U.S. patent application number 11/124020 was filed with the patent office on 2005-12-29 for snps of apolipoprotein b and modulation of their expression.
Invention is credited to Crooke, Rosanne M., Graham, Mark J., Mah, Steven.
Application Number | 20050287558 11/124020 |
Document ID | / |
Family ID | 35506289 |
Filed Date | 2005-12-29 |
United States Patent
Application |
20050287558 |
Kind Code |
A1 |
Crooke, Rosanne M. ; et
al. |
December 29, 2005 |
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.; (San Clemente,
CA) ; Mah, Steven; (San Diego, CA) |
Correspondence
Address: |
ISIS PHARMACEUTICALS, INC
1896 RUTHERFORD ROAD
CARLSBAD
CA
92008
US
|
Family ID: |
35506289 |
Appl. No.: |
11/124020 |
Filed: |
May 5, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60568409 |
May 5, 2004 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
514/44A; 536/23.1 |
Current CPC
Class: |
C12N 2310/321 20130101;
C12Q 2600/156 20130101; C12Q 2600/158 20130101; C12Q 2600/136
20130101; C12N 15/113 20130101; C12N 2320/30 20130101; C12N 2310/11
20130101; C12Q 1/6883 20130101; C12N 2310/3341 20130101; A61P 3/00
20180101; C12N 2310/111 20130101; C12N 2310/341 20130101; C12N
2310/315 20130101; C12Q 2600/106 20130101 |
Class at
Publication: |
435/006 ;
514/044; 536/023.1 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 048/00 |
Claims
What is claimed is:
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 at least one nucleobase selected from the group consisting
of: (a) C at position 27751 of SEQ ID NO: 1; (b) C at position
27735 of SEQ ID NO: 1; (c) G at position 27685 of SEQ ID NO: 1; (d)
G at position 27683 of SEQ ID NO: 1; (e) G at position 27679 of SEQ
ID NO: 1; (f) A at position 27634 of SEQ ID NO: 1; (g) T/U at
position 27627 of SEQ ID NO: 1; or (h) 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.
2. The antisense compound of claim 1 comprising at least one
nucleobase selected from the group consisting of: (a) G at position
15695 of SEQ ID NO: 2; (b) G at position 15711 of SEQ ID NO: 2; (c)
C at position 15761 of SEQ ID NO: 2; (d) C at position 15763 of SEQ
ID NO: 2; (e) C at position 15767 of SEQ ID NO: 2; (f) T/U at
position 15812 of SEQ ID NO: 2; (g) A at position 15819 of SEQ ID
NO: 2; or (h) C at position 15828 of SEQ ID NO: 2, wherein G is
guanine, C is cytosine, T is thymine, U is uracil, and A is
adenine.
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 the
antisense compound of claim 1 so that expression of apolipoprotein
B is inhibited.
15. A method of screening for a modulator of apolipoprotein B, the
method comprising the steps of: contacting a preferred target
segment of a nucleic acid molecule encoding apolipoprotein B with
one or more candidate modulators of apolipoprotein B, and
identifying one or more modulators of apolipoprotein B expression
which modulate the expression of apolipoprotein B, wherein said
preferred target segment comprises at least one of: (a) C at
position 27751 of SEQ ID NO: 1; (b) C at position 27735 of SEQ ID
NO: 1; (c) G at position 27685 of SEQ ID NO: 1; (d) G at position
27683 of SEQ ID NO: 1; (e) G at position 27679 of SEQ ID NO: 1; (f)
A at position 27634 of SEQ ID NO: 1; (g) T/U at position 27627 of
SEQ ID NO: 1; or (h) 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.
16. The method of claim 15 wherein the modulator of apolipoprotein
B expression comprises an oligonucleotide, an antisense
oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an
RNA oligonucleotide having at least a portion of said RNA
oligonucleotide capable of hybridizing with RNA to form an
oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
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
the antisense compound of claim 1 so that expression of
apolipoprotein B is inhibited.
18. The method of claim 21 wherein the disease or condition is a
disorder of lipid metabolism.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Application Ser. No. 60/568,409, filed May 5, 2004,
which is herein incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] A paper copy of the sequence listing and a computer-readable
form of the sequence listing, on diskette, containing the file
named BIOL0021USSEQ.txt, which is 129,536 bytes and was created on
May 5, 2005, are herein incorporated by reference.
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
[0008] A. Overview of the Invention
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] "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.
[0015] 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).
[0016] 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%.
[0017] B. Compounds of the Invention
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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).
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] Particularly preferred compounds are oligonucleotides from
about 12 to about 50 nucleobases, even more preferably those
comprising from about 15 to about 30 nucleobases.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] C. Targets of the Invention
[0033] "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.
[0034] 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.
[0035] 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).
[0036] 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.
[0037] 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.
[0038] Other target regions include the 5' untranslated region
(5UTR), 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 (3UTR), 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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:
[0049] 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.
[0050] This set of regions can be represented using the following
mathematical notation: 1 A = m S ( m ) where m N 8 m 80 and S ( m )
= { R n , n + m - 1 n { 1 , 2 , 3 , , L - m + 1 } }
[0051] where the mathematical operator .vertline. indicates "such
that",
[0052] 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),
[0053] where x is a variable,
[0054] where N indicates all natural numbers, defined as positive
integers,
[0055] and where the mathematical operator .orgate. indicates "the
union of sets".
[0056] 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.vertline.n.epsilon.{1, 2, 3, . . . , 93}}
[0057] 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.
[0058] 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.vertline.n.epsilon.{1, 2, 3, . . . , 81}}
[0059] 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,
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.
[0060] 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.1,80.vertline.n.epsilon.{1, 2, 3, . . . , 21}}
[0061] 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.
[0062] 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.
[0063] 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 2 A = m S ( m )
[0064] where .orgate. represents the union of the sets obtained by
combining all members of all sets.
[0065] 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.
[0066] D. Screening and Target Validation
[0067] 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.
[0068] 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.
[0069] 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).
[0070] 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.
[0071] E. Kits, Research Reagents, Diagnostics, and
Therapeutics
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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).
[0076] 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.
[0077] 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.
[0078] 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 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%.
[0079] 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.
[0080] 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.
[0081] F. Modifications
[0082] 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.
[0083] Modified Internucleoside Linkages (Backbones)
[0084] 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.
[0085] Preferred modified oligonucleotide backbones containing a
phosphorus atom therein include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkyl-phosphotriaminoalkylphosphotriesters, methyl and other
alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriest- ers,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3',5' to 5'
or 2' to 2' linkage. Preferred oligonucleotides having inverted
polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] Modified Sugar and Internucleoside Linkages-Mimetics
[0090] 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.
[0091] 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.
[0092] Modified Sugars
[0093] 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.su- b.3].sub.2, where n and
m are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-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.
[0094] 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.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.
[0095] 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.
[0096] Natural and Modified Nucleobases
[0097] 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 a 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[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-aminopropyl-adenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C. and
are presently preferred base substitutions, even more particularly
when combined with 2'-O-methoxyethyl sugar modifications.
[0098] 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.
[0099] Conjugates
[0100] 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-glyc- ero-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-triiodo-benzoic 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.
[0101] 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.
[0102] Chimeric Compounds
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] G. Formulations
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0121] 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).
[0122] 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.
[0123] 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. applications Ser. No.
09/108,673 (filed Jul. 1, 1998), U.S. 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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).
[0129] 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).
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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).
[0139] 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.
[0140] 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).
[0141] 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.
[0142] 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).
[0143] 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-glycer- ol), 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).
[0144] 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.
[0145] 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).
[0146] 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.
[0147] 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).
[0148] 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).
[0149] 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.
[0150] 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).
[0151] 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.).
[0152] 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.
[0153] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include but are not limited to cancer chemotherapeutic drugs such
as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,
mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine,
procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophospharnide, 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.
[0154] 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.
[0155] H. Dosing
[0156] 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 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.
[0157] 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.
[0158] I. Polymorphisms
[0159] 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).
[0160] 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.
[0161] 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.
1 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%.sup. 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%.sup. Substitution of G to A 27627 2% Substitution of T to C
27618 <1%.sup.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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:
[0169] C at position 27751 of SEQ ID NO: 1;
[0170] C at position 27735 of SEQ ID NO: 1;
[0171] G at position 27685 of SEQ ID NO: 1;
[0172] G at position 27683 of SEQ ID NO: 1;
[0173] G at position 27679 of SEQ ID NO: 1;
[0174] A at position 27634 of SEQ ID NO: 1;
[0175] T/U at position 27627 of SEQ ID NO: 1; or
[0176] 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.
[0177] 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:
[0178] G at position 15695 of SEQ ID NO: 2;
[0179] G at position 15711 of SEQ ID NO: 2;
[0180] C at position 15761 of SEQ ID NO: 2;
[0181] C at position 15763 of SEQ ID NO: 2;
[0182] C at position 15767 of SEQ ID NO: 2;
[0183] T/U at position 15812 of SEQ ID NO: 2;
[0184] A at position 15819 of SEQ ID NO: 2; or
[0185] C at position 15828 of SEQ ID NO: 2.
[0186] 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.
[0187] 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:
[0188] C at position 27751 of SEQ ID NO: 1;
[0189] C at position 27735 of SEQ ID NO: 1;
[0190] G at position 27685 of SEQ ID NO: 1;
[0191] G at position 27683 of SEQ ID NO: 1;
[0192] G at position 27679 of SEQ ID NO: 1;
[0193] A at position 27634 of SEQ ID NO: 1;
[0194] T/U at position 27627 of SEQ ID NO: 1; or
[0195] G at position 27618 of SEQ ID NO: 1.
[0196] 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:
[0197] G at position 15695 of SEQ ID NO: 2;
[0198] G at position 15711 of SEQ ID NO: 2;
[0199] C at position 15761 of SEQ ID NO: 2;
[0200] C at position 15763 of SEQ ID NO: 2;
[0201] C at position 15767 of SEQ ID NO: 2;
[0202] T/U at position 15812 of SEQ ID NO: 2;
[0203] A at position 15819 of SEQ ID NO: 2; or
[0204] C at position 15828 of SEQ ID NO: 2.
[0205] The invention further contemplates an oligonucleotide
comprising about 15 to 3o contiguous nucleobases of an allelic
variant of SEQ ID NO: 1, said allelic variant comprising at least
one of:
[0206] C at position 27751 of SEQ ID NO: 1;
[0207] C at position 27735 of SEQ ID NO: 1;
[0208] G at position 27685 of SEQ ID NO: 1;
[0209] G at position 27683 of SEQ ID NO: 1;
[0210] G at position 27679 of SEQ ID NO: 1;
[0211] A at position 27634 of SEQ ID NO: 1;
[0212] T/U at position 27627 of SEQ ID NO: 1; or
[0213] G at position 27618 of SEQ ID NO: 1.
[0214] The invention also contemplates an oligonucleotide
comprising about 15 to 3o contiguous nucleobases of an allelic
variant of SEQ ID NO: 2, said allelic variant comprising at least
one of:
[0215] G at position 15695 of SEQ ID NO: 2;
[0216] G at position 15711 of SEQ ID NO: 2;
[0217] C at position 15761 of SEQ ID NO: 2;
[0218] C at position 15763 of SEQ ID NO: 2;
[0219] C at position 15767 of SEQ ID NO: 2;
[0220] T/U at position 15812 of SEQ ID NO: 2;
[0221] A at position 15819 of SEQ ID NO: 2; or
[0222] C at position 15828 of SEQ ID NO: 2.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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. Altematively, 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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
[0238] Synthesis of Nucleoside Phosphoramidites
[0239] 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-methylcyt- idine
penultimate intermediate for 5-methyl dC amidite,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N4-benzoyl-5-methylcytidin-
-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-benzoyl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N--
diisopropylphosphoramnidite (MOE 5-Me-C amidite),
[5'-O-(4,4'-Dimethoxytri-
phenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.6-benzoyladenosin-3'-O-yl]-2-cya-
noethyl-N,N-diisopropylphosphoramidite (MOE A amdite),
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-isobu-
tyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites,
2'-(Dimethylaminooxyeth- oxy) nucleoside amidites,
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-- 5-methyluridine,
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-meth-
yluridine,
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methylu-
ridine,
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-me-
thyluridine, 5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N
dimethylaminooxyethyl]-5-methyluridine,
2'-O-(dimethylaminooxyethyl)-5-me- thyluridine,
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-dimethylaminoethoxy)-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
[0240] Oligonucleotide and Oligonucleoside Synthesis
[0241] 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.
[0242] 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.
[0243] 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-o- ne 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.
[0244] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863.
[0245] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050.
[0246] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
[0247] 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).
[0248] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925.
[0249] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243.
[0250] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0251] 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 amide4 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.
[0252] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and
5,264,564.
[0253] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618.
Example 3
[0254] RNA Synthesis
[0255] 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.
[0256] 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.
[0257] 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.
[0258] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethyl- ene-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.
[0259] 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.
[0260] 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,
43114314; 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).
[0261] 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
[0262] Synthesis of Chimeric Compounds
[0263] 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".
[0264] [2'-O-Me]--[2'-deoxy]--[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0265] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O- -phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spetrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry.
[0266] [2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)]
Chimeric Phosphorothioate Oligonucleotides
[0267] [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.
[0268] [2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy
Phosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0269] [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.
[0270] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065.
Example 5
[0271] Design and Screening of Duplexed Antisense Compounds
Targeting Apolipoprotein B
[0272] 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.
[0273] 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:
2 cgagaggcggacgggaccg Antisense Strand (SEQ ID NO: 3)
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline. gctctccgcctgccctggc
Complement (SEQ ID NO: 4)
[0274] 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:
3 cgagaggcggacgggaccgTT Antisense Strand (SEQ ID NO: 5)
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline. TTgctctccgcctgccctggc
Complement (SEQ ID NO: 6)
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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-MEMO.RTM. 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
[0279] Oligonucleotide Isolation
[0280] 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
[0281] Oligonucleotide Synthesis--96 Well Plate Format
[0282] 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.
[0283] 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
[0284] Oligonucleotide Analysis--96-Well Plate Format
[0285] 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
[0286] Cell Culture and Oligonucleotide Treatment
[0287] 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.
[0288] HepG2 Cells:
[0289] 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.
[0290] T-24 Cells:
[0291] 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.
[0292] A549 Cells:
[0293] 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.
[0294] NHDF Cells:
[0295] 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.
[0296] HEK Cells:
[0297] 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.
[0298] Treatment with Antisense Compounds:
[0299] 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.
[0300] 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
[0301] Analysis of Oligonucleotide Inhibition of Apolipoprotein B
Expression
[0302] 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.
[0303] 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.
[0304] 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
[0305] Poly(A)+ mRNA Isolation
[0306] 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.
[0307] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
[0308] Total RNA Isolation
[0309] 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.
[0310] 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
[0311] Real-Time Quantitative PCR Analysis of Apolipoprotein B mRNA
Levels
[0312] 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.
[0313] 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.
[0314] 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).
[0315] 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.
[0316] 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.
[0317] 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:
[0318] forward primer: TGCTAAAGGCACATATGGCCT (SEQ ID NO: 11)
[0319] 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.
[0320] For human GAPDH the PCR primers were:
[0321] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 14)
[0322] 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.
[0323] 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:
[0324] forward primer: CGTGGGCTCCAGCATTCTA (SEQ ID NO: 18)
[0325] 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:
[0326] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 21)
[0327] 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
[0328] Northern Blot Analysis of Apolipoprotein B mRNA Levels
[0329] 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.
[0330] 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.).
[0331] 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.).
[0332] 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
[0333] Western Blot Analysis of Apolipoprotein B Protein Levels
[0334] 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
[0335] Antisense Inhibition of Apolipoprotein B Expression
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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
[0340] Design of Phenotypic Assays for the use of Apolipoprotein B
Inhibitors
[0341] 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.).
[0342] 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.
[0343] 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.
[0344] 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
[0345] Activity in Animal Models
[0346] 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.
[0347] 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.
[0348] 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.
[0349] 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.
[0350] 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.
[0351] 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.
[0352] 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
[0353] Toxicity studies in mice revealed no severe toxic effects.
In vitro assays showed that ISIS 301012 does not possess
immunostimulatory activity.
[0354] 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.
4TABLE 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 +15 +8 +8 -11 -14 -21
concentration
[0355] 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
[0356] Identification of SNPs
[0357] 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.
[0358] 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.
5TABLE 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%.sup. 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%.sup. Substitution of G to A
27627 2% Substitution of T to C 27618 <1%.sup.
[0359] 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.
6 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%
[0360] 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.
[0361] 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.
[0362] The G/A substitution at position 27627 was found only in
four samples of African American origin.
[0363] The T/C substitution at position 27618 was found in one
African American sample.
