttta aaattagacc 36720caacctgata
agcctgcttg ggatgatctg tcagtgagcc taaggggcag gaggaacctc
36780gggcaccagt atttcacgcc aatccagggc ttcctatgta actagtcatg
gagctgactc 36840agtgatctgc tttgtatatg gtaggactgg tctctaacac
atgaagatga gtttcaaggg 36900ccactgctat cagctttcta aatcctcacc
agaaacaaca cttgcttggc ttcttctgtc 36960tctgggggaa ccaggagggc
agaaatgatg cccctcttga tgttcaggat gtaagtaggt 37020tcatctttct
ccgggtaaag gaaaacctgc ttcccttctg gaatggccag cttgagctca
37080tacctgtccc agagagagga tggtcacgga aatgtccttc tccattacaa
cttgctggaa 37140gtaagctggg tggcactgaa gtttcttttc tcatattttt
ttaaccaatt gttcttctga 37200catcatttat ttgtaaatat agaatatagc
tacacataga catacatttt caaaaaagta 37260tgcacatata cataagtttt
ggtgattctt tttttttctt tttttctttt ttctttttct 37320ttttttgaaa
tggagtctcg ctcggtcgcc cgggctggag tgcaatggcg caacctcagc
37380tcactgcaac ctccacctcc tgggatcaag tgattctcct gcctcagcct
ccggagtagc 37440taggactact ggcatgtgcc atcacaccca gctaattttt
gtatttttag tagagacagt 37500tttcaccatg ttggccaggc tggtctcaaa
ctcctgacct caggtgattc tccctcctcg 37560gcctcccaaa gtgctgggac
tacaggcatg agccaccagg tccagctgtg ttttaacttt 37620ttttgcttgg
ctcatccagc cctgctctgt tctccaccca tatcagtctc tttgcccatc
37680ctccccatac ggacacccga gtggttgttc catttgtttc tgcacactca
tacaatcctc 37740tgtaaacatg ggtgagtgca aacagagtca tacaaagatt
ttgccattgt ttgttttata 37800caagtggcat tgtattatgc atacttttat
cttgtttttc tcacttaatt atacttcaca 37860aaaatccttc tgagtcactt
ggtacagctc taacagattt ttttaaaagc tgcatgcaga 37920acattccatt
ccatggctaa atataattta ttcacacatt gccccatttg atggcatttg
37980ccctctctct ttctggattt tttttaaacc acacaaacaa taccaaaaca
aacatcctta 38040tgtgcctatc ttcacacatt actgctttta ttttcatggg
ctagagtccc aaagactaaa 38100atatctgtgt caaagggtat taagtatttt
aaattacagt agatattgcc tgatggctct 38160cgtttccacc agcagctcag
gacagcacac ttttcctcac tggcctgcca gcaacaggtg 38220ctatcactgt
tttccagttt tgcagtatga tgggtaaaag cagataaata tcttgttgtt
38280acattaattc gcatttcttg actaccagtg aattggagct tattttcata
cacttgttgg 38340acagtcattc ttttgtatgg tgtcaggcca tggctgaagc
ccatgctgac tttggtctta 38400gggatttccc actgggtact ctctgagttt
ctgaggacct ggctgcagcc actctggtta 38460catcaggagg aaaaggtgac
ggacttatct cccacagcct acactctctc agaaagttcc 38520caacacgaag
aaatgataaa tgtttgagat gatgggtatg ctaattactc tgatctgatc
38580accatacatt acatgtatca aaatatcact atgttctcaa taaatatgta
taattattgt 38640atgtcaattt ttaaaaaaga cagttaaagt catgaagatg
caaacatgga aagccctcct 38700tttcaagaac aggagtctct tctgtatttg
gagaagacca aggaccaaaa aataaaccac 38760tctttcctcc tgccaccata
cccctctccc tcatatacag ttaagtcttg gagaaaataa 38820agcataggaa
agaaagaagc ctcacagcct attttggcaa gaaaggactg gcatctgaat
38880ctaatgttta gcaaaactga aaaatgggca aggatggtga tatggtttgg
ctctgcgtcc 38940ccacccaaat ttcgtctcgt agctctcata attcccacgt
gttgtgggag ggacccgggg 39000ggaggtgatt gaatcatggg ggcaggtctt
tcccgtgctg ttctcgtgat tgcagatggg 39060tgtcgggaga tctgatggtt
ctaaaaacag gagtttctct gcacaagctc tctttgccag 39120ccaccatcta
cataagatgt gacttgctgt ccctcacctt ccgccatgat tgtgagacct
39180ccccagccac gtggaactgt gagtccaata aacctttctt ttgtaaattg
cccagtctca 39240ggtatgtctt tatcagcagc atgaaaacag actaatacag
acagagtttt ctgatgctac 39300caagttaaca actctatcct gcctgtattc
atacacacct aggaccctac aaactgtgat 39360tgaggatgag gcaggggtga
tgttgaaaat atttacaatg ggtgcgacat gggccctgac 39420cagtcagcag
agatgcagct gcagtggccg atcagcgtgc agtggctgaa tgccagccta
39480ggagggggag ccaccgaagc cttggtgctc ctctgccctg cggtgaacag
accctgcccc 39540gccatgtgcc ggccacagca gccagtgcct ctgggacccc
acaccaaaga ccaccaagca 39600ctagtcttga ctagttcttt aaataagaat
tcacttgttt aaacaaagca tctcagtttt 39660ataatctcaa gaagtttaaa
gcatgagatt tttagaaaca catgaaaatg ataattaaag 39720gaacctaact