Sequence CWU 1
1
23 1 43445 DNA Artificial Sequence Antisense Oligonucleotide 1
accaagacag cgctcaggac tggttctcct cgtggctccc aattcagtcc aggagaagca
60 gagattttgt ccccatggtg ggtcatctga agaaggcacc cctggtcagg
gcaggcttct 120 cagaccctga ggcgctggcc atggccccac tgagacacag
gaagggccgc gccagagcac 180 tgaagacgct tggggaaggg aacccacctg
ggacccagcc cctggtggct gcggctgcat 240 cccaggtggg ccccctcccc
gaggctcttc aaggctcaaa gagaagccag tgtagaaaag 300 caaacaggtc
aggcccggga ggcgcccttt ggaccttttg caatcctggc gctcttgcag 360
cctgggcttc ctataaatgg ggtgcgggcg ccggccgcgc attcccaccg ggacctgcgg
420 ggctgagtgc ccttctcggt tgctgccgct gaggagcccg cccagccagc
cagggccgcg 480 aggccgaggc caggccgcag cccaggagcc gccccaccgc
agctggcgat ggacccgccg 540 aggcccgcgc tgctggcgct gctggcgctg
cctgcgctgc tgctgctgct gctggcgggc 600 gccagggccg gtgagtgcgc
ggccgctctg cgggcgcaga gggagcggga gggagccggc 660 ggcacgaggt
tggccggggc agcctgggcc taggccagag ggagggcagc cacagggtcc 720
agggcgagtg gggggattgg accagctggc ggcccctgca ggctcaggat ggggggcgcg
780 ggatggaggg gctgaggagg gggtctccgg agcctgcctc cctcctgaaa
ggtgaaacct 840 gtgccggtgg tccccctgtc gggccctagc acccgctggg
aagacgtggg aagctcacag 900 atttctttct cctgtcttac agaagaggaa
atgctggaaa atgtcagcct ggtctgtcca 960 agtaaggcat ctgcgcatgg
ggcgtggaag ggcgcccagc cccgtgcact ctcctacacc 1020 cgggtccctg
agggcctccc actctacagg gctgagatgg catcgtggtg tgccttgctc 1080
tgaccccagg aagcaagttc cctgagcctc tgcccacacc caagggatgc caactctctt
1140 ctacctggcc ttctgttctg tcccaaaagt tcagcctggg ggcgggggag
ggaagggatt 1200 gtctctccgc tggcctgtgc acactttgaa gaaacatcac
tgtcctgttt atcagtgact 1260 agtcattgat tcgaagcatg tgagggtgag
gaaatactga ctttaacctt tgtgaagaaa 1320 tcgaacctcc acccccttcc
tatttacctg acccctgggg gttaaaggaa ctggcctcca 1380 agcgcgaccc
tgtgtgctgg agccgcgggg cggacttctg atggggcagc accgccatct 1440
agtggccgtc tgtcatcact gcagctggac tcaggaccca gatgttcttt ttcttcaatt
1500 gttcagaaaa ttcctctcaa ctacagtgga aacctccaga aattcttttc
taggagtttg 1560 ttaagttagt tacgcttaat gcttaatgaa ctttgcctta
agtatttggt agtcttagag 1620 tcacggaatt acggcgtgtt caagctaaaa
aagcattaga gatagtacta tttgcgtaat 1680 gttgtcatct cttaatttgc
cagagggtct ctcatgcaga ttttctgagc cccattactt 1740 gacacttgtc
actcccttcc ctgtgcctca gatgagatat tcaagacatg ccagccaatt 1800
taaacattag cctcagcaaa aacataatgg agaagtcaaa tctataaagg aaaattaagt
1860 ataaagtcaa ttaaaaaata atttgagttg aattaccatt tttaattctc
tatgccactg 1920 cccctctctg cccagaattg gctgtccttg ggagagctat
ttctgctatg tggctgacgt 1980 atttctcccc acgttagaag atgcgacccg
attcaagcac ctccggaagt acacatacaa 2040 ctatgaggct gagagttcca
gtggagtccc tgggactgct gattcaagaa gtgccaccag 2100 gatcaactgc
aaggtatgga ggatgcaggc aggagggacc tagagcccac agctttcccc 2160
cagccctgtt ccagcgggcg cccaacacgc gaccttcccg gagggtgtgt actgagcaaa
2220 cgcagaacat cccagaactg ttgtaatctg atcaaagcac tgggactttg
cctctgtttg 2280 taagtcagcc acattgctga gatgtggtct gcccccacca
aatttcgcaa gtcagaagta 2340 ttttcccgtt aacttcccag atgcaatagg
aatccatgat ctagattagc agcagtgtgg 2400 gtctgtagat ttcagcgtga
gagaggccca gtaggtgagc tatgggaggc aggcaactcg 2460 gaatcgcact
gtgaaatgca gtttttataa tttaagtcaa acagaatctg ttgctgaaaa 2520
atgaatggaa agaagaaaaa aatataaaca tacagtttgt tctaaaataa aactttgctt
2580 attattgaga ctggttgtac tcatgttaca tacatgtgga gcagatctac
aggctgctat 2640 tggggtttgg gtggggaaga gaagtcaagc tgagcagtca
ccttttttta gagagtaccg 2700 tagctcttgt atgtgctgtc caatatggta
gacatgagcc acattgggct atttaaatgg 2760 aatgaaatta aaaattcata
ttcgttgtca cattagctgc atttcaactg ctcaacagcc 2820 accctggcta
ctggctccca tattgaacag cacacatgta caacatttct ataaagttat 2880
ttgaatagtg ctggataata agtaggaatc cgttgaaact ccagctatat gcaaagctct
2940 aaataggccc taatagatat aaccagtttt ttgggtgaca ttaaggagac
atttgctgtg 3000 gaaacgaagg atggccctct tcctgctttc tgtttttctt
cttcactttc actcctagtc 3060 tgcagcgctt ctatttaacc acagctcttt
ataattaaag tgagtaactt tagaaccaat 3120 aaaaggacat cctccttccc
atgcctaggg gcaaacttaa gaaatgtgtt acccgggagg 3180 gggaaaacgt
cagcaatagg actaagtcta ggttggtgca cagagaaccc aggaggcatg 3240
ttgataaggc atgtggtgtt gaggcgcagg cagtggtgtt cccagcacca ttccctttgg
3300 tgctctgatt agagattaag ccctgggctt caggggccac ctctcattct
tgatagacaa 3360 cctcaatgct ctgctaccct gaattctcag gttgagctgg
aggttcccca gctctgcagc 3420 ttcatcctga agaccagcca gtgcaccctg
aaagaggtgt atggcttcaa ccctgagggc 3480 aaagccttgc tgaagaaaac
caagaactct gaggagtttg ctgcagccat gtccaggtaa 3540 gtcatgttgt
acatgagcac acgcatgtgt gtgtgtccgc tgaggtatga acttgtgtgt 3600
ttgcaccagg cacggatgtg actgtaagta tttgtattcc gtatccatcg tggatcaggg
3660 aattactgag ttttcacaat catcaaaaag agagaagcat tagttaacct
tccctagtta 3720 ggttccttta attatcattt tcatgtgttt ctaaaaatct
catgctttaa acttcttgag 3780 attataaaac tgagatgctt tgtttaaaca
agtgaattct tatttaaaga actagtcaag 3840 actagtgctt ggtggtcttt
ggtgtggggt cccagaggca ctggctgctg tggccggcac 3900 atggcggggc
agggtctgtt caccgcaggg cagaggagca ccaaggcttc ggtggctccc 3960
cctcctaggc tggcattcag ccactgcacg ctgatcggcc actgcagctg catctctgct
4020 gactggtcag ggcccatgtc gcacccattg taaatatttt caacatcacc
cctgcctcat 4080 cctcaatcac agtttgtagg gtcctaggtg tgtatgaata
caggcaggat agagttgtta 4140 acttggtagc atcagaaaac tctgtctgta
ttagtctgtt ttcatgctgc tgataaagac 4200 atacctgaga ctgggcaatt
tacaaaagaa aggtttattg gactcacagt tccacgtggc 4260 tggggaggtc
tcacaatcat ggcggaaggt gagggacagc aagtcacatc ttatgtagat 4320
ggtggctggc aaagagagct tgtgcagaga aactcctgtt tttagaacca tcagatctcc
4380 cgacacccat ctgcaatcac gagaacagca cgggaaagac ctgcccccat
gattcaatca 4440 cctccccccg ggtccctccc acaacacgtg ggaattatga
gagctacgag acgaaatttg 4500 ggtggggacg cagagccaaa ccatatcacc
atccttgccc atttttcagt tttgctaaac 4560 attagattca gatgccagtc
ctttcttgcc aaaataggct gtgaggcttc tttctttcct 4620 atgctttatt
ttctccaaga cttaactgta tatgagggag aggggtatgg tggcaggagg 4680
aaagagtggt ttattttttg gtccttggtc ttctccaaat acagaagaga ctcctgttct
4740 tgaaaaggag ggctttccat gtttgcatct tcatgacttt aactgtcttt
tttaaaaatt 4800 gacatacaat aattatacat atttattgag aacatagtga
tattttgata catgtaatgt 4860 atggtgatca gatcagagta attagcatac
ccatcatctc aaacatttat catttcttcg 4920 tgttgggaac tttctgagag
agtgtaggct gtgggagata agtccgtcac cttttcctcc 4980 tgatgtaacc
agagtggctg cagccaggtc ctcagaaact cagagagtac ccagtgggaa 5040
atccctaaga ccaaagtcag catgggcttc agccatggcc tgacaccata caaaagaatg
5100 actgtccaac aagtgtatga aaataagctc caattcactg gtagtcaaga
aatgcgaatt 5160 aatgtaacaa caagatattt atctgctttt acccatcata
ctgcaaaact ggaaaacagt 5220 gatagcacct gttgctggca ggccagtgag
gaaaagtgtg ctgtcctgag ctgctggtgg 5280 aaacgagagc catcaggcaa
tatctactgt aatttaaaat acttaatacc ctttgacaca 5340 gatattttag
tctttgggac tctagcccat gaaaataaaa gcagtaatgt gtgaagatag 5400
gcacataagg atgtttgttt tggtattgtt tgtgtggttt aaaaaaaatc cagaaagaga
5460 gagggcaaat gccatcaaat ggggcaatgt gtgaataaat tatatttagc
catggaatgg 5520 aatgttctgc atgcagcttt taaaaaaatc tgttagagct
gtaccaagtg actcagaagg 5580 atttttgtga agtataatta agtgagaaaa
acaagataaa agtatgcata atacaatgcc 5640 acttgtataa aacaaacaat
ggcaaaatct ttgtatgact ctgtttgcac tcacccatgt 5700 ttacagagga
ttgtatgagt gtgcagaaac aaatggaaca accactcggg tgtccgtatg 5760
gggaggatgg gcaaagagac tgatatgggt ggagaacaga gcagggctgg atgagccaag
5820 caaaaaaagt taaaacacag ctggacctgg tggctcatgc ctgtagtccc
agcactttgg 5880 gaggccgagg agggagaatc acctgaggtc aggagtttga
gaccagcctg gccaacatgg 5940 tgaaaactgt ctctactaaa aatacaaaaa
ttagctgggt gtgatggcac atgccagtag 6000 tcctagctac tccggaggct
gaggcaggag aatcacttga tcccaggagg tggaggttgc 6060 agtgagctga
ggttgcgcca ttgcactcca gcccgggcga ccgagcgaga ctccatttca 6120
aaaaaagaaa aagaaaaaag aaaaaaagaa aaaaaaagaa tcaccaaaac ttatgtatat
6180 gtgcatactt ttttgaaaat gtatgtctat gtgtagctat attctatatt
tacaaataaa 6240 tgatgtcaga agaacaattg gttaaaaaaa tatgagaaaa
gaaacttcag tgccacccag 6300 cttacttcca gcaagttgta atggagaagg
acatttccgt gaccatcctc tctctgggac 6360 aggtatgagc tcaagctggc
cattccagaa gggaagcagg ttttccttta cccggagaaa 6420 gatgaaccta
cttacatcct gaacatcaag aggggcatca tttctgccct cctggttccc 6480
ccagagacag aagaagccaa gcaagtgttg tttctggtga ggatttagaa agctgatagc
6540 agtggccctt gaaactcatc ttcatgtgtt agagaccagt cctaccatat
acaaagcaga 6600 tcactgagtc agctccatga ctagttacat aggaagccct
ggattggcgt gaaatactgg 6660 tgcccgaggt tcctcctgcc ccttaggctc
actgacagat catcccaagc aggcttatca 6720 ggttgggtct aattttaaaa
cagtcattga ggagtcctgg ccaccccacc cctgcttttg 6780 tttgatgctt
cacctgtgtt tgctgggtta tggtgtacac agtaaatcct gtgtgtattt 6840
taaacaccaa aaataatggg atctgttgct ggtctctttt acgaatttca ggtttcactg
6900 tgagacagaa ttcatttcac ctcagtccca tgagcacttt tgtgtgttct
aatttctcta 6960 cgacaccata atgggagaag acaccgatgc aacctgcgga
ggcctttctg cagacccacc 7020 tttaactggt tttctctctc ccaacttggg
ctggccaggc actagcaaga ccacactctg 7080 cataggaaga aaaagaaagt
ccctcccaaa gctagattcc ttctgctttt tctttcacga 7140 tccccacccc
atccctccca agtacccaag gatgttgccc gtgttgaata catgtggttg 7200
catcttcttc ctccatagga taccgtgtat ggaaactgct ccactcactt taccgtcaag
7260 acgaggaagg gcaatgtggc aacagaaata tccactgaaa gagacctggg
gcagtgtgat 7320 cgcttcaagc ccatccgcac aggcatcagc ccacttgctc
tcatcaaagg catggtaagt 7380 cccatgtcag cactgtcgtg cacagcaagg
agcatcctct tattaataca attccagaac 7440 ttttgagcta gtgggcacct
ttgaggacag cctgccctgg ctgtttttta tacagactag 7500 agataggacc
ctgagcaggc acgggaaggt ctgcccaggc ttcacggcct gggatcagtt 7560
gagccaaggc ttgagtcagg ctcctccctc ccagcccaga gctctgtctt tcctcctgtc
7620 cttctgtcac tggcaccaaa ctgcctctaa tctcatcact tgagagtaat
gactactcac 7680 ctctgagaag gttccgggga tggatgtagg gcagcaaaac
caccttctgt tcttttctgc 7740 acaaggactc cttgtgccag ctccaagcct
ctggcctttg aagaagtccc aagacctgtg 7800 ttctccccct ctccctcatc
ccatgaagtg gagtgactta gagtgctcca gcttcttgtc 7860 cttccacccc
cagtaccacc ctgaccaaac atggccccac tgccaccggc ctggagcacc 7920
ctctcctctc tgttaactgg ggccatggag caccatatta cctgagcctg cctgacccct
7980 gcaacatctt ccctgatatg agccccagcc tgtctcagtg aacatgaata
acttgggcaa 8040 tcactgtcat gctgggcgct gttcctggtc attgtcctta
gggttgaaaa cagggagtct 8100 gatgaccatg agtgccacag tcagaagagg
ataatgcact ggcttagggg tcttttctga 8160 gcatctgctg tttgctcaac
cccactctgg gcagcaccaa ggaagggaca gtggcagatg 8220 aaccatggac
cttcccctca ggatgcttcc agtctaatgc aggagccagg tcaataaagt 8280
atacgtggta tactcaataa ggtgataagc tgaacagtgc agacaagaag tcctgggcct
8340 gaccaggaag gagaaagaat tattcatgta gctcagcggg caacatttca
tggaagatgt 8400 ggagcaggaa cccaaaaaat gcaaagaata tgtaaatgaa
agagacatgt aagaatgggc 8460 ttttgggcaa agaaaagtta ctgagcaggt
gtgtgagggg ctatgtggtg ggatgggcat 8520 gtggaggata caaagtttag
acattgtcca gtgagggtgg aaaaagagga gtctacagct 8580 tgactcagct
ttggggatgc cgacttgttg caccccctgg tctaaatgtc aagtacccag 8640
ttatcttctt tctctgagtt tatctagtgg tacaggactc ctgctccctt ctaccttgaa
8700 ggtaaatgct tttaacagaa gatacaggga ctgatcaaaa tgctcgtctc
caatctcttt 8760 catagacccg ccccttgtca actctgatca gcagcagcca
gtcctgtcag tacacactgg 8820 acgctaagag gaagcatgtg gcagaagcca
tctgcaagga gcaacacctc ttcctgcctt 8880 tctcctacaa gtaggtcatg
tgatgcaccc ctgatttgtc atttaatggg tcagtgtgaa 8940 ctgaacactt
ctcaagtgct ctgttccagg caaacctgtg cctgggaggg aggaatggag 9000
agggataaaa tgccgcccct ccctgtcccc ctttttaagc gaacaggcca tttggcagaa
9060 aagtcctagg catgcaaaac aatccaagac caacaaaaga tatctaagac
ccattcttta 9120 agggctgtag atccagaaaa cctgaggatc actgcagggt
accctggtta gaaaaggttt 9180 catggaagat ttgggatact gactggaaac
ttgtgtatcc aaatccactt tgaaaactga 9240 taatcaatga atatatattg
agtaactgcc atattcttgg ctctatgttg tggaagatac 9300 gaaagaattt
tgagacattg cactagttcc tacctctggc cactccagac tagtggagag 9360
tataaggcac gcatgtcttt ttgatgggag gataactagc gtgaccagga agaggtggat
9420 gttattcatt cagggccaac aatggctgga tttacccatg ctttgaaaga
tgggcaggac 9480 ttgggtagat gcagagacag ggaaaacctt caacatggaa
agaatagtat gttctggcca 9540 tccgtgacat ggtgtgcttc cttggttacc
aggaataagt atgggatggt agcacaagtg 9600 acacagactt tgaaacttga
agacacacca aagatcaaca gccgcttctt tggtgaaggt 9660 aagagtttct
gtccacatag ttgctggaaa atctactcaa gatgtgccta tcatggctta 9720
gccacttgct gagccctgtt aaatgtctgc tgactaacaa gtgatacaga cactggtgtt
9780 ctggctacct ctagtgagaa agcaaactca tttcatgatg tcaagttgca
atggcataaa 9840 ggaaaagaag ttcccaaagc tacttaggca tttgtaaata
gaaaactgga atcctaagtt 9900 taacatgaca tatttgatag aactgacatc
acccatcctg tgataagatc cagagctgtc 9960 ccagacgagg tggaccaagt
gggagagaac cttcagagtc tggccagata gtaacctcag 10020 gagtcagtct
ttagaggtag aaggaactct aacaatctca agtccaaccc ttacccagta 10080
ttgtattgta tttatatctg tccaaattcc ttcttgtaca ttacctcatt gtcctttttg
10140 ctcatagcaa cctgtgatgt caggtggtag agatgtgatt ttatacctat
tctacagagg 10200 agacagtgac acagagaggc ttagagtttg atgtagtcaa
ggccgcagaa tattagaggg 10260 gggaaaataa gtgccaggtt gtaatctaag
ccaggactat tctcattaca ccacatttcc 10320 atgatgactt ttacctctct
tcctggcata ggtcacagta ggtggtggag aggatacaaa 10380 agtgtctccc
ctccccacaa gctgctggta gacccaatta gaagaaatgg tgataagcac 10440
ccatgtgcct ggtcccagtt gtaaccatgt caacagtagc acctcctcac caattatttc
10500 aagctaaggg taacctgatg atagactcag acaagtctgg attccacttt
agctctacct 10560 cttagaccct gagagctctt gggaaaccta agttgctcat
ctctgggtca cacttcctca 10620 tctctgggtc tcatctcttt gtctcatctc
tgggactcag agctgagatc cagggatgag 10680 caatttacat ggcccaaaaa
ctctgtgggt ctcagaagca gggctgaatt tatcattaaa 10740 ttgaacaata
atgccacccc acagggatag gatgatgagt cagtgaaaac aagtcaatca 10800
cctatggcag agccagatct agcaggcatt gaatacagga tagtttcttt cccttttccc
10860 ctgtgctgat actccacaat ttccagcttc cagtagacaa agatatggtt
gagatgaaga 10920 aagctagagt tcctttgaca ctttccatct tccaggtact
aagaagatgg gcctcgcatt 10980 tgagagcacc aaatccacat cacctccaaa
gcaggccgaa gctgttttga agactctcca 11040 ggaactgaaa aaactaacca
tctctgagca aaatatccag agagctaatc tcttcaataa 11100 gctggttact
gagctgagag gcctcagtga tgaagcagtc acatctctct tgccacagct 11160
gattgaggtg tccaggtatc taatggttac agctcaactt tttataaaac tgatggtaac
11220 tgactgaact ttcaaacctt ggccaaatgg agaatctcag ggaccatttg
gatatcaatc 11280 cagttaatca attagtcaat cagttcatga ttgctggata
gagaactatc agctgctgcg 11340 ctgagttcca tgaaacacac acgcgcatac
tgtgttcaag gcagctatgt atttgtgtgt 11400 taaaacagaa ggagaatagt
tcccacattt tgatgggtaa cttttaattc ctaggtctat 11460 tgcaggtgct
ctccagaagc ttataggctg gtggagagag aactcagacg aaaaatataa 11520
tatgatttct ctacccttca aggcactggc tttaagtgct atgaaggtga gagaagggac
11580 tgaggccagg aatgagaccc agctaatgtt ggccaggcat attctgtgtg
ctggccaaag 11640 gactgtgata acagtcttct tgttgctaca gatccacagt
cccctcttgg aacttttctc 11700 gattgggctt cttctgtggg taatattcct
aaggaaagca tcatggttct gagctccaag 11760 ttgggttttg aagttagatt
tgaatagtga atgaggtgat taagggctct cctggcagag 11820 gacacaccat
gagcaatatt ttatgtgccc tgaaggtggt ctgtataact ttatccatgt 11880
ctttcttctc agccccatca ctttacaagc cttggttcag tgtggacagc ctcagtgctc
11940 cactcacatc ctccagtggc tgaaacgtgt gcatgccaac ccccttctga
tagatgtggt 12000 cacctacctg gtggccctga tccccgagcc ctcagcacag
cagctgcgag agatcttcaa 12060 catggcgagg gatcagcgca gccgagccac
cttgtatgcg ctgagccacg cggtcaacaa 12120 gtgagtttcc acactgtatt
tctcctccta ggagcagagg aacatcttgc acctctgtgc 12180 atctctgtat
taaaactgaa cccctccttc cactttcaaa ctctgctcct tactcttgtg 12240
ttttttcttg atcatttttg gggtaatgac ttgaaataag aaatcagcaa acacaaattg
12300 aatttttaaa aatattttct ctacattata ttataaaagt ttttgaacat
agcaaagttg 12360 acagaatttc acagggaaaa cccctagaaa accagctatc
tcctactatt taagtgttat 12420 tatatttgct ttatcacata tacatccatc
cattaattca tcttattttc tgaagcattt 12480 caaagtaaat tgcaaacatc
aacacacttt cccctaagta ttacagcttg catattatta 12540 acttcagttc
aatattagtt agcagttttt tcctctgaat ttttttgttt gtttgttttg 12600
tttttttttg ttgttgttgt ttttttgaga tggtctcact gtgtcaccca ggctggagtg
12660 cagtgatgca gtcacggctc actgaagcct caaattcctg ggctgaagtg
atcctcccac 12720 ctcagcctcc tgagtagctg ggaccacagg tgcatgctac
catgccctgg ctaatttttg 12780 tattcttggt agatacaggg tttcaccatg
ttgctcaggc tagcaggttt ttcctttgat 12840 gaaatttttt ggctttttct
tttttacatt tttatataaa tttatgtgga acaagtgtaa 12900 ttttgttaca
tgaatagatt gtgcagtagt taagtcaggg ctttcagggt atccatcacc 12960
cagacaacat atagtgtacc cactaagtaa tttctcacca tccatctccc tccacttcca
13020 caccttctga gtctcaattg tctatcattc cacacactat gtccttgtgt
gcacattatt 13080 tcactcccac ttataaatga caacacgcaa tatttgtctt
tctgtgactg tcctgtttca 13140 cttaagacaa tgacctccag ttccatccat
gttgctgcaa atgacatgat tttattcttt 13200 ttatggccga atagtatttt
attgcctata catttcacat ttttaatcca atcgtccatt 13260 gatagacact
taggttgatt ccatgtcttt gctattgtga atagtgctgt gataaacata 13320
tgggtgcagg tttcctttgg atataatgat ttcttttcct ttaggtatat acccagtaat
13380 gggattgttg gatttattgg tagttctatt tttagttctt tgagaaatct
ctgtattgtt 13440 ttccatagtg gttgtactta tttacaatcc catcaacagt
gattaactgt ttccttttct 13500 ctgtatcctc accaacaact gttatttttt
gtcttttgaa taatggccct cctgactctt 13560 gtaagatgtt atctcattgt
ggttttaatt tacatttctc taatgattag taatgttatg 13620 cattttttca
tatgcctatt gccatttgta tgtcttcttt tgaaaaaaat gtctattcat 13680
gtcctttgcc tactttttaa tgggattatt tgggggattt ttttgttgag ttgtttgaat
13740 tgcttgtaca ttccggatat tagtacccca ttggatgaat agtttgcaaa
tattttctcc 13800 cattctgcag gttaccaccc tgttgattat ttgttttact
gtgcagaaac tttttacttt 13860 aattaagttc tatttgtcta ttttttgttt
ttgttgtctt tgcctttgag gtcttattca 13920 cgaattcttt gtctaggcca
atgtccagag aagttttccc taggttttct tcttgcattt 13980 ttatagtctc
aggtcttata tttaagtctt tgatccatct tgagttgatt tttttatatg 14040
gtgacagata ggagtccagt tttattcttc tgcatatggc aatccatctt tcccagcacc
14100 acttattgaa aagggtgtcc tttccctagt gtatgttttt gtcaattttg
tcaaagatcc 14160 gttgactgta agtatgtgac tttatttctg ggttcagtat
tctgttccat tgatctatgt 14220 gtctattttt atgccagtac catgctgttt
agattactat agccttgttg tataatctga 14280 agtcaggtaa tgtgatgcct
ccagctatgt tctttttgct taaaattgct tcagctattc 14340 aggctctttt
tggattccat atgaatttta taattatttt ttctaattca caagtttggg 14400
ttttaagaca aacctaactg gggttaccaa gtcctgactc tcttctctta ttctgtagct
14460 atcataagac aaaccctaca gggacccagg agctgctgga cattgctaat
tacctgatgg 14520 aacagattca agatgactgc actggggatg aagattacac
ctatttgatt ctgcgggtaa 14580 tctcagtctt ttatatgaca tacatcattt
cagaagcact tttcctggac accttttact 14640 tccctctcct gcaccctgat
gggttcttgt ttcttttctt caatgcaggt cattggaaat 14700 atgggccaaa
ccatggagca gttaactcca gaactcaagt cttcaatcct gaaatgtgtc 14760
caaagtacaa agccatcact gatgatccag aaagctgcca tccaggctct gcggaaaatg
14820 gagcctaaag acaaggtaaa gtccacaaga agaggtctga aagtgaaagt
ttattaacaa 14880 ggatttggaa ggtactaggg gaatgagact ctagatttca
tctactgact ttattctgct 14940 gtttctttcc tttccttcct tccttccttc
cttccttcct ccctccctcc ctttcttctt 15000 tccttccttc
cttccttctt tcgagatgga atctcactct attgcccagg ctggagtgca 15060
gtggcatgat ctcggctcac tgcaacttct gcctcctggg ttcaagcaat tctctctgcc
15120 tcagcctcct gagtaactgg gattacaggc atgtgccatt acacccagct
aatttttgta 15180 ttttttagta gagatggagt tttgccatgt tggccaggct
ggtcttgagc tcctgacctc 15240 aggtgatccg cctgcctcag ccttgcaaag
tgctgggatt acaggcgtga gccactgcac 15300 ctggcctcta ctgttttcta
attgcaaatt tcaacaagcc tattgacttg actgcctagc 15360 agtatgtgac
gtgagagaaa tacttgactt tgctgctatg tcaacatgca gaacgtgaga 15420
tgtttttgct tcctaccgtc cacctaccag attgaccatc cctctcatca tggaaaaaca
15480 tgcttaattt tcccccaata agcttaggct aggatagcca acttggcccc
ctcttaggtg 15540 caaagactcc agaactttgg aaactaccct atttattagc
cccaaactct tactacccct 15600 tctcatcttt atcctcacat taaaataact
tacgttaaaa caacttgatt ttcacttagt 15660 ggtggatctc caaacaaatc
acaacttggc cataatttat gtgttttaat ggaattgaat 15720 tcaacaggca
ttccacaggc tttttctggg aacccttact tgatagtgct ctaggaaaca 15780
ctggcaagaa gattcaatac cagcatttga agaacgatta cagagaaatt agacctgtgc
15840 ttaagaaaga gctagcagac aatgccagtg tttgccaggc atgttctgtg
ttctgaccac 15900 aggacagtga taaccatctc ctcttttgac tgcaggacca
ggaggttctt cttcagactt 15960 tccttgatga tgcttctccg ggagataagc
gactggctgc ctatcttatg ttgatgagga 16020 gtccttcaca ggcagatatt
aacaaaattg tccaaattct accatgggaa cagaatgagc 16080 aagtgaagaa
ctttgtggct tcccatattg ccaatatctt gaactcagaa gaattggata 16140
tccaagagta agtaagagct attcacccca tataccactg agggccctga gctggaattc
16200 caaccctagg ttttggcata gccactgtct gcccttgctt ctgaaacaaa
cacttgtgca 16260 aatgtgtagc agatctagac ccaaagactt agggtcaatg
aaatcaagac attttggtag 16320 tgattggaaa tccatattta cttggggtgc
aagagtcaaa ggataataac atggtgtgtc 16380 agctcaaaat atacttcttc
ttatctagtc tgaaaaagtt agtgaaagaa gctctgaaag 16440 aatctcaact
tccaactgtc atggacttca gaaaattctc tcggaactat caactctaca 16500
aatctgtttc tcttccatca cttgacccag cctcagccaa aatagaaggg aatcttatat
16560 ttgatccaaa taactacctt cctaaagaaa gcatgctgaa aactaccctc
actgcctttg 16620 gatttgcttc agctgacctc atcgaggtaa gtgtgaagag
tttgaggttc tctagcccat 16680 tttgtacagc atcataaaca gagagtccct
gggagccagg agctacccag aggaaaacta 16740 agaaccacca ggcacttcct
accatgattc tgaggctttc ttctttccct ccttccccgc 16800 cttcctctct
ccccgctagg ggtcacctga agcatgactt cttaacatta atagaaatgc 16860
aggcctggcg aggtggctca ctcctgtaat cccagcactt tgggaggccg aggcgggtgg
16920 atcatgaggt caggatatcg acaccatcct ggctaacacg gtgaaagccc
atctctacta 16980 aaaatacaaa aaattagccg ggcgtggtgg caggcacctg
tagtcccagc tacttgggag 17040 gatgaggcag gagaatggcg tgaacccagg
aggctgagct tgcagtgagc cgagagattg 17100 cgccactgcg ctccagcctg
ggcgacagag caagactcca tctcaaaaaa aaaaaaaaaa 17160 aaaaaaattg
aaatgcaaat gtctcgtctt taagtcccaa agccaaggaa gcatatgtgc 17220
tgcctagtca gatctgcttc aaatctcaaa tcactcccaa ctctgaatcc tttgttgaat
17280 tatttgtcct atctgaacct tagctgcctc ttctagaaaa aagcaagtaa
taaggtcaag 17340 attctagtga gattttaata aagcagctcc tgtgaaatgc
taaggtcagc tcctggcctg 17400 tggtattcaa atacttgttt agataaatgg
acatcaagag tggggactac taggctggca 17460 tacaacaaag aaacctgatg
ccattttctt gtctgatttt ctttctcaga ttggcttgga 17520 aggaaaaggc
tttgagccaa cattggaagc tctttttggg aagcaaggat ttttcccaga 17580
cagtgtcaac aaagctttgt actgggttaa tggtcaagtt cctgatggtg tctctaaggt
17640 cttagtggac cactttggct ataccaaaga tgataaacat gagcaggtgt
gtatttgtga 17700 agtatcttct taaggaaagc tttgggtctc aatgcaaaaa
caattctttt ctaagcatgg 17760 aagtcctcaa aatactatct aactgaaggg
ataactatgg tttttatcaa ccagacctgc 17820 tggggtaagg gccagtatcc
tctgcagtta aagatctcct gaattcagtg tgcccagaaa 17880 ccagactcac
aataagtact ctaggataac aagagtatga actctgggct gggtgtggtg 17940
gttcatgcct gtaatcccag cactttggga ggccaaggtg ggcagatcac aaggtcagga
18000 atttgagacc agcctggcca acatactgaa accccgtctc tactaaaaat
acaaaaaaac 18060 tagctgggca tggtagtggg tgcctgtaat cctagctact
cgggaggctg agacaggaga 18120 attgcttgaa cccgggaggt ggaggttgca
gtgagccgag atcacgccgt tacactccag 18180 cccgggtgac agtgtgagac
tgtatcttaa aaaaaaaaaa agtatgaact ctgggcatag 18240 atttaattct
aacttccctg tcttgaagct gtgcgcactt ggggaagttg gttgatatta 18300
tgtgtatctg tttctgtctg tatcccagac tactaataac agtccaaacc tcacaaggtt
18360 atttaaagac aatgaaataa ggcatctaaa atgccaagca cagtgcctga
tgctggcatt 18420 ggttgttcaa taagcagaca ctattacgag ttctaaatta
atattttcat tattattaac 18480 tgctgtcttt ggctctcact cccatcagtg
cactagcaaa tgagaccaaa cttccacttt 18540 gaagctagca atgagccccc
atttaaggag ggaaataggt tgtatgatct ggagcttatt 18600 cttgaatttt
ttgctaccca aagtgtggtc tggtcagaaa tacagcttct catgcttcac 18660
ccacaatcta ctgaatcaga agcgcatttt agcaagacct catgtgactt gtatgcacat
18720 tcaactttgc agagcaaggc agtaatttac ccctccaggc tcactgttga
gcacgagctc 18780 catcttctaa tttcctgacc cccacttgag gccgaggatc
tttgatctgc tttgagtctg 18840 tcagtttcac attttttttt tcccaatgcc
tgggcatcca tctctgagat tcttcttctc 18900 tctgagaaga acttgtctag
gatcaagtgt ttttcaaact tctggtgaat ttatataaca 18960 gctacatttt
cttaagaaac accttgtagt cttcactggt caaagaagag aaggctaagc 19020
agggaacggg tgggggatag aggatcttct aatcttgagg atcctggcat actggagaat
19080 agggacccct cctctcatcc caccacatct tactatgtct acagattttt
taattaagaa 19140 tagctttagg agtgccacta tccctgacaa gaccttagtt
ctttaatctc tgcttagagg 19200 aattagcctg gacttcagtg tctccctgtt
cctcacctgg agcatttttt aggcccatcc 19260 tggctgcatc agacaggtcc
cacattggga actgaaaggt gtttgacatt gctgacatct 19320 cactggccat
tttattacta aactctcagg atatggtaaa tggaataatg ctcagtgttg 19380
agaagctgat taaagatttg aaatccaaag aagtcccgga agccagagcc tacctccgca
19440 tcttgggaga ggagcttggt tttgccagtc tccatgacct ccagctcctg
ggaaagctgc 19500 ttctgatggg tgcccgcact ctgcagggga tcccccagat
ggtaagtcag caggccccac 19560 tgggggccca tgagaccaga cgttggtttt
tttttagatc gcccagactc ccttacgatc 19620 ccagctgcac aagcccgaaa
agatgcttgt actttcttca gagatggagg tttgccttga 19680 atttcactga
agatgactct tggatcacat ggaaatgtta acatttagaa attaagctat 19740
tcataatgtt agctgtattt ttaagagcat taatttattc atctggaaaa caatgttcgg
19800 tataccttcc tctacctttg ctgaaggtcc ttttattttt atttttattt
ttttaatttt 19860 ttgagatgga gtcttgctcc caggctggag tgcagtgata
caatctcggc tcactgcaac 19920 tctgccttcc gggttcaagc aattctcctg
cctcagcctc ccaagtagct gggactgtgg 19980 acgtgcacca gcatgcccgg
ctaatttgtg tatctttagt agagacaagc ctgttgacaa 20040 ccatgtcagg
ctggtttcga actcctgacc tcaagtgatc ctccagcctg ggcctcccac 20100
agtgctggaa taacaggtgt gagccactgc acctgacctg aaggtccttt taagattgaa
20160 atgatacaat gattataaaa gaaagtattt ggcaaactat aattcactat
ctaaatatgc 20220 tataattttt attattaatt cataaaagga aatatataaa
tgtactccta tggcttgatt 20280 aaaaaaatgt tgactttaag aaaacaggtc
tcaagctatt ttattgaaat attatttaaa 20340 aaataaaacc caatgcaaat
tgatatgtac atcatctcaa taggcctttg gtttcaaaaa 20400 attgatttta
tcataatata atacatttca agtacacctt cacttacagt cagactccag 20460
aacaccagaa ttaagccatg gcatatatga tacttaaagt ccataaagct ctgaggccca
20520 gcaatattct taagagcctt ctgagtccac ttgaaaatga catgatatct
atctagtgaa 20580 atttcttata tcctgattca ctgaaaacgg taaaaacatc
agtttgatct ttatttatca 20640 aactattcag ctcatcaaaa tatgctagtc