agggaaggtt aactaatgct tctctctttt tgatgattgt gaaaactcag
39780taattccctg atccacgatg gatacggaat acaaatactt acagtcacat
ccgtgcctgg 39840tgcaaacaca caagttcata cctcagcgga cacacacaca
tgcgtgtgct catgtacaac 39900atgacttacc tggacatggc tgcagcaaac
tcctcagagt tcttggtttt cttcagcaag 39960gctttgccct cagggttgaa
gccatacacc tctttcaggg tgcactggct ggtcttcagg 40020atgaagctgc
agagctgggg aacctccagc tcaacctgag aattcagggt agcagagcat
40080tgaggttgtc tatcaagaat gagaggtggc ccctgaagcc cagggcttaa
tctctaatca 40140gagcaccaaa gggaatggtg ctgggaacac cactgcctgc
gcctcaacac cacatgcctt 40200atcaacatgc ctcctgggtt ctctgtgcac
caacctagac ttagtcctat tgctgacgtt 40260ttccccctcc cgggtaacac
atttcttaag tttgccccta ggcatgggaa ggaggatgtc 40320cttttattgg
ttctaaagtt actcacttta attataaaga gctgtggtta aatagaagcg
40380ctgcagacta ggagtgaaag tgaagaagaa aaacagaaag caggaagagg
gccatccttc 40440gtttccacag caaatgtctc cttaatgtca cccaaaaaac
tggttatatc tattagggcc 40500tatttagagc tttgcatata gctggagttt
caacggattc ctacttatta tccagcacta 40560ttcaaataac tttatagaaa
tgttgtacat gtgtgctgtt caatatggga gccagtagcc 40620agggtggctg
ttgagcagtt gaaatgcagc taatgtgaca acgaatatga atttttaatt
40680tcattccatt taaatagccc aatgtggctc atgtctacca tattggacag
cacatacaag 40740agctacggta ctctctaaaa aaaggtgact gctcagcttg
acttctcttc cccacccaaa 40800ccccaatagc agcctgtaga tctgctccac
atgtatgtaa catgagtaca accagtctca 40860ataataagca aagttttatt
ttagaacaaa ctgtatgttt atattttttt cttctttcca 40920ttcatttttc
agcaacagat tctgtttgac ttaaattata aaaactgcat ttcacagtgc
40980gattccgagt tgcctgcctc ccatagctca cctactgggc ctctctcacg
ctgaaatcta 41040cagacccaca ctgctgctaa tctagatcat ggattcctat
tgcatctggg aagttaacgg 41100gaaaatactt ctgacttgcg aaatttggtg
ggggcagacc acatctcagc aatgtggctg 41160acttacaaac agaggcaaag
tcccagtgct ttgatcagat tacaacagtt ctgggatgtt 41220ctgcgtttgc
tcagtacaca ccctccggga aggtcgcgtg ttgggcgccc gctggaacag
41280ggctggggga aagctgtggg ctctaggtcc ctcctgcctg catcctccat
accttgcagt 41340tgatcctggt ggcacttctt gaatcagcag tcccagggac
tccactggaa ctctcagcct 41400catagttgta tgtgtacttc cggaggtgct
tgaatcgggt cgcatcttct aacgtgggga 41460gaaatacgtc agccacatag
cagaaatagc tctcccaagg acagccaatt ctgggcagag 41520aggggcagtg
gcatagagaa ttaaaaatgg taattcaact caaattattt tttaattgac
41580tttatactta attttccttt atagatttga cttctccatt atgtttttgc
tgaggctaat 41640gtttaaattg gctggcatgt cttgaatatc tcatctgagg
cacagggaag ggagtgacaa 41700gtgtcaagta atggggctca gaaaatctgc
atgagagacc ctctggcaaa ttaagagatg 41760acaacattac gcaaatagta
ctatctctaa tgctttttta gcttgaacac gccgtaattc 41820cgtgactcta
agactaccaa atacttaagg caaagttcat taagcattaa gcgtaactaa
41880cttaacaaac tcctagaaaa gaatttctgg aggtttccac tgtagttgag
aggaattttc 41940tgaacaattg aagaaaaaga acatctgggt cctgagtcca
gctgcagtga tgacagacgg 42000ccactagatg gcggtgctgc cccatcagaa
gtccgccccg cggctccagc acacagggtc 42060gcgcttggag gccagttcct
ttaaccccca ggggtcaggt aaataggaag ggggtggagg 42120ttcgatttct
tcacaaaggt taaagtcagt atttcctcac cctcacatgc ttcgaatcaa
42180tgactagtca ctgataaaca ggacagtgat gtttcttcaa agtgtgcaca
ggccagcgga 42240gagacaatcc cttccctccc ccgcccccag gctgaacttt
tgggacagaa cagaaggcca 42300ggtagaagag agttggcatc ccttgggtgt
gggcagaggc tcagggaact tgcttcctgg 42360ggtcagagca aggcacacca
cgatgccatc tcagccctgt agagtgggag gccctcaggg 42420acccgggtgt
aggagagtgc acggggctgg gcgcccttcc acgccccatg cgcagatgcc
42480ttacttggac agaccaggct gacattttcc agcatttcct cttctgtaag
acaggagaaa 42540gaaatctgtg agcttcccac gtcttcccag cgggtgctag
ggcccgacag ggggaccacc 42600ggcacaggtt tcacctttca ggagggaggc
aggctccgga gaccccctcc tcagcccctc 42660catcccgcgc cccccatcct
gagcctgcag gggccgccag ctggtccaat ccccccactc 42720gccctggacc
ctgtggctgc cctccctctg gcctaggccc aggctgcccc ggccaacctc