cttcctttcc agataaagag gaattactct 20700 ccaatgtatg ggaggttgta
attaacaaaa ccgactttaa aaagacttac ttttatttgc 20760 tctcccttgt
tgggtctaca gattggagag gtcatcagga agggctcaaa gaatgacttt 20820
tttcttcact acatcttcat ggagaatgcc tttgaactcc ccactggagc tggattacag
20880 ttgcaaatat cttcatctgg agtcattgct cccggagcca aggctggagt
aaaactggaa 20940 gtagccaacg taagattctg tttgcctttt gatttcttag
gttattactt tcttccaggg 21000 tgcatttctt gttaaaacat atttaaaaat
gtgtttccac ttcaagacaa aatgcttcat 21060 cattgtaatc acctcattat
ttttttatga aaaacttcaa gcttccacca gaatgcacta 21120 cctcactagc
tccagtagtg gtatggccat aagacaagaa ctcagttctc tcaacaaatg 21180
agtattccta tcatcttttt aatctggttt tgcctcacgt taactcaggt gctttctagt
21240 tctgggtagt atactccaac tctagagaac tgagaactcg ctttccttct
tccaaacaaa 21300 tcccagtaat gtttccaaag gtctgagtta tccaggaaat
ctttgcccgg aggtgagaaa 21360 gggtggttga tctgactgac aggggactga
agtatttaat gaatctgaat aggttgtttt 21420 ctgacttata gatgcaggct
gaactggtgg caaaaccctc cgtgtctgtg gagtttgtga 21480 caaatatggg
catcatcatt ccggacttcg ctaggagtgg ggtccagatg aacaccaact 21540
tcttccacga gtcgggtctg gaggctcatg ttgccctaaa agctgggaag ctgaagttta
21600 tcattccttc cccaaagaga ccagtcaagc tgctcagtgg agggtaattc
tttcagccaa 21660 gtctgcctag ccagtttgaa agagagaaca gagaatgtac
ctgcagaatt ttgccaggct 21720 aaacagttga ttgagatcat tcaggtcctg
aggaagcagg agaggagtag aaaggaaaga 21780 ttccgggtta cctattttaa
ttctagccta gacttactac ataactacat aattaccttt 21840 cttctacttt
tcacatttta ctaaactgtc ctttatcttt ctgctttgag acttattaag 21900
acctactgct taattagttt ttattaagtt gtgatttttt gttatctatt tgttttgaga
21960 atgaagaaac aatagctctg gagagatcat ctttggaaaa ttaatatttt
cccccccaaa 22020 aaatacctaa gaacatattg atttgaggta gctaggtagg
taaagcatga aactcctaac 22080 ctcgtgataa tggaatacag cctcttttgg
agagttccat tttaagtggc accctcaacc 22140 attgatttgc cttagttttc
atattttaga cacattcatg tgttcattca aaaataatat 22200 ttaattggcc
agccacggtg gttcatgcct gtaatcctag cactttggga gccccaggtg 22260
gatggatcgc ttgagccctg gtgtttggat accagcctgg gcaacatggc aaaaccccat
22320 ctctacaaaa aaaattaaat aaataacaaa attagccagt cgtggtggca
catgcctgta 22380 gctccagcta ctcagaaggc tgagatggga ggatcaactg
agcccaagag ttcaagcctt 22440 cagtgaacca tgcttgcacc actgcactcc
agcctgggag acagagcaag atcctgtctc 22500 acaaaaaaca aaaaatagta
tatttaattg cctaatatat accacgtatg ttgagtgaga 22560 cacacaaggt
ccctgacctt tgaacgctta cattttataa gggagacaca caattaagca 22620
agcagtaatc atagagtaag ggctaagtta tagaaagtat tagagtacca tgaaatttta
22680 tatcatgtag cctgtgctag tcagggaatg cattctgaag caagtgtact
tgacctgata 22740 actgaggact gtgtcagagt catttaggca aaggagaaag
gagtgagtgt tccaggcaaa 22800 aggaaaagca tgtaatggcc tgaaggtaaa
ggaatatggt tcaaggaact ggaagaagtg 22860 cagaatggta aggggctcag
agatgatggg gagaggtagg caggggagag agcatgccca 22920 gctgcgaaag
ccatcctaag gagtttggac tcttttgaag gcacaggagt tgaaaagggg 22980
agcagaaata agataggggt gatgttttag aagaaatact ctgactctag tgtggaagat
23040 gggtgagaag gaggcacagc tggacacgaa gagaccattg gacatctctt
acgatcctat 23100 gtggctaaga gctgataatg gcctgcagtg gagaaaagcc
aggtatagaa aggagtgagc 23160 agattctaca actttctaag aggcagaatc
ataagtactg ggtgattaac tgggtatggg 23220 gacaaggcaa aagaaagaag
aaaagaggaa ggaggcgccc ttcattttaa taagaactac 23280 agtgggagag
cttctggttt caaggaaagt gacaaattca gttttggatg tgctgtattt 23340
gatgtcctcc tatgaaacaa ccagtttaga aatctagctg tcaaatagac ctatggatct
23400 gagcccagta aagaggcttg ggctccacat atggatttgg gaatcattag
tatacagagg 23460 ttgttgtggt taaacagcaa ctggtataga gtgagacatg
agagatgagg acagaaatat 23520 ggagaagaca aacatataaa ggaagaaggg
gaataaccag caatgagtta gaagaagtga 23580 ccagagaagc agaaggagaa
ccaaagccat aaaaggtcac agaagccaaa gagcagccac 23640 aggggagatc
accccatggg taggcgaaag ctggcattag gactccagca catcagcaaa 23700
gcttggtctt gtggcacccc caacttggag aaacaatact tggaggaaaa tgtgctattt
23760 caaagaaagc atccttagaa aaaaccaggc caatgttgaa ctttcttaca
tgtactaagt 23820 ttttaagtac acacttggaa ggaaggtgcc atcatctctt
cagatgtgag aggctccagc 23880 gtcttagtct ggtcatgagt gcgcaactct
atggaaggct tctgggaggt caaggaagat 23940 gaaacctaaa tatgcccatt
ggatgtagga gcaaggaggg cattagagac attgatgaaa 24000 gcattttcag
gagatggagt gagcagtcag agcacattgg gaggaagtag agactgcaaa 24060
ggcagacaac tcttgatggt gaggaagatg agaaagcaag aaaagaaaga aaggagcata
24120 ggggaggggc acaggggaag agacttgagc gtgcttaatg caggtggaag
gaagcaggta 24180 gagagtagga gatttcatat gaaagagaca gtttctcttg
ccctgcattg taggaaggaa 24240 ggggcacact gaagttcagc cccagtgatc
agctatttaa catctctgag cctctgcttc 24300 tgtaaaatga gaaccataag
cctactgttg tggggattac aggtaacaga tggaaagaac 24360 tcagccagaa
gcttcagagt cactctcatg gcttgtcatg ttgatgttct ttctaatatt 24420
atttgtttct cagtaaatta aatagttaga gataggtgtg gactgaggga agacaggagg
24480 ataagggggt atttgcaccc tgagaatttg tgatgtccat tttgattcat
gacttggcaa 24540 taactcaggt atttttgttc ttcaccagca acacattaca
tttggtctct accaccaaaa 24600 cggaggtgat cccacctctc attgagaaca
ggcagtcctg gtcagtttgc aagcaagtct 24660 ttcctggcct gaattactgc
acctcaggcg cttactccaa cgccagctcc acagactccg 24720 cctcctacta
tccgctgacc ggggacacca ggttagagat gctcagtgcc tgacccagca 24780
ttttctcacc ttccacatca tggccaccta gcatggcaca ggaaaaaata ctctgtgttg
24840 taagaccctg tcactagcct tctgggtttg caccatcttt gggtatttaa
agcagggtcc 24900 tctggccaac acattgggtg tcaccttttg cttccttgtg
catgggatgg gatcacagca 24960 cagatcccaa tttgctccta attcagtgtc
catgtttctg agcctccaga cccatcgcta 25020 tgagcttcct ggagcccacc
aatgtgcttg aagccttcac cgtacttagg tggctccctg 25080 tcttcagccc
ccaagttcca gtgcttgttc tcagctttgc tgaaacaacc agccaactcc 25140
tgctctgctt gtccaaagtc ttgggaatcc tggtgtctgc ccttgccttg ggttcttgta
25200 ggactgaggg atcaaaaaga tcatcttagt taagggcaag agacaatgtt
aaaataagga 25260 ccatattttt gttgcatttg aggctgaatt gttttgggaa
cataatcacc atccttgaaa 25320 gctctaacat tatgcactgt cttcattgta
atgtctttag attagagctg gaactgaggc 25380 ctacaggaga gattgagcag
tattctgtca gcgcaaccta tgagctccag agagaggaca 25440 gagccttggt
ggataccctg aagtttgtaa ctcaagcaga aggtgagtat tcaaaacaca 25500
gctgcctcat ctctgctcgc agtctcaggt tcagaattca tgaggagaag acatgtaatt
25560 taacctattt aacaaatagg ttaactgagt acccactaag cggcaggcct
attctaagac 25620 ctgggttaac tgagtaccca ataagcggca ggcctattct
aagacctggg gctagaacag 25680 tgaacaatgg agtctctgcc ttcatggaag
ttacagtgaa caaccaaaca agttaatatt 25740 tggaatatca gataagtact
gaggaggaaa acagagcgta gactggtcta tggagggcta 25800 ggagtaggag
ggaggaagaa gggcagggaa agcagtgcat ttggaataat aagggaaagt 25860
ctccctggta aagtgagcat aaggagacct atcagaaata agaggagaag ccgtgtggta
25920 agactgttaa caggcagagg gaccagcaag tgcaaaggcc ctgaggctga
cacactacta 25980 ccatgtttca aggaaaggaa ggaagacagt atggctggag
cagaaagacc agggagaaaa 26040 gaggtagaag atgaggacag agagatatgg
agaggtgaag gaaggataat ctcataggcc 26100 atggtaagaa ctttggcttt
ttctatgaat taaacgaaag ccattgggga gtcctcatga 26160 tttgatttat
gtttatgttg agaaaagact atgggcagac aagggcagag aaactaatat 26220
gtaggttatc acaataatcc aggcaggaat cagtgttgtt ttggatcagg gcaatggcag
26280 aagagatatg agaaggggat ggattctggc catattttga agattaggct
gacaagattt 26340 gctgatacag tggatgttga gtgtaagagg aaaaggggaa
tgaagacaaa cctaaggttt 26400 ttggcccggg caactgaaaa atggaacttc
catttattga gatggaaagg gctactggag 26460 gagcaggttt tagggaatgg
gagaaattta ggtgttcact ttggaaaaaa aattatatag 26520 ggatagcgag
gagcaggttt tagggaatgg ggcacattta ggtgttcact ttggaaaaat 26580
ttttatatag ggatagcata tcacagaatt aaactaggaa gaaaatccca tgatagaaag
26640 cactggagga gcagggcacg ctggggaaat agtgtttggt aaacattgtt
ttacgaagga 26700 tataaaatgg accagcctat ggattgaagg acgcccggga
atcttgttac aaagaaaggg 26760 ggagttgggg agatggagcc cagggcaagg
gcagcaagga accaggacag gcatcttggg 26820 tagaaagtaa tatagagatg
tcgtgtcttc ctggcccaga agggctgcga gcctttgctg 26880 ttccacaaac
aagctaagtg ctccccattt cagggccttt gcattcctga ccttctgcct 26940
ggaatgtgct cctcccagaa ctcagcgtgg ctccaacctc ttttcattct ggtctctgcc
27000 cacatgtgcc cttatcagag agaatttctc tgaccaccaa gtatgaaata
acacttcttc 27060 tatccctttc ttttatcctt gtatccagtt ttactcttct
tcataacatt cattaccatc 27120 tgacatgagc aagttacttg tttattgcct
gtacacctcc cccactagaa ggtaagcccc 27180 atgaaagcaa ggattcccca
gtaccaagag cagtgcccag cacacaatag gctcataaca 27240 ggcaatccat
aaagacttgc atacatgaac acaactgagt ttaaaattat cagtaaatga 27300
gacccattaa aaaattttaa tgagaaaaaa aaaattcagt aaaatcctga actgtgtttt
27360 tgtttaagca cattgattcc ttggagtttc tctacctttt cctctctttc
cttccaaaac 27420 atagcttctt tatttattta tttatttatt tgtttgttta
tttatttatt tatttattta 27480 tttatttttt gagatggagt ctcgctcttt
tgcccaggct gcagtgcagt ggtgccatct 27540 cggctcactg caagccccgc
ctcccgggtt catgccattc tcctgcctca gcctcctgag 27600 tagctgggac
tacaggcacc caccaacgcg cccggctaat tttttgtatt tttagtagag 27660
acggggtttc accatgttag ccagaatggt cttgatctcc tgacctcatg atctgcccgc
27720 cttggcctcc caaagtgctg ggattacagg tgtgagccac cgcacccggc
ccaaaacata 27780 gcttcttacc acacatctct tgattctctt atacactcgt
ccaggtgcga agcagactga 27840 ggctaccatg acattcaaat ataatcggca
gagtatgacc ttgtccagtg aagtccaaat 27900 tccggatttt gatgttgacc
tcggaacaat cctcagagtt aatgatgaat ctactgaggg 27960 caaaacgtct
tacagactca ccctggacat tcagaacaag aaaattactg aggtcgccct 28020
catgggccac ctaaggtaaa gaaggccgag ggtcatctga cctgcactgc aggcctgggt
28080 ggttcttttc attattcctc ttccacttca tacctgacca agccatgttc
tcccctagtc 28140 tacaatcaga gtggcagaga gagccctcaa caattttttt
tttttttgag atggagtctc 28200 actctgtcac caggctggag tgcagtggca
caatctcggc tcactgcaac ctccgcctcc 28260 cgagttcaag tgattctcct
gcttaagcct cccaaggagc tggaactata ggtgcatgcc 28320 accacaccca
gctaattttt atatttttag tagagacagg gtttcaccat attgaccagg 28380
atggtctcga tctcctgacc tcgtgatcca cctgccttgg cctcccaaag tgctgggatt
28440 acaggtgtaa gccactgcac ccggccaagc tctcaacatt ttaaccctct
gcgcatgtcc 28500 agttggattt tcctaccatt tatcaggcac ttactattca
tgtatcaagc acagtgctgg 28560 gtgctttaaa gaaattatct cggtcctcac
aataaactgc gaggtcactg tgagttttcc 28620 tgtttcatgg ataaggaaat
ggtagctcag aggggttaaa tcatttggtc aaaatcacag 28680 agctagtaaa
tagcagagca ggattcaaac agttttcaaa aaacttctct ttctcctaaa 28740
cctgtttgca aagtccttaa tttgtgctga atgttggctt tagaagttga tgagtttgat
28800 ctgtggctgt ttctctgaac catccttgta tctggttttg atcaccacaa
atggaacttc 28860 tgtttaatcc tgcatatctc cattgaaagg acaaaatcat
tggtgccaac tgattttctt 28920 taccatagtt gtgacacaaa ggaagaaaga
aaaatcaagg gtgttatttc cataccccgt 28980 ttgcaagcag aagccagaag
tgagatcctc gcccactggt cgcctgccaa actgcttctc 29040 caaatggact
catctgctac agcttatggc tccacagttt ccaagagggt ggcatggcat 29100
tatggtatgt gtctcttccc ctgtgtgagc acttccaaag taatgcaggt gttgagacct
29160 gtggttacag gctgaactag taccattcac aactatttcc tacgtatttt
cagatgaaga 29220 gaagattgaa tttgaatgga acacaggcac caatgtagat
accaaaaaaa tgacttccaa 29280 tttccctgtg gatctctccg attatcctaa
gagcttgcat atgtatgcta atagactcct 29340 ggatcacaga gtccctcaaa
cagacatgac tttccggcac gtgggttcca aattaatagt 29400 tgtaagtatg
agtctgccag tcaataaata catggatata agtgctaatt acatcctcaa 29460
ctctgagcta ggtgcaggaa ggtttccaaa gatgtataag gcatgcttcc ttccccccag
29520 ggaattcttg gggagaaaaa aaaactttca caagtgtgta gttacccagt
tacacaaagc 29580 tgaatgtgat acatatcaaa gagatgctac taagtagaac
agttctttgc ctagtggtat 29640 caaaggaagc ttcaggacac cagctaggag
gctgactatg ttagacattc cttttataaa 29700 tatggacagt gatcagtgac
tggcaacgaa gattcataat tttctgttat ttatttttaa 29760 ctttcagtgc
attgtccagc ttaataatta acttgtcaaa tcggtatttt tgcctaatgt 29820
tcattgctct ttgaggctca tccaagccca ttaccttaaa aatctcctgt cattttgtag
29880 gcaatgagct catggcttca gaaggcatct gggagtcttc cttataccca
gactttgcaa 29940 gaccacctca atagcctgaa ggagttcaac ctccagaaca
tgggattgcc agacttccac 30000 atcccagaaa acctcttctt aaaaaggtaa
aagaagaaag cagcaaggct tcttgaacca 30060 tgcaaagtaa
atgaaagatt ttacatagca tgatttagac atttttttaa atttttaaag 30120
gaaataattt aagcatttta aggagattaa taactatagc acaaacactg tggcatcttt
30180 gcattagtaa acatgagaac accaaccctg tcaggaagaa tctaagaaag
tcattagagg 30240 attctggtac tttcacccta agatatttta ttcagtacaa
cctgttataa gcaaattctc 30300 cctctgactg tgaagaattc agaatggcta
gaggcgttat tgactacagg cttgctgtta 30360 agctagagag agtcagaaca
gccattgagc actaaatgga ggcagcattc tgagaaaata 30420 ctttaaccca
ggcttactga cttccatacc tatgttcttt ccacaaatca agttgtctca 30480
attcagttta gcaaatttgt atcaagtatc ccctatgtgc aaaatgctag actaggtaca
30540 gtgagaagat agaaactggg taaggtatag ccttttcttt caagaagata
ccatggagac 30600 atcaacaaat gagaaataat taattatata agcaaaatta
tgacatgctc tttgagaaag 30660 gtgcaaggga ctatgtaact gtaagaatga
gacaaattgg ctatgactta ggtgggatgg 30720 taatgataag gagtggccct
tagaagagct ttgtcaggat ttgagtgttt gacaggtgga 30780 ggtaaaagca
aaggggtcca ggcataggag tagcacaaag aaaagtgcag agtggctttg 30840
ggaatggggc aagtacaata ttgttgtgaa ggtcagaggc agagaacttt gaatgactga
30900 tgtctgactg tggggatgtt atctttgttg ttcatttcag cgatggccgg
gtcaaatata 30960 ccttgaacaa gaacagtttg aaaattgaga ttcctttgcc