42780gtgccgccgg ctccctcccg ctccctctgc gcccgcagag cggccgcgca
ctcaccggcc 42840ctggcgcccg ccagcagcag cagcagcagc gcaggcagcg
ccagcagcgc cagcagcgcg 42900ggcctcggcg ggtccatcgc cagctgcggt
ggggcggctc ctgggctgcg gcctggcctc 42960ggcctcgcgg ccctggctgg
ctgggcgggc tcctcagcgg cagcaaccga gaagggcact 43020cagccccgca
ggtcccggtg ggaatgcgcg gccggcgccc gcaccccatt tataggaagc
43080ccaggctgca agagcgccag gattgcaaaa ggtccaaagg gcgcctcccg
ggcctgacct 43140gtttgctttt ctacactggc ttctctttga gccttgaaga
gcctcgggga gggggcccac 43200ctgggatgca gccgcagcca ccaggggctg
ggtcccaggt gggttccctt ccccaagcgt 43260cttcagtgct ctggcgcggc
ccttcctgtg tctcagtggg gccatggcca gcgcctcagg 43320gtctgagaag
cctgccctga ccaggggtgc cttcttcaga tgacccacca tggggacaaa
43380atctctgctt ctcctggact gaattgggag ccacgaggag aaccagtcct
gagcgctgtc 43440ttggt 43445319RNAArtificial SequenceAntisense
Oligonucleotide 3cgagaggcgg acgggaccg 19419RNAArtificial
SequenceAntisense Oligonucleotide 4gcucuccgcc ugcccuggc
19521DNAArtificial SequenceAntisense Oligonucleotide 5cgagaggcgg
acgggaccgt t 21621DNAArtificial SequenceAntisense Oligonucleotide
6ttgcucuccg ccugcccugg c 21720DNAArtificial SequenceAntisense
Oligonucleotide 7tccgtcatcg ctcctcaggg 20820DNAArtificial
SequenceAntisense Oligonucleotide 8gtgcgcgcga gcccgaaatc
20920DNAArtificial SequenceAntisense Oligonucleotide 9atgcattctg
cccccaagga 201014121DNAH. sapiens 10attcccaccg ggacctgcgg
ggctgagtgc ccttctcggt tgctgccgct gaggagcccg 60cccagccagc cagggccgcg
aggccgaggc caggccgcag cccaggagcc gccccaccgc 120agctggcgat
ggacccgccg aggcccgcgc tgctggcgct gctggcgctg cctgcgctgc
180tgctgctgct gctggcgggc gccagggccg aagaggaaat gctggaaaat
gtcagcctgg 240tctgtccaaa agatgcgacc cgattcaagc acctccggaa
gtacacatac aactatgagg 300ctgagagttc cagtggagtc cctgggactg
ctgattcaag aagtgccacc aggatcaact 360gcaaggttga gctggaggtt
ccccagctct gcagcttcat cctgaagacc agccagtgca 420ccctgaaaga
ggtgtatggc ttcaaccctg agggcaaagc cttgctgaag aaaaccaaga
480actctgagga gtttgctgca gccatgtcca ggtatgagct caagctggcc
attccagaag 540ggaagcaggt tttcctttac ccggagaaag atgaacctac
ttacatcctg aacatcaaga 600ggggcatcat ttctgccctc ctggttcccc
cagagacaga agaagccaag caagtgttgt 660ttctggatac cgtgtatgga
aactgctcca ctcactttac cgtcaagacg aggaagggca 720atgtggcaac
agaaatatcc actgaaagag acctggggca gtgtgatcgc ttcaagccca
780tccgcacagg catcagccca cttgctctca tcaaaggcat gacccgcccc
ttgtcaactc 840tgatcagcag cagccagtcc tgtcagtaca cactggacgc
taagaggaag catgtggcag 900aagccatctg caaggagcaa cacctcttcc
tgcctttctc ctacaacaat aagtatggga 960tggtagcaca agtgacacag
actttgaaac ttgaagacac accaaagatc aacagccgct 1020tctttggtga
aggtactaag aagatgggcc tcgcatttga gagcaccaaa tccacatcac
1080ctccaaagca ggccgaagct gttttgaaga ctctccagga actgaaaaaa
ctaaccatct 1140ctgagcaaaa tatccagaga gctaatctct tcaataagct
ggttactgag ctgagaggcc 1200tcagtgatga agcagtcaca tctctcttgc
cacagctgat tgaggtgtcc agccccatca 1260ctttacaagc cttggttcag
tgtggacagc ctcagtgctc cactcacatc ctccagtggc 1320tgaaacgtgt
gcatgccaac ccccttctga tagatgtggt cacctacctg gtggccctga
1380tccccgagcc ctcagcacag cagctgcgag agatcttcaa catggcgagg
gatcagcgca 1440gccgagccac cttgtatgcg ctgagccacg cggtcaacaa
ctatcataag acaaacccta 1500cagggaccca ggagctgctg gacattgcta
attacctgat ggaacagatt caagatgact 1560gcactgggga tgaagattac
acctatttga ttctgcgggt cattggaaat atgggccaaa 1620ccatggagca
gttaactcca gaactcaagt cttcaatcct caaatgtgtc caaagtacaa
1680agccatcact gatgatccag aaagctgcca tccaggctct gcggaaaatg
gagcctaaag 1740acaaggacca ggaggttctt cttcagactt tccttgatga
tgcttctccg ggagataagc 1800gactggctgc ctatcttatg ttgatgagga
gtccttcaca ggcagatatt aacaaaattg 1860tccaaattct accatgggaa
cagaatgagc aagtgaagaa ctttgtggct tcccatattg 1920ccaatatctt
gaactcagaa gaattggata tccaagatct gaaaaagtta gtgaaagaag
1980ctctgaaaga atctcaactt ccaactgtca tggacttcag aaaattctct
cggaactatc 2040aactctacaa atctgtttct cttccatcac ttgacccagc
ctcagccaaa atagaaggga 2100atcttatatt tgatccaaat aactaccttc
ctaaagaaag catgctgaaa actaccctca 2160ctgcctttgg atttgcttca
gctgacctca tcgagattgg cttggaagga aaaggctttg 2220agccaacatt
ggaagctctt tttgggaagc aaggattttt cccagacagt gtcaacaaag
2280ctttgtactg ggttaatggt caagttcctg atggtgtctc taaggtctta
gtggaccact 2340ttggctatac caaagatgat aaacatgagc aggatatggt
aaatggaata
atgctcagtg 2400ttgagaagct gattaaagat ttgaaatcca aagaagtccc
ggaagccaga gcctacctcc 2460gcatcttggg agaggagctt ggttttgcca
gtctccatga cctccagctc ctgggaaagc 2520tgcttctgat gggtgcccgc
actctgcagg ggatccccca gatgattgga gaggtcatca 2580ggaagggctc
aaagaatgac ttttttcttc actacatctt catggagaat gcctttgaac
2640tccccactgg agctggatta cagttgcaaa tatcttcatc tggagtcatt
gctcccggag 2700ccaaggctgg agtaaaactg gaagtagcca acatgcaggc
tgaactggtg gcaaaaccct 2760ccgtgtctgt ggagtttgtg acaaatatgg
gcatcatcat tccggacttc gctaggagtg 2820gggtccagat gaacaccaac
ttcttccacg agtcgggtct ggaggctcat gttgccctaa 2880aagctgggaa
gctgaagttt atcattcctt ccccaaagag accagtcaag ctgctcagtg
2940gaggcaacac attacatttg gtctctacca ccaaaacgga ggtgatccca
cctctcattg 3000agaacaggca gtcctggtca gtttgcaagc aagtctttcc
tggcctgaat tactgcacct 3060caggcgctta ctccaacgcc agctccacag
actccgcctc ctactatccg ctgaccgggg 3120acaccagatt agagctggaa
ctgaggccta caggagagat tgagcagtat tctgtcagcg 3180caacctatga
gctccagaga gaggacagag ccttggtgga taccctgaag tttgtaactc
3240aagcagaagg tgcgaagcag actgaggcta ccatgacatt caaatataat
cggcagagta 3300tgaccttgtc cagtgaagtc caaattccgg attttgatgt
tgacctcgga acaatcctca 3360gagttaatga tgaatctact gagggcaaaa
cgtcttacag actcaccctg gacattcaga 3420acaagaaaat tactgaggtc
gccctcatgg gccacctaag ttgtgacaca aaggaagaaa 3480gaaaaatcaa
gggtgttatt tccatacccc gtttgcaagc agaagccaga agtgagatcc
3540tcgcccactg gtcgcctgcc aaactgcttc tccaaatgga ctcatctgct
acagcttatg 3600gctccacagt ttccaagagg gtggcatggc attatgatga
agagaagatt gaatttgaat 3660ggaacacagg caccaatgta gataccaaaa
aaatgacttc caatttccct gtggatctct 3720ccgattatcc taagagcttg
catatgtatg ctaatagact cctggatcac agagtccctg 3780aaacagacat
gactttccgg cacgtgggtt ccaaattaat agttgcaatg agctcatggc
3840ttcagaaggc atctgggagt cttccttata cccagacttt gcaagaccac
ctcaatagcc 3900tgaaggagtt caacctccag aacatgggat tgccagactt
ccacatccca gaaaacctct 3960tcttaaaaag cgatggccgg gtcaaatata
ccttgaacaa gaacagtttg aaaattgaga 4020ttcctttgcc ttttggtggc
aaatcctcca gagatctaaa gatgttagag actgttagga 4080caccagccct
ccacttcaag tctgtgggat tccatctgcc atctcgagag ttccaagtcc
4140ctacttttac cattcccaag ttgtatcaac tgcaagtgcc tctcctgggt
gttctagacc 4200tctccacgaa tgtctacagc aacttgtaca actggtccgc
ctcctacagt ggtggcaaca 4260ccagcacaga ccatttcagc cttcgggctc
gttaccacat gaaggctgac tctgtggttg 4320acctgctttc ctacaatgtg
caaggatctg gagaaacaac atatgaccac aagaatacgt 4380tcacactatc
atgtgatggg tctctacgcc acaaatttct agattcgaat atcaaattca
4440gtcatgtaga aaaacttgga aacaacccag tctcaaaagg tttactaata
ttcgatgcat 4500ctagttcctg gggaccacag atgtctgctt cagttcattt
ggactccaaa aagaaacagc 4560atttgtttgt caaagaagtc aagattgatg
ggcagttcag agtctcttcg ttctatgcta 4620aaggcacata tggcctgtct
tgtcagaggg atcctaacac tggccggctc aatggagagt 4680ccaacctgag
gtttaactcc tcctacctcc aaggcaccaa ccagataaca ggaagatatg
4740aagatggaac cctctccctc acctccacct ctgatctgca aagtggcatc
attaaaaata 4800ctgcttccct aaagtatgag aactacgagc tgactttaaa
atctgacacc aatgggaagt 4860ataagaactt tgccacttct aacaagatgg
atatgacctt ctctaagcaa aatgcactgc 4920tgcgttctga atatcaggct
gattacgagt cattgaggtt cttcagcctg ctttctggat 4980cactaaattc