ttttggtggc aaatcctcca 31020 gagatctaaa gatgttagag actgttagga
caccagccct ccacttcaag tctgtgggat 31080 tccatctgcc atctcgagag
ttccaagtcc ctacttttac cattcccaag ttgtatcaac 31140 tgcaagtgcc
tctcctgggt gttctagacc tctccacgaa tgtctacagc aacttgtaca 31200
actggtccgc ctcctacagt ggtggcaaca ccagcacaga ccatttcagc cttcgggctc
31260 gttaccacat gaaggctgac tctgtggttg acctgctttc ctacaatgtg
caaggtgagc 31320 tatgctcagg taaagggtgc accgggctag ttcatggcag
gctctaagag gagagcctcc 31380 tccagggagg aaaggacttt ggctttctag
cagataatct tccttgctac ttggaagtct 31440 tttattttat tcaacaaata
gaaatattta ttaaacatat cacgtgtatt aaatattcta 31500 gtaggcagta
acagaaagta gacagataag ccagcaatta taattcagtg tgagaggtgc 31560
tatgataaag tgtagtatat aagtataagg tagagtggaa gcactcaaca agggaaccta
31620 aacaaagcct gtggtggtca ggcaaggctt cctggaggaa tgccttttgc
tatcagattt 31680 tatctttgca ttacagatgg aggagtctat tgcacaattg
gcccagaaaa atggggcttt 31740 attattgaaa gactttcaac atagagattg
ctctggaaat gtactgctta atttaaccaa 31800 tgtcttttca tttttatgtt
aggatctgga gaaacaacat atgaccacaa gaatacgttc 31860 acactatcat
atgatgggtc tctacgccac aaatttctag attcgaatat caaattcagt 31920
catgtagaaa aacttggaaa caacccagtc tcaaaaggtt tactaatatt cgatgcatct
31980 agttcctggg gaccacagat gtctgcttca gttcatttgg actccaaaaa
gaaacagcat 32040 ttgtttgtca aagaagtcaa gattgatggg cagttcagag
tctcttcgtt ctatgctaaa 32100 ggcacatatg gcctgtcttg tcagagggat
cctaacactg gccggctcaa tggagagtcc 32160 aacctgaggt ttaactcctc
ctacctccaa ggcaccaacc agataacagg aagatatgaa 32220 gatggaaccc
tctccctcac ctccacctct gatctgcaaa gtggcatcat taaaaatact 32280
gcttccctaa agtatgagaa ctacgagctg actttaaaat ctgacaccaa tgggaagtat
32340 aagaactttg ccacttctaa caagatggat atgaccttct ctaagcaaaa
tgcactgctg 32400 cgttctgaat atcaggctga ttacgagtca ttgaggttct
tcagcctgct ttctggatca 32460 ctaaattccc atggtcttga gttaaatgct
gacatcttag gcactgacaa aattaatagt 32520 ggtgctcaca aggcgacact
aaggattggc caagatggaa tatctaccag tgcaacgacc 32580 aacttgaagt
gtagtctcct ggtgctggag aatgagctga atgcagagct tggcctctct 32640
ggggcatcta tgaaattaac aacaaatggc cgcttcaggg aacacaatgc aaaattcagt
32700 ctggatggga aagccgccct cacagagcta tcactgggaa gtgcttatca
ggccatgatt 32760 ctgggtgtcg acagcaaaaa cattttcaac ttcaaggtca
gtcaagaagg acttaagctc 32820 tcaaatgaca tgatgggctc atatgctgaa
atgaaatttg accacacaaa cagtctgaac 32880 attgcaggct tatcactgga
cttctcttca aaacttgaca acatttacag ctctgacaag 32940 ttttataagc
aaactgttaa tttacagcta cagccctatt ctctggtaac tactttaaac 33000
agtgacctga aatacaatgc tctggatctc accaacaatg ggaaactacg gctagaaccc
33060 ctgaagctgc atgtggctgg taacctaaaa ggagcctacc aaaataatga
aataaaacac 33120 atctatgcca tctcttctgc tgccttatca gcaagctata
aagcagacac tgttgctaag 33180 gttcagggtg tggagtttag ccatcggctc
aacacagaca tcgctgggct ggcttcagcc 33240 attgacatga gcacaaacta
taattcagac tcactgcatt tcagcaatgt cttccgttct 33300 gtaatggccc
cgtttaccat gaccatcgat gcacatacaa atggcaatgg gaaactcgct 33360
ctctggggag aacatactgg gcagctgtat agcaaattcc tgttgaaagc agaacctctg
33420 gcatttactt tctctcatga ttacaaaggc tccacaagtc atcatctcgt
gtctaggaaa 33480 agcatcagtg cagctcttga acacaaagtc agtgccctgc
ttactccagc tgagcagaca 33540 ggcacctgga aactcaagac ccaatttaac
aacaatgaat acagccagga cttggatgct 33600 tacaacacta aagataaaat
tggcgtggag cttactggac gaactctggc tgacctaact 33660 ctactagact
ccccaattaa agtgccactt ttactcagtg agcccatcaa tatcattgat 33720
gctttagaga tgagagatgc cgttgagaag ccccaagaat ttacaattgt tgcttttgta
33780 aagtatgata aaaaccaaga tgttcactcc attaacctcc cattttttga
gaccttgcaa 33840 gaatattttg agaggaatcg acaaaccatt atagttgtac
tggaaaacgt acagagaaac 33900 ctgaagcaca tcaatattga tcaatttgta
agaaaataca gagcagccct gggaaaactc 33960 ccacagcaag ctaatgatta
tctgaattca ttcaattggg agagacaagt ttcacatgcc 34020 aaggagaaac
tgactgctct cacaaaaaag tatagaatta cagaaaatga tatacaaatt 34080
gcattagatg atgccaaaat caactttaat gaaaaactat ctcaactgca gacatatatg
34140 atacaatttg atcagtatat taaagatagt tatgatttac atgatttgaa
aatagctatt 34200 gctaatatta ttgatgaaat cattgaaaaa ttaaaaagtc
ttgatgagca ctatcatatc 34260 cgtgtaaatt tagtaaaaac aatccatgat
ctacatttgt ttattgaaaa tattgatttt 34320 aacaaaagtg gaagtagtac
tgcatcctgg attcaaaatg tggatactaa gtaccaaatc 34380 agaatccaga
tacaagaaaa actgcagcag cttaagagac acatacagaa tatagacatc 34440
cagcacctag ctggaaagtt aaaacaacac attgaggcta ttgatgttag agtgctttta
34500 gatcaattgg gaactacaat ttcatttgaa agaataaatg acattcttga
gcatgtcaaa 34560 cactttgtta taaatcttat tggggatttt gaagtagctg
agaaaatcaa tgccttcaga 34620 gccaaagtcc atgagttaat cgagaggtat
gaagtagacc aacaaatcca ggttttaatg 34680 gataaattag tagagttggc
ccaccaatac aagttgaagg agactattca gaagctaagc 34740 aatgtcctac
aacaagttaa gataaaagat tactttgaga aattggttgg atttattgat 34800
gatgctgtca agaagcttaa tgaattatct tttaaaacat tcattgaaga tgttaacaaa
34860 ttccttgaca tgttgataaa gaaattaaag tcatttgatt accaccagtt
tgtagatgaa 34920 accaatgaca aaatccgtga ggtgactcag agactcaatg
gtgaaattca ggctctggaa 34980 ctaccacaaa aagctgaagc attaaaactg
tttttagagg aaaccaaggc cacagttgca 35040 gtgtatctgg aaagcctaca
ggacaccaaa ataaccttaa tcatcaattg gttacaggag 35100 gctttaagtt
cagcatcttt ggctcacatg aaggccaaat tccgagagac cctagaagat 35160
acacgagacc gaatgtatca aatggacatt cagcaggaac ttcaacgata cctgtctctg
35220 gtaggccagg tttatagcac acttgtcacc tacatttctg attggtggac
tcttgctgct 35280 aagaacctta ctgactttgc agagcaatat tctatccaag
attgggctaa acgtatgaaa 35340 gcattggtag agcaagggtt cactgttcct
gaaatcaaga ccatccttgg gaccatgcct 35400 gcctttgaag tcagtcttca
ggctcttcag aaagctacct tccagacacc tgattttata 35460 gtccccctaa
cagatttgag gattccatca gttcagataa acttcaaaga cttaaaaaat 35520
ataaaaatcc catccaggtt ttccacacca gaatttacca tccttaacac cttccacatt
35580 ccttccttta caattgactt tgtagaaatg aaagtaaaga tcatcagaac
cattgaccag 35640 atgctgaaca gtgagctgca gtggcccgtt ccagatatat
atctcaggga tctgaaggtg 35700 gaggacattc ctctagcgag aatcaccctg
ccagacttcc gtttaccaga aatcgcaatt 35760 ccagaattca taatcccaac
tctcaacctt aatgattttc aagttcctga ccttcacata 35820 ccagaattcc
agcttcccca catctcacac acaattgaag tacctacttt tggcaagcta 35880
tacagtattc tgaaaatcca atctcctctt ttcacattag atgcaaatgc tgacataggg
35940 aatggaacca cctcagcaaa cgaagcaggt atcgcagctt ccatcactgc
caaaggagag 36000 tccaaattag aagttctcaa ttttgatttt caagcaaatg
cacaactctc aaaccctaag 36060 attaatccgc tggctctgaa ggagtcagtg
aagttctcca gcaagtacct gagaacggag 36120 catgggagtg aaatgctgtt
ttttggaaat gctattgagg gaaaatcaaa cacagtggca 36180 agtttacaca
cagaaaaaaa tacactggag cttagtaatg gagtgattgt caagataaac 36240
aatcagctta ccctggatag caacactaaa tacttccaca aattgaacat ccccaaactg
36300 gacttctcta gtcaggctga cctgcgcaac gagatcaaga cactgttgaa
agctggccac 36360 atagcatgga cttcttctgg aaaagggtca tggaaatggg
cctgccccag attctcagat 36420 gagggaacac atgaatcaca aattagtttc
accatagaag gacccctcac ttcctttgga 36480 ctgtccaata agatcaatag
caaacaccta agagtaaacc aaaacttggt ttatgaatct 36540 ggctccctca
acttttctaa acttgaaatt caatcacaag tcgattccca gcatgtgggc 36600
cacagtgttc taactgctaa aggcatggca ctgtttggag aagggaaggc agagtttact
36660 gggaggcatg atgctcattt aaatggaaag gttattggaa ctttgaaaaa
ttctcttttc 36720 ttttcagccc agccatttga gatcacggca tccacaaaca
atgaagggaa tttgaaagtt 36780 cgttttccat taaggttaac agggaagata
gacttcctga ataactatgc actgtttctg 36840 agtcccagtg cccagcaagc
aagttggcaa gtaagtgcta ggttcaatca gtataagtac 36900 aaccaaaatt
tctctgctgg aaacaacgag aacattatgg aggcccatgt aggaataaat 36960
ggagaagcaa atctggattt cttaaacatt cctttaacaa ttcctgaaat gcgtctacct
37020 tacacaataa tcacaactcc tccactgaaa gatttctctc tatgggaaaa
aacaggcttg 37080 aaggaattct tgaaaacgac aaagcaatca tttgatttaa
gtgtaaaagc tcagtataag 37140 aaaaacaaac acaggcattc catcacaaat
cctttggctg tgctttgtga gtttatcagt 37200 cagagcatca aatcctttga
caggcatttt gaaaaaaaca gaaacaatgc attagatttt 37260 gtcaccaaat
cctataatga aacaaaaatt aagtttgata agtacaaagc tgaaaaatct 37320
cacgacgagc tccccaggac ctttcaaatt cctggataca ctgttccagt tgtcaatgtt
37380 gaagtgtctc cattcaccat agagatgtcg gcattcggct atgtgttccc
aaaagcagtc 37440 agcatgccta gtttctccat cctaggttct gacgtccgtg
tgccttcata cacattaatc 37500 ctgccatcat tagagctgcc agtccttcat
gtccctagaa atctcaagct ttctcttcca 37560 gatttcaagg aattgtgtac
cataagccat atttttattc ctgccatggg caatattacc 37620 tatgatttct
cctttaaatc aagtgtcatc acactgaata ccaatgctga actttttaac 37680
cagtcagata ttgttgctca tctcctttct tcatcttcat ctgtcattga tgcactgcag
37740 tacaaattag agggcaccac aagattgaca agaaaaaggg gattgaagtt
agccacagct 37800 ctgtctctga gcaacaaatt tgtggagggt agtcataaca
gtactgtgag cttaaccacg 37860 aaaaatatgg aagtgtcagt ggcaacaacc
acaaaagccc aaattccaat tttgagaatg 37920 aatttcaagc aagaacttaa
tggaaatacc aagtcaaaac ctactgtctc ttcctccatg 37980 gaatttaagt
atgatttcaa ttcttcaatg ctgtactcta ccgctaaagg agcagttgac 38040
cacaagctta gcttggaaag cctcacctct tacttttcca ttgagtcatc taccaaagga
38100 gatgtcaagg gttcggttct ttctcgggaa tattcaggaa ctattgctag
tgaggccaac 38160 acttacttga attccaagag cacacggtct tcagtgaagc
tgcagggcac ttccaaaatt 38220 gatgatatct ggaaccttga agtaaaagaa
aattttgctg gagaagccac actccaacgc 38280 atatattccc tctgggagca
cagtacgaaa aaccacttac agctagaggg cctctttttc 38340 accaacggag
aacatacaag caaagccacc ctggaactct ctccatggca aatgtcagct 38400
cttgttcagg tccatgcaag tcagcccagt tccttccatg atttccctga ccttggccag
38460 gaagtggccc tgaatgctaa cactaagaac cagaagatca gatggaaaaa
tgaagtccgg 38520 attcattctg ggtctttcca gagccaggtc gagctttcca
atgaccaaga aaaggcacac 38580 cttgacattg caggatcctt agaaggacac
ctaaggttcc tcaaaaatat catcctacca 38640 gtctatgaca agagcttatg
ggatttccta aagctggatg taaccaccag cattggtagg 38700 agacagcatc
ttcgtgtttc aactgccttt gtgtacacca aaaaccccaa tggctattca 38760
ttctccatcc ctgtaaaagt tttggctgat aaattcatta ttcctgggct gaaactaaat
38820 gatctaaatt cagttcttgt catgcctacg ttccatgtcc catttacaga
tcttcaggtt 38880 ccatcgtgca aacttgactt cagagaaata caaatctata
agaagctgag aacttcatca 38940 tttgccctca acctaccaac actccccgag
gtaaaattcc ctgaagttga tgtgttaaca 39000 aaatattctc aaccagaaga
ctccttgatt cccttttttg agataaccgt gcctgaatct 39060 cagttaactg
tgtcccagtt cacgcttcca aaaagtgttt cagatggcat tgctgctttg 39120
gatctaaatg cagtagccaa caagatcgca gactttgagt tgcccaccat catcgtgcct
39180 gagcagacca ttgagattcc ctccattaag ttctctgtac ctgctggaat
tgtcattcct 39240 tcctttcaag cactgactgc acgctttgag gtagactctc
ccgtgtataa tgccacttgg 39300 agtgccagtt tgaaaaacaa agcagattat
gttgaaacag tcctggattc cacatgcagc 39360 tcaaccgtac agttcctaga
atatgaacta aatggtaaga aatatcctgc ctcctctcct 39420 agatactgta
tattttcaat gagagttatg agtaaataat tatgtattta gttgtgagta 39480
gatgtacaat tactcaatgt cacaaaattt taagtaagaa aagagataca tgtataccct
39540 acacgtaaaa accaaactgt agaaaatcta gtgtcattca agacaaacag
ctttaaagaa 39600 aatggatttt tctgtaatta ttttaggact aacaatgtct
tttaactatt tattttaaaa 39660 taagtgtgag ctgtacattg catattttaa
acacaagtga aatatctggt taggatagaa 39720 ttctcccagt tttcacaatg
aaaacatcaa cgtcctactg ttatgaatct aataaaatac 39780 aaaatctctc
ctatacagtt ttgggaacac acaaaatcga agatggtacg ttagcctcta 39840
agactaaagg aacatttgca caccgtgact tcagtgcaga atatgaagaa gatggcaaat
39900 atgaaggact tcagtatgga gcttttattg aattgaaacc ttataccttt
tgaaaactca 39960 ttgtgatttt cttcatctcc ataccccttt cgtgatagct
catctgtttt tctgctttca 40020 gggaatggga aggaaaagcg cacctcaata
tcaaaagccc agcgttcacc gatctccatc 40080 tgcgctacca gaaagacaag
aaaggcatct ccacctcagc agcctcccca gccgtaggca 40140 ccgtgggcat
ggatatggat gaagatgacg acttttctaa atggaacttc tactacagcc 40200
ctcaggtaaa taccacctaa tgagtgacac gcccccaaga gcgagtggag aattggggca
40260 gatacattta attcaggacc aaatattcag agattcccca aactaggtga
aagacaggcg 40320 gtaagcaact tcttctctga ggaaatattc tctagaaagt
attacaatga gtccttgatt 40380 gattttaatg tttagatgca cacatgacat
cccatcagca ctattattta ttaattctgg 40440 gcaaatccag gaagatgagg
gttatacctc atcatctaaa tcataggcaa gctcagccat 40500 aggcagggta
tatttttcag agaggactgg tttctgtagt atttaaaact ttaaaattct 40560
tccccacaat agaattgcta gatgagatac atcaaattcc tctcatgtca tttacaagct
40620 ctgccagggc caaatcaagg gtgacattac cagaggagaa gaccaaacat
ggttctatga 40680 ctgttactaa aagtttgtca tgggcttgga gaatgcgtac
tgatgttggg attctgggtc 40740 tctgcagggt gggctccaac ttgccttttt
tgctatttct tcttttccta tctgtcattt 40800 cctgactctt cttctctctc
ctcttctttc tcttcccccc actcctcttc cagttttcag 40860 tcctaggaag
gctttaattt taagtgtcac aatgtaaatg acaaacagca agcgtttttg 40920
ttaaatcctt tctggggcat gtgataaaga gaaattaaca acagtagact tatttaacca
40980 taaaacaaac acatgaactg acatatgaaa gataaatccc tttcagtata
tgaaagattc 41040 tctgatcttt atttttaact gctaatgaag ttttagtgta
ctatattgtg taattggagt 41100 aattgaaaac atgttatttt tttttttctc
tctgtttagt cctctccaga taaaaaactc 41160 accatattca aaactgagtt
gagggtccgg gaatctgatg aggaaactca gatcaaagtt 41220 aattgggaag
aagaggcagc ttctggcttg ctaacctctc tgaaagacaa cgtgcccaag 41280