ccatggtctt gagttaaatg ctgacatctt aggcactgac aaaattaata
5040gtggtgctca caaggcgaca ctaaggattg gccaagatgg aatatctacc
agtgcaacga 5100ccaacttgaa gtgtagtctc ctggtgctgg agaatgagct
gaatgcagag cttggcctct 5160ctggggcatc tatgaaatta acaacaaatg
gccgcttcag ggaacacaat gcaaaattca 5220gtctggatgg gaaagccgcc
ctcacagagc tatcactggg aagtgcttat caggccatga 5280ttctgggtgt
cgacagcaaa aacattttca acttcaaggt cagtcaagaa ggacttaagc
5340tctcaaatga catgatgggc tcatatgctg aaatgaaatt tgaccacaca
aacagtctga 5400acattgcagg cttatcactg gacttctctt caaaacttga
caacatttac agctctgaca 5460agttttataa gcaaactgtt aatttacagc
tacagcccta ttctctggta actactttaa 5520acagtgacct gaaatacaat
gctctggatc tcaccaacaa tgggaaacta cggctagaac 5580ccctgaagct
gcatgtggct ggtaacctaa aaggagccta ccaaaataat gaaataaaac
5640acatctatgc catctcttct gctgccttat cagcaagcta taaagcagac
actgttgcta 5700aggttcaggg tgtggagttt agccatcggc tcaacacaga
catcgctggg ctggcttcag 5760ccattgacat gagcacaaac tataattcag
actcactgca tttcagcaat gtcttccgtt 5820ctgtaatggc cccgtttacc
atgaccatcg atgcacatac aaatggcaat gggaaactcg 5880ctctctgggg
agaacatact gggcagctgt atagcaaatt cctgttgaaa gcagaacctc
5940tggcatttac tttctctcat gattacaaag gctccacaag tcatcatctc
gtgtctagga 6000aaagcatcag tgcagctctt gaacacaaag tcagtgccct
gcttactcca gctgagcaga 6060caggcacctg gaaactcaag acccaattta
acaacaatga atacagccag gacttggatg 6120cttacaacac taaagataaa
attggcgtgg agcttactgg acgaactctg gctgacctaa 6180ctctactaga
ctccccaatt aaagtgccac ttttactcag tgagcccatc aatatcattg
6240atgctttaga gatgagagat gccgttgaga agccccaaga atttacaatt
gttgcttttg 6300taaagtatga taaaaaccaa gatgttcact ccattaacct
cccatttttt gagaccttgc 6360aagaatattt tgagaggaat cgacaaacca
ttatagttgt agtggaaaac gtacagagaa 6420acctgaagca catcaatatt
gatcaatttg taagaaaata cagagcagcc ctgggaaaac 6480tcccacagca
agctaatgat tatctgaatt cattcaattg ggagagacaa gtttcacatg
6540ccaaggagaa actgactgct ctcacaaaaa agtatagaat tacagaaaat
gatatacaaa 6600ttgcattaga tgatgccaaa atcaacttta atgaaaaact
atctcaactg cagacatata 6660tgatacaatt tgatcagtat attaaagata
gttatgattt acatgatttg aaaatagcta 6720ttgctaatat tattgatgaa
atcattgaaa aattaaaaag tcttgatgag cactatcata 6780tccgtgtaaa
tttagtaaaa acaatccatg atctacattt gtttattgaa aatattgatt
6840ttaacaaaag tggaagtagt actgcatcct ggattcaaaa tgtggatact
aagtaccaaa 6900tcagaatcca gatacaagaa aaactgcagc agcttaagag
acacatacag aatatagaca 6960tccagcacct agctggaaag ttaaaacaac
acattgaggc tattgatgtt agagtgcttt 7020tagatcaatt gggaactaca
atttcatttg aaagaataaa tgatgttctt gagcatgtca 7080aacactttgt
tataaatctt attggggatt ttgaagtagc tgagaaaatc aatgccttca
7140gagccaaagt ccatgagtta atcgagaggt atgaagtaga ccaacaaatc
caggttttaa 7200tggataaatt agtagagttg acccaccaat acaagttgaa
ggagactatt cagaagctaa 7260gcaatgtcct acaacaagtt aagataaaag
attactttga gaaattggtt ggatttattg 7320atgatgctgt gaagaagctt
aatgaattat cttttaaaac attcattgaa gatgttaaca 7380aattccttga
catgttgata aagaaattaa agtcatttga ttaccaccag tttgtagatg
7440aaaccaatga caaaatccgt gaggtgactc agagactcaa tggtgaaatt
caggctctgg 7500aactaccaca aaaagctgaa gcattaaaac tgtttttaga
ggaaaccaag gccacagttg 7560cagtgtatct ggaaagccta caggacacca
aaataacctt aatcatcaat tggttacagg 7620aggctttaag ttcagcatct
ttggctcaca tgaaggccaa attccgagag actctagaag 7680atacacgaga
ccgaatgtat caaatggaca ttcagcagga acttcaacga tacctgtctc
7740tggtaggcca ggtttatagc acacttgtca cctacatttc tgattggtgg
actcttgctg 7800ctaagaacct tactgacttt gcagagcaat attctatcca
agattgggct aaacgtatga 7860aagcattggt agagcaaggg ttcactgttc
ctgaaatcaa gaccatcctt gggaccatgc 7920ctgcctttga agtcagtctt
caggctcttc agaaagctac cttccagaca cctgatttta 7980tagtccccct
aacagatttg aggattccat cagttcagat aaacttcaaa gacttaaaaa
8040atataaaaat cccatccagg ttttccacac cagaatttac catccttaac
accttccaca 8100ttccttcctt tacaattgac tttgtcgaaa tgaaagtaaa
gatcatcaga accattgacc 8160agatgcagaa cagtgagctg cagtggcccg
ttccagatat atatctcagg gatctgaagg 8220tggaggacat tcctctagcg
agaatcaccc tgccagactt ccgtttacca gaaatcgcaa 8280ttccagaatt
cataatccca actctcaacc ttaatgattt tcaagttcct gaccttcaca
8340taccagaatt ccagcttccc cacatctcac acacaattga agtacctact
tttggcaagc 8400tatacagtat tctgaaaatc caatctcctc ttttcacatt
agatgcaaat gctgacatag 8460ggaatggaac cacctcagca aacgaagcag
gtatcgcagc ttccatcact gccaaaggag 8520agtccaaatt agaagttctc
aattttgatt ttcaagcaaa tgcacaactc tcaaacccta 8580agattaatcc
gctggctctg aaggagtcag tgaagttctc cagcaagtac ctgagaacgg
8640agcatgggag tgaaatgctg ttttttggaa atgctattga gggaaaatca
aacacagtgg 8700caagtttaca cacagaaaaa aatacactgg agcttagtaa
tggagtgatt gtcaagataa 8760acaatcagct taccctggat agcaacacta
aatacttcca caaattgaac atccccaaac 8820tggacttctc tagtcaggct
gacctgcgca acgagatcaa gacactgttg aaagctggcc 8880acatagcatg
gacttcttct ggaaaagggt catggaaatg ggcctgcccc agattctcag
8940atgagggaac acatgaatca caaattagtt tcaccataga aggacccctc
acttcctttg 9000gactgtccaa taagatcaat agcaaacacc taagagtaaa
ccaaaacttg gtttatgaat 9060ctggctccct caacttttct aaacttgaaa
ttcaatcaca agtcgattcc cagcatgtgg 9120gccacagtgt tctaactgct
aaaggcatgg cactgtttgg agaagggaag gcagagttta 9180ctgggaggca
tgatgctcat ttaaatggaa aggttattgg aactttgaaa aattctcttt
9240tcttttcagc ccagccattt gagatcacgg catccacaaa caatgaaggg
aatttgaaag 9300ttcgttttcc attaaggtta acagggaaga tagacttcct
gaataactat gcactgtttc 9360tgagtcccag tgcccagcaa gcaagttggc
aagtaagtgc taggttcaat cagtataagt 9420acaaccaaaa tttctctgct
ggaaacaacg agaacattat ggaggcccat gtaggaataa 9480atggagaagc
aaatctggat ttcttaaaca ttcctttaac aattcctgaa atgcgtctac
9540cttacacaat aatcacaact cctccactga aagatttctc tctatgggaa
aaaacaggct 9600tgaaggaatt cttgaaaacg acaaagcaat catttgattt
aagtgtaaaa gctcagtata 9660agaaaaacaa acacaggcat tccatcacaa
atcctttggc tgtgctttgt gagtttatca 9720gtcagagcat caaatccttt
gacaggcatt ttgaaaaaaa cagaaacaat gcattagatt 9780ttgtcaccaa
atcctataat gaaacaaaaa ttaagtttga taagtacaaa gctgaaaaat
9840ctcacgacga gctccccagg acctttcaaa ttcctggata cactgttcca
gttgtcaatg 9900ttgaagtgtc tccattcacc atagagatgt cggcattcgg
ctatgtgttc ccaaaagcag 9960tcagcatgcc tagtttctcc atcctaggtt
ctgacgtccg tgtgccttca tacacattaa 10020tcctgccatc attagagctg
ccagtccttc atgtccctag aaatctcaag ctttctcttc 10080cacatttcaa
ggaattgtgt accataagcc atatttttat tcctgccatg ggcaatatta
10140cctatgattt ctcctttaaa tcaagtgtca tcacactgaa taccaatgct
gaacttttta 10200accagtcaga tattgttgct catctccttt cttcatcttc
atctgtcatt gatgcactgc 10260agtacaaatt agagggcacc acaagattga
caagaaaaag gggattgaag ttagccacag 10320ctctgtctct gagcaacaaa
tttgtggagg gtagtcataa cagtactgtg agcttaacca 10380cgaaaaatat
ggaagtgtca gtggcaaaaa ccacaaaagc cgaaattcca attttgagaa
10440tgaatttcaa gcaagaactt aatggaaata ccaagtcaaa acctactgtc
tcttcctcca 10500tggaatttaa gtatgatttc aattcttcaa tgctgtactc
taccgctaaa ggagcagttg 10560accacaagct tagcttggaa agcctcacct
cttacttttc cattgagtca tctaccaaag 10620gagatgtcaa gggttcggtt
ctttctcggg aatattcagg aactattgct agtgaggcca 10680acacttactt
gaattccaag agcacacggt cttcagtgaa gctgcagggc acttccaaaa
10740ttgatgatat ctggaacctt gaagtaaaag aaaattttgc tggagaagcc
acactccaac 10800gcatatattc cctctgggag cacagtacga aaaaccactt
acagctagag ggcctctttt 10860tcaccaacgg agaacataca agcaaagcca
ccctggaact ctctccatgg caaatgtcag 10920ctcttgttca ggtccatgca
agtcagccca gttccttcca tgatttccct gaccttggcc 10980aggaagtggc
cctgaatgct aacactaaga accagaagat cagatggaaa aatgaagtcc
11040ggattcattc tgggtctttc cagagccagg tcgagctttc caatgaccaa