gccacagggg tcctttatga ttatgtcaac aagtaccact gggaacacac agggctcacc
41340 ctgagagaag tgtcttcaaa gctgagaaga aatctgcaga acaatgctga
gtgggtttat 41400 caaggggcca ttaggcaaat tgatgatatc gacgtgaggt
tccagaaagc agccagtggc 41460 accactggga cctaccaaga gtggaaggac
aaggcccaga atctgtacca ggaactgttg 41520 actcaggaag gccaagccag
tttccaggga ctcaaggata acgtgtttga tggcttggta 41580 cgagttactc
aagaattcca tatgaaagtc aagcatctga ttgactcact cattgatttt 41640
ctgaacttcc ccagattcca gtttccgggg aaacctggga tatacactag ggaggaactt
41700 tgcactatgt tcataaggga ggtagggacg gtactgtccc aggtatattc
gaaagtccat 41760 aatggttcag aaatactgtt ttcctatttc caagacctag
tgattacact tcctttcgag 41820 ttaaggaaac ataaactaat agatgtaatc
tcgatgtata gggaactgtt gaaagattta 41880 tcaaaagaag cccaagaggt
atttaaagcc attcagtctc tcaagaccac agaggtgcta 41940 cgtaatcttc
aggacctttt acaattcatt ttccaactaa tagaagataa cattaaacag 42000
ctgaaagaga tgaaatttac ttatcttatt aattatatcc aagatgagat caacacaatc
42060 ttcagtgatt atatcccata tgtttttaaa ttgttgaaag aaaacctatg
ccttaatctt 42120 cataagttca atgaatttat tcaaaacgag cttcaggaag
cttctcaaga gttacagcag 42180 atccatcaat acattatggc ccttcgtgaa
gaatattttg atccaagtat agttggctgg 42240 acagtgaaat attatgaact
tgaagaaaag atagtcagtc tgatcaagaa cctgttagtt 42300 gctcttaagg
acttccattc tgaatatatt gtcagtgcct ctaactttac ttcccaactc 42360
tcaagtcaag ttgagcaatt tctgcacaga aatattcagg aatatcttag catccttacc
42420 gatccagatg gaaaagggaa agagaagatt gcagagcttt ctgccactgc
tcaggaaata 42480 attaaaagcc aggccattgc gacgaagaaa ataatttctg
attaccacca gcagtttaga 42540 tataaactgc aagatttttc agaccaactc
tctgattact atgaaaaatt tattgctgaa 42600 tccaaaagat tgattgacct
gtccattcaa aactaccaca catttctgat atacatcacg 42660 gagttactga
aaaagctgca atcaaccaca gtcatgaacc cctacatgaa gcttgctcca 42720
ggagaactta ctatcatcct ctaatttttt aaaagaaatc ttcatttatt cttcttttcc
42780 aattgaactt tcacatagca cagaaaaaat tcaaactgcc tatattgata
aaaccataca 42840 gtgagccagc cttgcagtag gcagtagact ataagcagaa
gcacatatga actggacctg 42900 caccaaagct ggcaccaggg ctcggaaggt
ctctgaactc agaaggatgg cattttttgc 42960 aagttaaaga aaatcaggat
ctgagttatt ttgctaaact tgggggagga ggaacaaata 43020 aatggagtct
ttattgtgta tcataccact gaatgtggct catttgtatt gaaagacagt 43080
gaaacgaggg cattgataaa atgttctggc acagcaaaac ctctagaaca catagtgtga
43140 tttaagtaac agaataaaaa tggaaacgga gaaattatgg agggaaatat
tttgcaaaaa 43200 tatttaaaaa gatgaggtaa ttgtgttttt ataattaaat
attttataat taaaatattt 43260 ataattaaaa tatttataat taaatatttt
ataattaaaa tatttataat taaatatttt 43320 ataattaaag tatttataat
taaatatttt ataattaaaa tatttataat taaatatttt 43380 ataattaaaa
tatttataat taaatatttt ataattaaaa tatttataat taaatatttt 43440 ataat
43445 2 43445 DNA Artificial Sequence Antisense Oligonucleotide 2
attataaaat atttaattat aaatatttta attataaaat atttaattat aaatatttta
60 attataaaat atttaattat aaatatttta attataaaat atttaattat
aaatacttta 120 attataaaat atttaattat aaatatttta attataaaat
atttaattat aaatatttta 180 attataaata ttttaattat aaaatattta
attataaaaa cacaattacc tcatcttttt 240 aaatattttt gcaaaatatt
tccctccata atttctccgt ttccattttt attctgttac 300 ttaaatcaca
ctatgtgttc tagaggtttt gctgtgccag aacattttat caatgccctc 360
gtttcactgt ctttcaatac aaatgagcca cattcagtgg tatgatacac aataaagact
420 ccatttattt gttcctcctc ccccaagttt agcaaaataa ctcagatcct
gattttcttt 480 aacttgcaaa aaatgccatc cttctgagtt cagagacctt
ccgagccctg gtgccagctt 540 tggtgcaggt ccagttcata tgtgcttctg
cttatagtct actgcctact gcaaggctgg 600 ctcactgtat ggttttatca
atataggcag tttgaatttt ttctgtgcta tgtgaaagtt 660 caattggaaa
agaagaataa atgaagattt cttttaaaaa attagaggat gatagtaagt 720
tctcctggag caagcttcat gtaggggttc atgactgtgg ttgattgcag ctttttcagt
780 aactccgtga tgtatatcag aaatgtgtgg tagttttgaa tggacaggtc
aatcaatctt 840 ttggattcag caataaattt ttcatagtaa tcagagagtt
ggtctgaaaa atcttgcagt 900 ttatatctaa actgctggtg gtaatcagaa
attattttct tcgtcgcaat ggcctggctt 960 ttaattattt cctgagcagt
ggcagaaagc tctgcaatct tctctttccc ttttccatct 1020 ggatcggtaa
ggatgctaag atattcctga atatttctgt gcagaaattg ctcaacttga 1080
cttgagagtt gggaagtaaa gttagaggca ctgacaatat attcagaatg gaagtcctta
1140 agagcaacta acaggttctt gatcagactg actatctttt cttcaagttc
ataatatttc 1200 actgtccagc caactatact tggatcaaaa tattcttcac
gaagggccat aatgtattga 1260 tggatctgct gtaactcttg agaagcttcc
tgaagctcgt tttgaataaa ttcattgaac 1320 ttatgaagat taaggcatag
gttttctttc aacaatttaa aaacatatgg gatataatca 1380 ctgaagattg
tgttgatctc atcttggata taattaataa gataagtaaa tttcatctct 1440
ttcagctgtt taatgttatc ttctattagt tggaaaatga attgtaaaag gtcctgaaga
1500 ttacgtagca cctctgtggt cttgagagac tgaatggctt taaatacctc
ttgggcttct 1560 tttgataaat ctttcaacag
ttccctatac atcgagatta catctattag tttatgtttc 1620 cttaactcga
aaggaagtgt aatcactagg tcttggaaat aggaaaacag tatttctgaa 1680
ccattatgga ctttcgaata tacctgggac agtaccgtcc ctacctccct tatgaacata
1740 gtgcaaagtt cctccctagt gtatatccca ggtttccccg gaaactggaa
tctggggaag 1800 ttcagaaaat caatgagtga gtcaatcaga tgcttgactt
tcatatggaa ttcttgagta 1860 actcgtacca agccatcaaa cacgttatcc
ttgagtccct ggaaactggc ttggccttcc 1920 tgagtcaaca gttcctggta
cagattctgg gccttgtcct tccactcttg gtaggtccca 1980 gtggtgccac
tggctgcttt ctggaacctc acgtcgatat catcaatttg cctaatggcc 2040
ccttgataaa cccactcagc attgttctgc agatttcttc tcagctttga agacacttct
2100 ctcagggtga gccctgtgtg ttcccagtgg tacttgttga cataatcata
aaggacccct 2160 gtggccttgg gcacgttgtc tttcagagag gttagcaagc
cagaagctgc ctcttcttcc 2220 caattaactt tgatctgagt ttcctcatca
gattcccgga ccctcaactc agttttgaat 2280 atggtgagtt ttttatctgg
agaggactaa acagagagaa aaaaaaaaat aacatgtttt 2340 caattactcc
aattacacaa tatagtacac taaaacttca ttagcagtta aaaataaaga 2400
tcagagaatc tttcatatac tgaaagggat ttatctttca tatgtcagtt catgtgtttg
2460 ttttatggtt aaataagtct actgttgtta atttctcttt atcacatgcc
ccagaaagga 2520 tttaacaaaa acgcttgctg tttgtcattt acattgtgac
acttaaaatt aaagccttcc 2580 taggactgaa aactggaaga ggagtggggg
gaagagaaag aagaggagag agaagaagag 2640 tcaggaaatg acagatagga
aaagaagaaa tagcaaaaaa ggcaagttgg agcccaccct 2700 gcagagaccc
agaatcccaa catcagtacg cattctccaa gcccatgaca aacttttagt 2760
aacagtcata gaaccatgtt tggtcttctc ctctggtaat gtcacccttg atttggccct
2820 ggcagagctt gtaaatgaca tgagaggaat ttgatgtatc tcatctagca
attctattgt 2880 ggggaagaat tttaaagttt taaatactac agaaaccagt
cctctctgaa aaatataccc 2940 tgcctatggc tgagcttgcc tatgatttag
atgatgaggt ataaccctca tcttcctgga 3000 tttgcccaga attaataaat
aatagtgctg atgggatgtc atgtgtgcat ctaaacatta 3060 aaatcaatca
aggactcatt gtaatacttt ctagagaata tttcctcaga gaagaagttg 3120
cttaccgcct gtctttcacc tagtttgggg aatctctgaa tatttggtcc tgaattaaat
3180 gtatctgccc caattctcca ctcgctcttg ggggcgtgtc actcattagg
tggtatttac 3240 ctgagggctg tagtagaagt tccatttaga aaagtcgtca
tcttcatcca tatccatgcc 3300 cacggtgcct acggctgggg aggctgctga
ggtggagatg cctttcttgt ctttctggta 3360 gcgcagatgg agatcggtga
acgctgggct tttgatattg aggtgcgctt ttccttccca 3420 ttccctgaaa
gcagaaaaac agatgagcta tcacgaaagg ggtatggaga tgaagaaaat 3480
cacaatgagt tttcaaaagg tataaggttt caattcaata aaagctccat actgaagtcc
3540 ttcatatttg ccatcttctt catattctgc actgaagtca cggtgtgcaa
atgttccttt 3600 agtcttagag gctaacgtac catcttcgat tttgtgtgtt
cccaaaactg tataggagag 3660 attttgtatt ttattagatt cataacagta
ggacgttgat gttttcattg tgaaaactgg 3720 gagaattcta tcctaaccag
atatttcact tgtgtttaaa atatgcaatg tacagctcac 3780 acttatttta
aaataaatag ttaaaagaca ttgttagtcc taaaataatt acagaaaaat 3840
ccattttctt taaagctgtt tgtcttgaat gacactagat tttctacagt ttggttttta
3900 cgtgtagggt atacatgtat ctcttttctt acttaaaatt ttgtgacatt
gagtaattgt 3960 acatctactc acaactaaat acataattat ttactcataa
ctctcattga aaatatacag 4020 tatctaggag aggaggcagg atatttctta
ccatttagtt catattctag gaactgtacg 4080 gttgagctgc atgtggaatc
caggactgtt tcaacataat ctgctttgtt tttcaaactg 4140 gcactccaag
tggcattata cacgggagag tctacctcaa agcgtgcagt cagtgcttga 4200
aaggaaggaa tgacaattcc agcaggtaca gagaacttaa tggagggaat ctcaatggtc
4260 tgctcaggca cgatgatggt gggcaactca aagtctgcga tcttgttggc
tactgcattt 4320 agatccaaag cagcaatgcc atctgaaaca ctttttggaa
gcgtgaactg ggacacagtt 4380 aactgagatt caggcacggt tatctcaaaa
aagggaatca aggagtcttc tggttgagaa 4440 tattttgtta acacatcaac
ttcagggaat tttacctcgg ggagtgttgg taggttgagg 4500 gcaaatgatg
aagttctcag cttcttatag atttgtattt ctctgaagtc aagtttgcac 4560
gatggaacct gaagatctgt aaatgggaca tggaacgtag gcatgacaag aactgaattt
4620 agatcattta gtttcagccc aggaataatg aatttatcag ccaaaacttt
tacagggatg 4680 gagaatgaat agccattggg gtttttggtg tacacaaagg
cagttgaaac acgaagatgc 4740 tgtctcctac caatgctggt ggttacatcc
agctttagga aatcccataa gctcttgtca 4800 tagactggta ggatgatatt
tttgaggaac cttaggtgtc cttctaagga tcctgcaatg 4860 tcaaggtgtg
ccttttcttg gtcattggaa agctcgacct ggctctggaa agacccagaa 4920
tgaatccgga cttcattttt ccatctgatc ttctggttct tagtgttagc attcagggcc
4980 acttcctggc caaggtcagg gaaatcatgg aaggaactgg gctgacttgc
atggacctga 5040 acaagagctg acatttgcca tggagagagt tccagggtgg
ctttgcttgt atgttctccg 5100 ttggtgaaaa agaggccctc tagctgtaag
tggtttttcg tactgtgctc ccagagggaa 5160 tatatgcgtt ggagtgtggc
ttctccagca aaattttctt ttacttcaag gttccagata 5220 tcatcaattt
tggaagtgcc ctgcagcttc actgaagacc gtgtgctctt ggaattcaag 5280
taagtgttgg cctcactagc aatagttcct gaatattccc gagaaagaac cgaacccttg
5340 acatctcctt tggtagatga ctcaatggaa aagtaagagg tgaggctttc
caagctaagc 5400 ttgtggtcaa ctgctccttt agcggtagag tacagcattg
aagaattgaa atcatactta 5460 aattccatgg aggaagagac agtaggtttt
gacttggtat ttccattaag ttcttgcttg 5520 aaattcattc tcaaaattgg
aatttgggct tttgtggttg ttgccactga cacttccata 5580 tttttcgtgg
ttaagctcac agtactgtta tgactaccct ccacaaattt gttgctcaga 5640
gacagagctg tggctaactt caatcccctt tttcttgtca atcttgtggt gccctctaat
5700 ttgtactgca gtgcatcaat gacagatgaa gatgaagaaa ggagatgagc
aacaatatct 5760 gactggttaa aaagttcagc attggtattc agtgtgatga
cacttgattt aaaggagaaa 5820 tcataggtaa tattgcccat ggcaggaata
aaaatatggc ttatggtaca caattccttg 5880 aaatctggaa gagaaagctt
gagatttcta gggacatgaa ggactggcag ctctaatgat 5940 ggcaggatta
atgtgtatga aggcacacgg acgtcagaac ctaggatgga gaaactaggc 6000
atgctgactg cttttgggaa cacatagccg aatgccgaca tctctatggt gaatggagac
6060 acttcaacat tgacaactgg aacagtgtat ccaggaattt gaaaggtcct
ggggagctcg 6120 tcgtgagatt tttcagcttt gtacttatca aacttaattt
ttgtttcatt ataggatttg 6180 gtgacaaaat ctaatgcatt gtttctgttt
ttttcaaaat gcctgtcaaa ggatttgatg 6240 ctctgactga taaactcaca
aagcacagcc aaaggatttg tgatggaatg cctgtgtttg 6300 tttttcttat
actgagcttt tacacttaaa tcaaatgatt gctttgtcgt tttcaagaat 6360
tccttcaagc ctgttttttc ccatagagag aaatctttca gtggaggagt tgtgattatt
6420 gtgtaaggta gacgcatttc aggaattgtt aaaggaatgt ttaagaaatc
cagatttgct 6480 tctccattta ttcctacatg ggcctccata atgttctcgt
tgtttccagc agagaaattt 6540 tggttgtact tatactgatt gaacctagca
cttacttgcc aacttgcttg ctgggcactg 6600 ggactcagaa acagtgcata
gttattcagg aagtctatct tccctgttaa ccttaatgga 6660 aaacgaactt
tcaaattccc ttcattgttt gtggatgccg tgatctcaaa tggctgggct 6720
gaaaagaaaa gagaattttt caaagttcca ataacctttc catttaaatg agcatcatgc
6780 ctcccagtaa actctgcctt cccttctcca aacagtgcca tgcctttagc
agttagaaca 6840 ctgtggccca catgctggga atcgacttgt gattgaattt
caagtttaga aaagttgagg 6900 gagccagatt cataaaccaa gttttggttt
actcttaggt gtttgctatt gatcttattg 6960 gacagtccaa aggaagtgag
gggtccttct atggtgaaac taatttgtga ttcatgtgtt 7020 ccctcatctg
agaatctggg gcaggcccat ttccatgacc cttttccaga agaagtccat 7080
gctatgtggc cagctttcaa cagtgtcttg atctcgttgc gcaggtcagc ctgactagag
7140 aagtccagtt tggggatgtt caatttgtgg aagtatttag tgttgctatc
cagggtaagc 7200 tgattgttta tcttgacaat cactccatta ctaagctcca
gtgtattttt ttctgtgtgt 7260 aaacttgcca ctgtgtttga ttttccctca
atagcatttc caaaaaacag catttcactc 7320 ccatgctccg ttctcaggta
cttgctggag aacttcactg actccttcag agccagcgga 7380 ttaatcttag
ggtttgagag ttgtgcattt gcttgaaaat caaaattgag aacttctaat 7440
ttggactctc ctttggcagt gatggaagct gcgatacctg cttcgtttgc tgaggtggtt
7500 ccattcccta tgtcagcatt tgcatctaat gtgaaaagag gagattggat
tttcagaata 7560 ctgtatagct tgccaaaagt aggtacttca attgtgtgtg
agatgtgggg aagctggaat 7620 tctggtatgt gaaggtcagg aacttgaaaa
tcattaaggt tgagagttgg gattatgaat 7680 tctggaattg cgatttctgg
taaacggaag tctggcaggg tgattctcgc tagaggaatg 7740 tcctccacct
tcagatccct gagatatata tctggaacgg gccactgcag ctcactgttc 7800
agcatctggt caatggttct gatgatcttt actttcattt ctacaaagtc aattgtaaag
7860 gaaggaatgt ggaaggtgtt aaggatggta aattctggtg tggaaaacct
ggatgggatt 7920 tttatatttt ttaagtcttt gaagtttatc tgaactgatg
gaatcctcaa atctgttagg 7980 gggactataa aatcaggtgt ctggaaggta
gctttctgaa gagcctgaag actgacttca 8040 aaggcaggca tggtcccaag
gatggtcttg atttcaggaa cagtgaaccc ttgctctacc 8100 aatgctttca
tacgtttagc ccaatcttgg atagaatatt gctctgcaaa gtcagtaagg 8160
ttcttagcag caagagtcca ccaatcagaa atgtaggtga caagtgtgct ataaacctgg
8220 cctaccagag acaggtatcg ttgaagttcc tgctgaatgt ccatttgata
cattcggtct 8280 cgtgtatctt ctagggtctc tcggaatttg gccttcatgt
gagccaaaga tgctgaactt 8340 aaagcctcct gtaaccaatt gatgattaag
gttattttgg tgtcctgtag gctttccaga 8400 tacactgcaa ctgtggcctt
ggtttcctct aaaaacagtt ttaatgcttc agctttttgt 8460 ggtagttcca
gagcctgaat ttcaccattg agtctctgag tcacctcacg gattttgtca 8520
ttggtttcat ctacaaactg gtggtaatca aatgacttta atttctttat caacatgtca
8580 aggaatttgt taacatcttc aatgaatgtt ttaaaagata attcattaag
cttcttgaca 8640 gcatcatcaa taaatccaac caatttctca aagtaatctt
ttatcttaac ttgttgtagg 8700 acattgctta gcttctgaat agtctccttc
aacttgtatt ggtgggccaa ctctactaat 8760 ttatccatta aaacctggat
ttgttggtct acttcatacc tctcgattaa ctcatggact 8820 ttggctctga
aggcattgat tttctcagct acttcaaaat ccccaataag atttataaca 8880
aagtgtttga catgctcaag aatgtcattt attctttcaa atgaaattgt agttcccaat
8940 tgatctaaaa gcactctaac atcaatagcc tcaatgtgtt gttttaactt
tccagctagg 9000 tgctggatgt ctatattctg tatgtgtctc ttaagctgct
gcagtttttc ttgtatctgg 9060 attctgattt ggtacttagt atccacattt
tgaatccagg atgcagtact acttccactt 9120 ttgttaaaat caatattttc
aataaacaaa tgtagatcat ggattgtttt tactaaattt 9180 acacggatat
gatagtgctc atcaagactt tttaattttt caatgatttc atcaataata 9240
ttagcaatag ctattttcaa atcatgtaaa tcataactat ctttaatata ctgatcaaat
9300 tgtatcatat atgtctgcag ttgagatagt ttttcattaa agttgatttt
ggcatcatct 9360 aatgcaattt gtatatcatt ttctgtaatt ctatactttt
ttgtgagagc agtcagtttc 9420 tccttggcat gtgaaacttg tctctcccaa
ttgaatgaat tcagataatc attagcttgc 9480 tgtgggagtt ttcccagggc
tgctctgtat tttcttacaa attgatcaat attgatgtgc 9540 ttcaggtttc
tctgtacgtt ttccagtaca actataatgg tttgtcgatt cctctcaaaa 9600
tattcttgca aggtctcaaa aaatgggagg ttaatggagt gaacatcttg gtttttatca
9660 tactttacaa aagcaacaat tgtaaattct tggggcttct caacggcatc
tctcatctct 9720 aaagcatcaa tgatattgat gggctcactg agtaaaagtg
gcactttaat tggggagtct 9780 agtagagtta ggtcagccag agttcgtcca
gtaagctcca cgccaatttt atctttagtg 9840 ttgtaagcat ccaagtcctg
gctgtattca ttgttgttaa attgggtctt gagtttccag 9900 gtgcctgtct
gctcagctgg agtaagcagg gcactgactt tgtgttcaag agctgcactg 9960
atgcttttcc tagacacgag atgatgactt gtggagcctt tgtaatcatg agagaaagta
10020 aatgccagag gttctgcttt caacaggaat ttgctataca gctgcccagt
atgttctccc 10080 cagagagcga gtttcccatt gccatttgta tgtgcatcga
tggtcatggt aaacggggcc 10140 attacagaac ggaagacatt gctgaaatgc
agtgagtctg aattatagtt tgtgctcatg 10200 tcaatggctg aagccagccc
agcgatgtct gtgttgagcc gatggctaaa ctccacaccc 10260 tgaaccttag
caacagtgtc tgctttatag cttgctgata aggcagcaga agagatggca 10320
tagatgtgtt ttatttcatt attttggtag gctcctttta ggttaccagc cacatgcagc
10380 ttcaggggtt ctagccgtag tttcccattg ttggtgagat ccagagcatt
gtatttcagg 10440 tcactgttta aagtagttac cagagaatag ggctgtagct
gtaaattaac agtttgctta 10500 taaaacttgt cagagctgta aatgttgtca
agttttgaag agaagtccag tgataagcct 10560 gcaatgttca gactgtttgt
gtggtcaaat ttcatttcag catatgagcc catcatgtca 10620 tttgagagct
taagtccttc ttgactgacc ttgaagttga aaatgttttt gctgtcgaca 10680
cccagaatca tggcctgata agcacttccc agtgatagct ctgtgagggc ggctttccca
10740 tccagactga attttgcatt gtgttccctg aagcggccat ttgttgttaa
tttcatagat 10800 gccccagaga ggccaagctc tgcattcagc tcattctcca
gcaccaggag actacacttc 10860 aagttggtcg ttgcactggt agatattcca
tcttggccaa tccttagtgt cgccttgtga 10920 gcaccactat taattttgtc
agtgcctaag atgtcagcat ttaactcaag accatgggaa 10980 tttagtgatc
cagaaagcag gctgaagaac ctcaatgact cgtaatcagc ctgatattca 11040
gaacgcagca gtgcattttg cttagagaag gtcatatcca tcttgttaga agtggcaaag
11100 ttcttatact tcccattggt gtcagatttt aaagtcagct cgtagttctc
atactttagg 11160 gaagcagtat ttttaatgat gccactttgc agatcagagg
tggaggtgag ggagagggtt 11220 ccatcttcat atcttcctgt tatctggttg
gtgccttgga ggtaggagga gttaaacctc 11280 aggttggact ctccattgag
ccggccagtg ttaggatccc tctgacaaga caggccatat 11340 gtgcctttag
catagaacga agagactctg aactgcccat caatcttgac ttctttgaca 11400
aacaaatgct gtttcttttt ggagtccaaa tgaactgaag cagacatctg tggtccccag
11460 gaactagatg catcgaatat tagtaaacct tttgagactg ggttgtttcc
aagtttttct 11520 acatgactga atttgatatt cgaatctaga aatttgtggc
gtagagaccc atcatatgat 11580 agtgtgaacg tattcttgtg gtcatatgtt
gtttctccag atcctaacat aaaaatgaaa 11640 agacattggt taaattaagc
agtacatttc cagagcaatc tctatgttga aagtctttca 11700 ataataaagc
cccatttttc tgggccaatt gtgcaataga ctcctccatc tgtaatgcaa 11760
agataaaatc tgatagcaaa aggcattcct ccaggaagcc ttgcctgacc accacaggct
11820 ttgtttaggt tcccttgttg agtgcttcca ctctacctta tacttatata
ctacacttta 11880 tcatagcacc tctcacactg aattataatt gctggcttat
ctgtctactt tctgttactg 11940 cctactagaa tatttaatac acgtgatatg
tttaataaat atttctattt gttgaataaa 12000 ataaaagact tccaagtagc
aaggaagatt atctgctaga aagccaaagt cctttcctcc 12060 ctggaggagg
ctctcctctt agagcctgcc atgaactagc ccggtgcacc ctttacctga 12120
gcatagctca ccttgcacat tgtaggaaag caggtcaacc acagagtcag ccttcatgtg
12180 gtaacgagcc cgaaggctga aatggtctgt gctggtgttg ccaccactgt
aggaggcgga 12240 ccagttgtac aagttgctgt agacattcgt ggagaggtct
agaacaccca ggagaggcac 12300 ttgcagttga tacaacttgg gaatggtaaa
agtagggact tggaactctc gagatggcag 12360 atggaatccc acagacttga
agtggagggc tggtgtccta acagtctcta acatctttag 12420 atctctggag
gatttgccac caaaaggcaa aggaatctca attttcaaac tgttcttgtt 12480
caaggtatat ttgacccggc catcgctgaa atgaacaaca aagataacat ccccacagtc
12540 agacatcagt cattcaaagt tctctgcctc tgaccttcac aacaatattg
tacttgcccc 12600 attcccaaag ccactctgca cttttctttg tgctactcct
atgcctggac ccctttgctt 12660 ttacctccac ctgtcaaaca ctcaaatcct
gacaaagctc ttctaagggc cactccttat 12720 cattaccatc ccacctaagt
catagccaat ttgtctcatt cttacagtta catagtccct 12780 tgcacctttc
tcaaagagca tgtcataatt ttgcttatat aattaattat ttctcatttg 12840
ttgatgtctc catggtatct tcttgaaaga aaaggctata ccttacccag tttctatctt
12900 ctcactgtac ctagtctagc attttgcaca taggggatac ttgatacaaa
tttgctaaac 12960 tgaattgaga caacttgatt tgtggaaaga acataggtat
ggaagtcagt aagcctgggt 13020 taaagtattt tctcagaatg ctgcctccat
ttagtgctca atggctgttc tgactctctc 13080 tagcttaaca gcaagcctgt
agtcaataac gcctctagcc attctgaatt cttcacagtc 13140 agagggagaa
tttgcttata acaggttgta ctgaataaaa tatcttaggg tgaaagtacc 13200
agaatcctct aatgactttc ttagattctt cctgacaggg ttggtgttct catgtttact
13260 aatgcaaaga tgccacagtg tttgtgctat agttattaat ctccttaaaa
tgcttaaatt 13320 atttccttta aaaatttaaa aaaatgtcta aatcatgcta
tgtaaaatct ttcatttact 13380 ttgcatggtt caagaagcct tgctgctttc
ttcttttacc tttttaagaa gaggttttct 13440 gggatgtgga agtctggcaa
tcccatgttc tggaggttga actccttcag gctattgagg 13500 tggtcttgca
aagtctgggt ataaggaaga ctcccagatg ccttctgaag ccatgagctc 13560
attgcctaca aaatgacagg agatttttaa ggtaatgggc ttggatgagc ctcaaagagc
13620 aatgaacatt aggcaaaaat accgatttga caagttaatt attaagctgg
acaatgcact 13680 gaaagttaaa aataaataac agaaaattat gaatcttcgt
tgccagtcac tgatcactgt 13740 ccatatttat aaaaggaatg tctaacatag
tcagcctcct agctggtgtc ctgaagcttc 13800 ctttgatacc actaggcaaa
gaactgttct acttagtagc atctctttga tatgtatcac 13860 attcagcttt
gtgtaactgg gtaactacac acttgtgaaa gttttttttt ctccccaaga 13920
attccctggg gggaaggaag catgccttat acatctttgg aaaccttcct gcacctagct
13980 cagagttgag gatgtaatta gcacttatat ccatgtattt attgactggc
agactcatac 14040 ttacaactat taatttggaa cccacgtgcc ggaaagtcat
gtctgtttga gggactctgt 14100 gatccaggag tctattagca tacatatgca
agctcttagg ataatcggag agatccacag 14160 ggaaattgga agtcattttt
ttggtatcta cattggtgcc tgtgttccat tcaaattcaa 14220 tcttctcttc
atctgaaaat acgtaggaaa tagttgtgaa tggtactagt tcagcctgta 14280
accacaggtc tcaacacctg cattactttg gaagtgctca cacaggggaa gagacacata
14340 ccataatgcc atgccaccct cttggaaact gtggagccat aagctgtagc
agatgagtcc 14400 atttggagaa gcagtttggc aggcgaccag tgggcgagga
tctcacttct ggcttctgct 14460 tgcaaacggg gtatggaaat aacacccttg
atttttcttt cttcctttgt gtcacaacta 14520 tggtaaagaa aatcagttgg
caccaatgat tttgtccttt caatggagat atgcaggatt 14580 aaacagaagt
tccatttgtg gtgatcaaaa ccagatacaa ggatggttca gagaaacagc 14640
cacagatcaa actcatcaac ttctaaagcc aacattcagc acaaattaag gactttgcaa
14700 acaggtttag gagaaagaga agttttttga aaactgtttg aatcctgctc
tgctatttac 14760 tagctctgtg attttgacca aatgatttaa cccctctgag
ctaccatttc cttatccatg 14820 aaacaggaaa actcacagtg acctcgcagt
ttattgtgag gaccgagata atttctttaa 14880 agcacccagc actgtgcttg
atacatgaat agtaagtgcc tgataaatgg taggaaaatc 14940 caactggaca
tgcgcagagg gttaaaatgt tgagagcttg gccgggtgca gtggcttaca 15000
cctgtaatcc cagcactttg ggaggccaag gcaggtggat cacgaggtca ggagatcgag
15060 accatcctgg tcaatatggt gaaaccctgt ctctactaaa aatataaaaa
ttagctgggt 15120 gtggtggcat gcacctatag ttccagctcc ttgggaggct
taagcaggag aatcacttga 15180 actcgggagg cggaggttgc agtgagccga
gattgtgcca ctgcactcca gcctggtgac 15240 agagtgagac tccatctcaa
aaaaaaaaaa aattgttgag ggctctctct gccactctga 15300 ttgtagacta
ggggagaaca tggcttggtc aggtatgaag tggaagagga ataatgaaaa 15360
gaaccaccca ggcctgcagt gcaggtcaga tgaccctcgg ccttctttac cttaggtggc
15420 ccatgagggc gacctcagta attttcttgt tctgaatgtc cagggtgagt
ctgtaagacg 15480 ttttgccctc agtagattca tcattaactc tgaggattgt
tccgaggtca acatcaaaat 15540 ccggaatttg gacttcactg gacaaggtca
tactctgccg attatatttg aatgtcatgg 15600 tagcctcagt ctgcttcgca
cctggacgag tgtataagag aatcaagaga tgtgtggtaa 15660 gaagctatgt
tttgggccgg gtgcggtggc tcacacctgt aatcccagca ctttgggagg 15720
ccaaggcggg cagatcatga ggtcaggaga tcaagaccat tctggctaac atggtgaaac
15780 cccgtctcta ctaaaaatac aaaaaattag ccgggcgcgt tggtgggtgc
ctgtagtccc 15840 agctactcag gaggctgagg caggagaatg gcatgaaccc
gggaggcggg gcttgcagtg 15900 agccgagatg gcaccactgc actgcagcct
gggcaaaaga gcgagactcc atctcaaaaa 15960 ataaataaat aaataaataa
ataaataaac aaacaaataa ataaataaat aaataaagaa 16020 gctatgtttt
ggaaggaaag agaggaaaag gtagagaaac tccaaggaat caatgtgctt 16080
aaacaaaaac acagttcagg attttactga attttttttt tctcattaaa attttttaat
16140 gggtctcatt tactgataat tttaaactca gttgtgttca tgtatgcaag
tctttatgga 16200 ttgcctgtta tgagcctatt gtgtgctggg cactgctctt
ggtactgggg aatccttgct 16260 ttcatggggc ttaccttcta gtgggggagg
tgtacaggca ataaacaagt aacttgctca 16320 tgtcagatgg taatgaatgt
tatgaagaag agtaaaactg gatacaagga taaaagaaag 16380 ggatagaaga
agtgttattt catacttggt ggtcagagaa attctctctg ataagggcac 16440
atgtgggcag agaccagaat gaaaagaggt tggagccacg ctgagttctg ggaggagcac
16500 attccaggca gaaggtcagg aatgcaaagg ccctgaaatg gggagcactt
agcttgtttg 16560 tggaacagca aaggctcgca gcccttctgg gccaggaaga
cacgacatct ctatattact 16620 ttctacccaa gatgcctgtc
ctggttcctt gctgcccttg ccctgggctc catctcccca 16680 actccccctt
tctttgtaac aagattcccg ggcgtccttc aatccatagg ctggtccatt 16740
ttatatcctt cgtaaaacaa tgtttaccaa acactatttc cccagcgtgc cctgctcctc
16800 cagtgctttc tatcatggga ttttcttcct agtttaattc tgtgatatgc
tatccctata 16860 taaaaatttt tccaaagtga acacctaaat gtgccccatt
ccctaaaacc tgctcctcgc 16920 tatccctata taattttttt tccaaagtga
acacctaaat ttctcccatt ccctaaaacc 16980 tgctcctcca gtagcccttt
ccatctcaat aaatggaagt tccatttttc agttgcccgg 17040 gccaaaaacc
ttaggtttgt cttcattccc cttttcctct tacactcaac atccactgta 17100
tcagcaaatc ttgtcagcct aatcttcaaa atatggccag aatccatccc cttctcatat
17160 ctcttctgcc attgccctga tccaaaacaa cactgattcc tgcctggatt
attgtgataa 17220 cctacatatt agtttctctg cccttgtctg cccatagtct
tttctcaaca taaacataaa 17280 tcaaatcatg aggactcccc aatggctttc
gtttaattca tagaaaaagc caaagttctt 17340 accatggcct atgagattat
ccttccttca cctctccata tctctctgtc ctcatcttct 17400 acctcttttc
tccctggtct ttctgctcca gccatactgt cttccttcct ttccttgaaa 17460
catggtagta gtgtgtcagc ctcagggcct ttgcacttgc tggtccctct gcctgttaac
17520 agtcttacca cacggcttct cctcttattt ctgataggtc tccttatgct
cactttacca 17580 gggagacttt cccttattat tccaaatgca ctgctttccc
tgcccttctt cctccctcct 17640 actcctagcc ctccatagac cagtctacgc
tctgttttcc tcctcagtac ttatctgata 17700 ttccaaatat taacttgttt
ggttgttcac tgtaacttcc atgaaggcag agactccatt 17760 gttcactgtt
ctagccccag gtcttagaat aggcctgccg cttattgggt actcagttaa 17820
cccaggtctt agaataggcc tgccgcttag tgggtactca gttaacctat ttgttaaata
17880 ggttaaatta catgtcttct cctcatgaat tctgaacctg agactgcgag
cagagatgag 17940 gcagctgtgt tttgaatact caccttctgc ttgagttaca
aacttcaggg tatccaccaa 18000 ggctctgtcc tctctctgga gctcataggt
tgcgctgaca gaatactgct caatctctcc 18060 tgtaggcctc agttccagct
ctaatctaaa gacattacaa tgaagacagt gcataatgtt 18120 agagctttca
aggatggtga ttatgttccc aaaacaattc agcctcaaat gcaacaaaaa 18180
tatggtcctt attttaacat tgtctcttgc ccttaactaa gatgatcttt ttgatccctc
18240 agtcctacaa gaacccaagg caagggcaga caccaggatt cccaagactt
tggacaagca 18300 gagcaggagt tggctggttg tttcagcaaa gctgagaaca
agcactggaa cttgggggct 18360 gaagacaggg agccacctaa gtacggtgaa
ggcttcaagc acattggtgg gctccaggaa 18420 gctcatagcg atgggtctgg
aggctcagaa acatggacac tgaattagga gcaaattggg 18480 atctgtgctg
tgatcccatc ccatgcacaa ggaagcaaaa ggtgacaccc aatgtgttgg 18540