gaaaaggcac 11100accttgacat tgcaggatcc ttagaaggac acctaaggtt
cctcaaaaat atcatcctac 11160cagtctatga caagagctta tgggatttcc
taaagctgga tgtaaccacc agcattggta 11220ggagacagca tcttcgtgtt
tcaactgcct ttgtgtacac caaaaacccc aatggctatt 11280cattctccat
ccctgtaaaa gttttggctg ataaattcat tactcctggg ctgaaactaa
11340atgatctaaa ttcagttctt gtcatgccta cgttccatgt cccatttaca
gatcttcagg 11400ttccatcgtg caaacttgac ttcagagaaa tacaaatcta
taagaagctg agaacttcat 11460catttgccct caacctacca acactccccg
aggtaaaatt ccctgaagtt gatgtgttaa 11520caaaatattc tcaaccagaa
gactccttga ttcccttttt tgagataacc gtgcctgaat 11580ctcagttaac
tgtgtcccag ttcacgcttc caaaaagtgt ttcagatggc attgctgctt
11640tggatctaaa tgcagtagcc aacaagatcg cagactttga gttgcccacc
atcatcgtgc 11700ctgagcagac cattgagatt ccctccatta agttctctgt
acctgctgga attgtcattc 11760cttcctttca agcactgact gcacgctttg
aggtagactc tcccgtgtat aatgccactt 11820ggagtgccag tttgaaaaac
aaagcagatt atgttgaaac agtcctggat tccacatgca 11880gctcaaccgt
acagttccta gaatatgaac taaatgtttt gggaacacac aaaatcgaag
11940atggtacgtt agcctctaag actaaaggaa cacttgcaca ccgtgacttc
agtgcagaat 12000atgaagaaga tggcaaattt gaaggacttc aggaatggga
aggaaaagcg cacctcaata 12060tcaaaagccc agcgttcacc gatctccatc
tgcgctacca gaaagacaag aaaggcatct 12120ccacctcagc agcctcccca
gccgtaggca ccgtgggcat ggatatggat gaagatgacg 12180acttttctaa
atggaacttc tactacagcc ctcagtcctc tccagataaa aaactcacca
12240tattcaaaac tgagttgagg gtccgggaat ctgatgagga aactcagatc
aaagttaatt 12300gggaagaaga ggcagcttct ggcttgctaa cctctctgaa
agacaacgtg cccaaggcca 12360caggggtcct ttatgattat gtcaacaagt
accactggga acacacaggg ctcaccctga 12420gagaagtgtc ttcaaagctg
agaagaaatc tgcagaacaa tgctgagtgg gtttatcaag 12480gggccattag
gcaaattgat gatatcgacg tgaggttcca gaaagcagcc agtggcacca
12540ctgggaccta ccaagagtgg aaggacaagg cccagaatct gtaccaggaa
ctgttgactc 12600aggaaggcca agccagtttc cagggactca aggataacgt
gtttgatggc ttggtacgag 12660ttactcaaaa attccatatg aaagtcaagc
atctgattga ctcactcatt gattttctga 12720acttccccag attccagttt
ccggggaaac ctgggatata cactagggag gaactttgca 12780ctatgttcat
aagggaggta gggacggtac tgtcccaggt atattcgaaa gtccataatg
12840gttcagaaat actgttttcc tatttccaag acctagtgat tacacttcct
ttcgagttaa 12900ggaaacataa actaatagat gtaatctcga tgtataggga
actgttgaaa gatttatcaa 12960aagaagccca agaggtattt aaagccattc
agtctctcaa gaccacagag gtgctacgta 13020atcttcagga ccttttacaa
ttcattttcc aactaataga agataacatt aaacagctga 13080aagagatgaa
atttacttat cttattaatt atatccaaga tgagatcaac acaatcttca
13140atgattatat cccatatgtt tttaaattgt tgaaagaaaa cctatgcctt
aatcttcata 13200agttcaatga atttattcaa aacgagcttc aggaagcttc
tcaagagtta cagcagatcc 13260atcaatacat tatggccctt cgtgaagaat
attttgatcc aagtatagtt ggctggacag 13320tgaaatatta tgaacttgaa
gaaaagatag tcagtctgat caagaacctg ttagttgctc 13380ttaaggactt
ccattctgaa tatattgtca gtgcctctaa ctttacttcc caactctcaa
13440gtcaagttga gcaatttctg cacagaaata ttcaggaata tcttagcatc
cttaccgatc 13500cagatggaaa agggaaagag aagattgcag agctttctgc
cactgctcag gaaataatta 13560aaagccaggc cattgcgacg aagaaaataa
tttctgatta ccaccagcag tttagatata 13620aactgcaaga tttttcagac
caactctctg attactatga aaaatttatt gctgaatcca 13680aaagattgat
tgacctgtcc attcaaaact accacacatt tctgatatac atcacggagt
13740tactgaaaaa gctgcaatca accacagtca tgaaccccta catgaagctt
gctccaggag 13800aacttactat catcctctaa ttttttaaaa gaaatcttca
tttattcttc ttttccaatt 13860gaactttcac atagcacaga aaaaattcaa
actgcctata ttgataaaac catacagtga 13920gccagccttg cagtaggcag
tagactataa gcagaagcac atatgaactg gacctgcacc 13980aaagctggca
ccagggctcg gaaggtctct gaactcagaa ggatggcatt ttttgcaagt
14040taaagaaaat caggatctga gttattttgc taaacttggg ggaggaggaa
caaataaatg 14100gagtctttat tgtgtatcat a 141211121DNAArtificial
SequencePCR Primer 11tgctaaaggc acatatggcc t 211223DNAArtificial
SequencePCR Primer 12ctcaggttgg actctccatt gag 231328DNAArtificial
SequencePCR Probe 13cttgtcagag ggatcctaac actggccg
281419DNAArtificial SequencePCR Primer 14gaaggtgaag gtcggagtc
191520DNAArtificial SequencePCR Primer 15gaagatggtg atgggatttc
201620DNAArtificial SequencePCR Probe 16caagcttccc gttctcagcc
20172354DNAM. musculus 17gaattccaac ttcctcacct ctcacataca
attgaaatac ctgcttttgg caaactgcat 60agcatcctta agatccaatc tcctctcttt
atattagatg ctaatgccaa catacagaat 120gtaacaactt cagggaacaa
agcagagatt gtggcttctg tcactgctaa aggagagtcc 180caatttgaag
ctctcaattt tgattttcaa gcacaagctc aattcctgga gttaaatcct
240catcctccag tcctgaagga atccatgaac ttctccagta agcatgtgag
aatggagcat 300gagggtgaga tagtatttga tggaaaggcc attgagggga
aatcagacac agtcgcaagt 360ttacacacag agaaaaatga agtagagttt
aataatggta tgactgtcaa agtaaacaat 420cagctcaccc ttgacagtca
cacaaagtac ttccacaagt tgagtgttcc taggctggac 480ttctccagta
aggcttctct taataatgaa atcaagacac tattagaagc tggacatgtg
540gcattgacat cttcagggac agggtcatgg aactgggcct gtcccaactt
ctcggatgaa 600ggcatacatt cgtcccaaat tagctttact gtggatggtc
ccattgcttt tgttggacta 660tccaataaca taaatggcaa acacttacgg
gtcatccaaa aactgactta tgaatctggc 720ttcctcaact attctaagtt
tgaagttgag tcaaaagttg aatctcagca cgtgggctcc 780agcattctaa
cagccaatgg tcgggcactg ctcaaggacg caaaggcaga aatgactggt
840gagcacaatg ccaacttaaa tggaaaagtt attggaactt tgaaaaattc
tctcttcttt 900tcagcacaac catttgagat tactgcatcc acaaataatg
aaggaaattt gaaagtgggt 960tttccactaa agctgactgg gaaaatagac
ttcctgaata actatgcatt gtttctgagt 1020ccccgtgccc aacaagcaag
ctggcaagcg agtaccagat tcaatcagta caaatacaat 1080caaaactttt
ctgctataaa caatgaacac aacatagaag ccagtatagg aatgaatgga
1140gatgccaacc tggatttctt aaacatacct ttaacaattc ctgaaattaa
cttgccttac 1200acggagttca aaactccctt actgaaggat ttctccatat
gggaagaaac aggcttgaaa 1260gaatttttga agacaacaaa gcaatcattt
gatttgagtg taaaggctca atataaaaag 1320aacagtgaca agcattccat
tgttgtccct ctgggtatgt tttatgaatt tattctcaac 1380aatgtcaatt
cgtgggacag aaaatttgag aaagtcagaa acaatgcttt acattttctt
1440accacctcct ataatgaagc aaaaattaag gttgataagt acaaaactga
aaattccctt 1500aatcagccct ctgggacctt tcaaaatcat ggctacacta
tcccagttgt caacattgaa 1560gtatctccat ttgctgtaga gacactggct
tccaggcatg tgatccccac agcaataagc 1620accccaagtg tcacaatccc
tggtcctaac atcatggtgc cttcatacaa gttagtgctg 1680ccacccctgg
agttgccagt tttccatggt cctgggaatc tattcaagtt tttcctccca
1740gatttcaagg gattcaacac tattgacaat atttatattc cagccatggg
caactttacc 1800tatgactttt cttttaaatc aagtgtcatc acactgaata
ccaatgctgg actttataac 1860caatcagata tcgttgccca tttcctttct
tcctcttcat ttgtcactga cgccctgcag 1920tacaaattag agggaacatc
acgtctgatg cgaaaaaggg gattgaaact agccacagct 1980gtctctctaa
ctaacaaatt tgtaaagggc agtcatgaca gcaccattag tttaaccaag
2040aaaaacatgg aagcatcagt gagaacaact gccaacctcc atgctcccat
attctcaatg 2100aacttcaagc aggaacttaa tggaaatacc aagtcaaaac
ccactgtttc atcatccatt 2160gaactaaact atgacttcaa ttcctcaaag
ctgcactcta ctgcaacagg aggcattgat 2220cacaagttca gcttagaaag
tctcacttcc tacttttcca ttgagtcatt caccaaagga 2280aatatcaaga
gttccttcct ttctcaggaa tattcaggaa gtgttgccaa tgaagccaat
2340gtatatctga attc 23541819DNAArtificial SequencePCR Primer
18cgtgggctcc agcattcta 191921DNAArtificial SequencePCR Primer
19agtcatttct gcctttgcgt c 212022DNAArtificial SequencePCR Probe
20ccaatggtcg ggcactgctc aa 222120DNAArtificial SequencePCR
Primer 21ggcaaattca acggcacagt 202220DNAArtificial SequencePCR
Primer 22gggtctcgct cctggaagat 202327DNAArtificial SequencePCR
Probe 23aaggccgaga atgggaagct tgtcatc 27
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