ccagaggacc ctgctttaaa tacccaaaga tggtgcaaac ccagaaggct agtgacaggg
18600 tcttacaaca cagagtattt tttcctgtgc catgctaggt ggccatgatg
tggaaggtga 18660 gaaaatgctg ggtcaggcac tgagcatctc taacctggtg
tccccggtca gcggatagta 18720 ggaggcggag tctgtggagc tggcgttgga
gtaagcgcct gaggtgcagt aattcaggcc 18780 aggaaagact tgcttgcaaa
ctgaccagga ctgcctgttc tcaatgagag gtgggatcac 18840 ctccgttttg
gtggtagaga ccaaatgtaa tgtgttgctg gtgaagaaca aaaatacctg 18900
agttattgcc aagtcatgaa tcaaaatgga catcacaaat tctcagggtg caaatacccc
18960 cttatcctcc tgtcttccct cagtccacac ctatctctaa ctatttaatt
tactgagaaa 19020 caaataatat tagaaagaac atcaacatga caagccatga
gagtgactct gaagcttctg 19080 gctgagttct ttccatctgt tacctgtaat
ccccacaaca gtaggcttat ggttctcatt 19140 ttacagaagc agaggctcag
agatgttaaa tagctgatca ctggggctga acttcagtgt 19200 gccccttcct
tcctacaatg cagggcaaga gaaactgtct ctttcatatg aaatctccta 19260
ctctctacct gcttccttcc acctgcatta agcacgctca agtctcttcc cctgtgcccc
19320 tcccctatgc tcctttcttt cttttcttgc tttctcatct tcctcaccat
caagagttgt 19380 ctgcctttgc agtctctact tcctcccaat gtgctctgac
tgctcactcc atctcctgaa 19440 aatgctttca tcaatgtctc taatgccctc
cttgctccta catccaatgg gcatatttag 19500 gtttcatctt ccttgacctc
ccagaagcct tccatagagt tgcgcactca tgaccagact 19560 aagacgctgg
agcctctcac atctgaagag atgatggcac cttccttcca agtgtgtact 19620
taaaaactta gtacatgtaa gaaagttcaa cattggcctg gttttttcta aggatgcttt
19680 ctttgaaata gcacattttc ctccaagtat tgtttctcca agttgggggt
gccacaagac 19740 caagctttgc tgatgtgctg gagtcctaat gccagctttc
gcctacccat ggggtgatct 19800 cccctgtggc tgctctttgg cttctgtgac
cttttatggc tttggttctc cttctgcttc 19860 tctggtcact tcttctaact
cattgctggt tattcccctt cttcctttat atgtttgtct 19920 tctccatatt
tctgtcctca tctctcatgt ctcactctat accagttgct gtttaaccac 19980
aacaacctct gtatactaat gattcccaaa tccatatgtg gagcccaagc ctctttactg
20040 ggctcagatc cataggtcta tttgacagct agatttctaa actggttgtt
tcataggagg 20100 acatcaaata cagcacatcc aaaactgaat ttgtcacttt
ccttgaaacc agaagctctc 20160 ccactgtagt tcttattaaa atgaagggcg
cctccttcct cttttcttct ttcttttgcc 20220 ttgtccccat acccagttaa
tcacccagta cttatgattc tgcctcttag aaagttgtag 20280 aatctgctca
ctcctttcta tacctggctt ttctccactg caggccatta tcagctctta 20340
gccacatagg atcgtaagag atgtccaatg gtctcttcgt gtccagctgt gcctccttct
20400 cacccatctt ccacactaga gtcagagtat ttcttctaaa acatcacccc
tatcttattt 20460 ctgctcccct tttcaactcc tgtgccttca aaagagtcca
aactccttag gatggctttc 20520 gcagctgggc atgctctctc ccctgcctac
ctctccccat catctctgag ccccttacca 20580 ttctgcactt cttccagttc
cttgaaccat attcctttac cttcaggcca ttacatgctt 20640 ttccttttgc
ctggaacact cactcctttc tcctttgcct aaatgactct gacacagtcc 20700
tcagttatca ggtcaagtac acttgcttca gaatgcattc cctgactagc acaggctaca
20760 tgatataaaa tttcatggta ctctaatact ttctataact tagcccttac
tctatgatta 20820 ctgcttgctt aattgtgtgt ctcccttata aaatgtaagc
gttcaaaggt cagggacctt 20880 gtgtgtctca ctcaacatac gtggtatata
ttaggcaatt aaatatacta ttttttgttt 20940 tttgtgagac aggatcttgc
tctgtctccc aggctggagt gcagtggtgc aagcatggtt 21000 cactgaaggc
ttgaactctt gggctcagtt gatcctccca tctcagcctt ctgagtagct 21060
ggagctacag gcatgtgcca ccacgactgg ctaattttgt tatttattta attttttttg
21120 tagagatggg gttttgccat gttgcccagg ctggtatcca aacaccaggg
ctcaagcgat 21180 ccatccacct ggggctccca aagtgctagg attacaggca
tgaaccaccg tggctggcca 21240 attaaatatt atttttgaat gaacacatga
atgtgtctaa aatatgaaaa ctaaggcaaa 21300 tcaatggttg agggtgccac
ttaaaatgga actctccaaa agaggctgta ttccattatc 21360 acgaggttag
gagtttcatg ctttacctac ctagctacct caaatcaata tgttcttagg 21420
tattttttgg gggggaaaat attaattttc caaagatgat ctctccagag ctattgtttc
21480 ttcattctca aaacaaatag ataacaaaaa atcacaactt aataaaaact
aattaagcag 21540 taggtcttaa taagtctcaa agcagaaaga taaaggacag
tttagtaaaa tgtgaaaagt 21600 agaagaaagg taattatgta gttatgtagt
aagtctaggc tagaattaaa ataggtaacc 21660 cggaatcttt cctttctact
cctctcctgc ttcctcagga cctgaatgat ctcaatcaac 21720 tgtttagcct
ggcaaaattc tgcaggtaca ttctctgttc tctctttcaa actggctagg 21780
cagacttggc tgaaagaatt accctccact gagcagcttg actggtctct ttggggaagg
21840 aatgataaac ttcagcttcc cagcttttag ggcaacatga gcctccagac
ccgactcgtg 21900 gaagaagttg gtgttcatct ggaccccact cctagcgaag
tccggaatga tgatgcccat 21960 atttgtcaca aactccacag acacggaggg
ttttgccacc agttcagcct gcatctataa 22020 gtcagaaaac aacctattca
gattcattaa atacttcagt cccctgtcag tcagatcaac 22080 caccctttct
cacctccggg caaagatttc ctggataact cagacctttg gaaacattac 22140
tgggatttgt ttggaagaag gaaagcgagt tctcagttct ctagagttgg agtatactac
22200 ccagaactag aaagcacctg agttaacgtg aggcaaaacc agattaaaaa
gatgatagga 22260 atactcattt gttgagagaa ctgagttctt gtcttatggc
cataccacta ctggagctag 22320 tgaggtagtg cattctggtg gaagcttgaa
gtttttcata aaaaaataat gaggtgatta 22380 caatgatgaa gcattttgtc
ttgaagtgga aacacatttt taaatatgtt ttaacaagaa 22440 atgcaccctg
gaagaaagta ataacctaag aaatcaaaag gcaaacagaa tcttacgttg 22500
gctacttcca gttttactcc agccttggct ccgggagcaa tgactccaga tgaagatatt
22560 tgcaactgta atccagctcc agtggggagt tcaaaggcat tctccatgaa
gatgtagtga 22620 agaaaaaagt cattctttga gcccttcctg atgacctctc
caatctgtag acccaacaag 22680 ggagagcaaa taaaagtaag tctttttaaa
gtcggttttg ttaattacaa cctcccatac 22740 attggagagt aattcctctt
tatctggaaa ggaaggacta gcatattttg atgagctgaa 22800 tagtttgata
aataaagatc aaactgatgt ttttaccgtt ttcagtgaat caggatataa 22860
gaaatttcac tagatagata tcatgtcatt ttcaagtgga ctcagaaggc tcttaagaat
22920 attgctgggc ctcagagctt tatggacttt aagtatcata tatgccatgg
cttaattctg 22980 gtgttctgga gtctgactgt aagtgaaggt gtacttgaaa
tgtattatat tatgataaaa 23040 tcaatttttt gaaaccaaag gcctattgag
atgatgtaca tatcaatttg cattgggttt 23100 tattttttaa ataatatttc
aataaaatag cttgagacct gttttcttaa agtcaacatt 23160 tttttaatca
agccatagga gtacatttat atatttcctt ttatgaatta ataataaaaa 23220
ttatagcata tttagatagt gaattatagt ttgccaaata ctttctttta taatcattgt
23280 atcatttcaa tcttaaaagg accttcaggt caggtgcagt ggctcacacc
tgttattcca 23340 gcactgtggg aggcccaggc tggaggatca cttgaggtca
ggagttcgaa accagcctga 23400 catggttgtc aacaggcttg tctctactaa
agatacacaa attagccggg catgctggtg 23460 cacgtccaca gtcccagcta
cttgggaggc tgaggcagga gaattgcttg aacccggaag 23520 gcagagttgc
agtgagccga gattgtatca ctgcactcca gcctgggagc aagactccat 23580
ctcaaaaaat taaaaaaata aaaataaaaa taaaaggacc ttcagcaaag gtagaggaag
23640 gtataccgaa cattgttttc cagatgaata aattaatgct cttaaaaata
cagctaacat 23700 tatgaatagc ttaatttcta aatgttaaca tttccatgtg
atccaagagt catcttcagt 23760 gaaattcaag gcaaacctcc atctctgaag
aaagtacaag catcttttcg ggcttgtgca 23820 gctgggatcg taagggagtc
tgggcgatct aaaaaaaaac caacgtctgg tctcatgggc 23880 ccccagtggg
gcctgctgac ttaccatctg ggggatcccc tgcagagtgc gggcacccat 23940
cagaagcagc tttcccagga gctggaggtc atggagactg gcaaaaccaa gctcctctcc
24000 caagatgcgg aggtaggctc tggcttccgg gacttctttg gatttcaaat
ctttaatcag 24060 cttctcaaca ctgagcatta ttccatttac catatcctga
gagtttagta ataaaatggc 24120 cagtgagatg tcagcaatgt caaacacctt
tcagttccca atgtgggacc tgtctgatgc 24180 agccaggatg ggcctaaaaa
atgctccagg tgaggaacag ggagacactg aagtccaggc 24240 taattcctct
aagcagagat taaagaacta aggtcttgtc agggatagtg gcactcctaa 24300
agctattctt aattaaaaaa tctgtagaca tagtaagatg tggtgggatg agaggagggg
24360 tccctattct ccagtatgcc aggatcctca agattagaag atcctctatc
ccccacccgt 24420 tccctgctta gccttctctt ctttgaccag tgaagactac
aaggtgtttc ttaagaaaat 24480 gtagctgtta tataaattca ccagaagttt
gaaaaacact tgatcctaga caagttcttc 24540 tcagagagaa gaagaatctc
agagatggat gcccaggcat tgggaaaaaa aaaatgtgaa 24600 actgacagac
tcaaagcaga tcaaagatcc tcggcctcaa gtgggggtca ggaaattaga 24660
agatggagct cgtgctcaac agtgagcctg gaggggtaaa ttactgcctt gctctgcaaa
24720 gttgaatgtg catacaagtc acatgaggtc ttgctaaaat gcgcttctga
ttcagtagat 24780 tgtgggtgaa gcatgagaag ctgtatttct gaccagacca
cactttgggt agcaaaaaat 24840 tcaagaataa gctccagatc atacaaccta
tttccctcct taaatggggg ctcattgcta 24900 gcttcaaagt ggaagtttgg
tctcatttgc tagtgcactg atgggagtga gagccaaaga 24960 cagcagttaa
taataatgaa aatattaatt tagaactcgt aatagtgtct gcttattgaa 25020
caaccaatgc cagcatcagg cactgtgctt ggcattttag atgccttatt tcattgtctt
25080 taaataacct tgtgaggttt ggactgttat tagtagtctg ggatacagac
agaaacagat 25140 acacataata tcaaccaact tccccaagtg cgcacagctt
caagacaggg aagttagaat 25200 taaatctatg cccagagttc atactttttt
tttttttaag atacagtctc acactgtcac 25260 ccgggctgga gtgtaacggc
gtgatctcgg ctcactgcaa cctccacctc ccgggttcaa 25320 gcaattctcc
tgtctcagcc tcccgagtag ctaggattac aggcacccac taccatgccc 25380
agctagtttt tttgtatttt tagtagagac ggggtttcag tatgttggcc aggctggtct
25440 caaattcctg accttgtgat ctgcccacct tggcctccca aagtgctggg
attacaggca 25500 tgaaccacca cacccagccc agagttcata ctcttgttat
cctagagtac ttattgtgag 25560 tctggtttct gggcacactg aattcaggag
atctttaact gcagaggata ctggccctta 25620 ccccagcagg tctggttgat
aaaaaccata gttatccctt cagttagata gtattttgag 25680 gacttccatg
cttagaaaag aattgttttt gcattgagac ccaaagcttt ccttaagaag 25740
atacttcaca aatacacacc tgctcatgtt tatcatcttt ggtatagcca aagtggtcca
25800 ctaagacctt agagacacca tcaggaactt gaccattaac ccagtacaaa
gctttgttga 25860 cactgtctgg gaaaaatcct tgcttcccaa aaagagcttc
caatgttggc tcaaagcctt 25920 ttccttccaa gccaatctga gaaagaaaat
cagacaagaa aatggcatca ggtttctttg 25980 ttgtatgcca gcctagtagt
ccccactctt gatgtccatt tatctaaaca agtatttgaa 26040 taccacaggc
caggagctga ccttagcatt tcacaggagc tgctttatta aaatctcact 26100
agaatcttga ccttattact tgcttttttc tagaagaggc agctaaggtt cagataggac
26160 aaataattca acaaaggatt cagagttggg agtgatttga gatttgaagc
agatctgact 26220 aggcagcaca tatgcttcct tggctttggg acttaaagac
gagacatttg catttcaatt 26280 tttttttttt tttttttttt tgagatggag
tcttgctctg tcgcccaggc tggagcgcag 26340 tggcgcaatc tctcggctca
ctgcaagctc agcctcctgg gttcacgcca ttctcctgcc 26400 tcatcctccc
aagtagctgg gactacaggt gcctgccacc acgcccggct aattttttgt 26460
atttttagta gagatgggct ttcaccgtgt tagccaggat ggtgtcgata tcctgacctc
26520 atgatccacc cgcctcggcc tcccaaagtg ctgggattac aggagtgagc
cacctcgcca 26580 ggcctgcatt tctattaatg ttaagaagtc atgcttcagg
tgacccctag cggggagaga 26640 ggaaggcggg gaaggaggga aagaagaaag
cctcagaatc atggtaggaa gtgcctggtg 26700 gttcttagtt ttcctctggg
tagctcctgg ctcccaggga ctctctgttt atgatgctgt 26760 acaaaatggg
ctagagaacc tcaaactctt cacacttacc tcgatgaggt cagctgaagc 26820
aaatccaaag gcagtgaggg tagttttcag catgctttct ttaggaaggt agttatttgg
26880 atcaaatata agattccctt ctattttggc tgaggctggg tcaagtgatg
gaagagaaac 26940 agatttgtag agttgatagt tccgagagaa ttttctgaag
tccatgacag ttggaagttg 27000 agattctttc agagcttctt tcactaactt
tttcagacta gataagaaga agtatatttt 27060 gagctgacac accatgttat
tatcctttga ctcttgcacc ccaagtaaat atggatttcc 27120 aatcactacc
aaaatgtctt gatttcattg accctaagtc tttgggtcta gatctgctac 27180
acatttgcac aagtgtttgt ttcagaagca agggcagaca gtggctatgc caaaacctag
27240 ggttggaatt ccagctcagg gccctcagtg gtatatgggg tgaatagctc
ttacttactc 27300 ttggatatcc aattcttctg agttcaagat attggcaata
tgggaagcca caaagttctt 27360 cacttgctca ttctgttccc atggtagaat
ttggacaatt ttgttaatat ctgcctgtga 27420 aggactcctc atcaacataa
gataggcagc cagtcgctta tctcccggag aagcatcatc 27480 aaggaaagtc
tgaagaagaa cctcctggtc ctgcagtcaa aagaggagat ggttatcact 27540
gtcctgtggt cagaacacag aacatgcctg gcaaacactg gcattgtctg ctagctcttt
27600 cttaagcaca ggtctaattt ctctgtaatc gttcttcaaa tgctggtatt
gaatcttctt 27660 gccagtgttt cctagagcac tatcaagtaa gggttcccag
aaaaagcctg tggaatgcct 27720 gttgaattca attccattaa aacacataaa
ttatggccaa gttgtgattt gtttggagat 27780 ccaccactaa gtgaaaatca
agttgtttta acgtaagtta ttttaatgtg aggataaaga 27840 tgagaagggg
tagtaagagt ttggggctaa taaatagggt agtttccaaa gttctggagt 27900
ctttgcacct aagagggggc caagttggct atcctagcct aagcttattg ggggaaaatt
27960 aagcatgttt ttccatgatg agagggatgg tcaatctggt aggtggacgg
taggaagcaa 28020 aaacatctca cgttctgcat gttgacatag cagcaaagtc
aagtatttct ctcacgtcac 28080 atactgctag gcagtcaagt caataggctt
gttgaaattt gcaattagaa aacagtagag 28140 gccaggtgca gtggctcacg
cctgtaatcc cagcactttg caaggctgag gcaggcggat 28200 cacctgaggt
caggagctca agaccagcct ggccaacatg gcaaaactcc atctctacta 28260
aaaaatacaa aaattagctg ggtgtaatgg cacatgcctg taatcccagt tactcaggag
28320 gctgaggcag agagaattgc ttgaacccag gaggcagaag ttgcagtgag
ccgagatcat 28380 gccactgcac tccagcctgg gcaatagagt gagattccat
ctcgaaagaa ggaaggaagg 28440 aaggaaagaa gaaagggagg gagggaggaa
ggaaggaagg aaggaaggaa ggaaaggaaa 28500 gaaacagcag aataaagtca
gtagatgaaa tctagagtct cattccccta gtaccttcca 28560 aatccttgtt
aataaacttt cactttcaga cctcttcttg tggactttac cttgtcttta 28620
ggctccattt tccgcagagc ctggatggca gctttctgga tcatcagtga tggctttgta
28680 ctttggacac atttcaggat tgaagacttg agttctggag ttaactgctc
catggtttgg 28740 cccatatttc caatgacctg cattgaagaa aagaaacaag
aacccatcag ggtgcaggag 28800 agggaagtaa aaggtgtcca ggaaaagtgc
ttctgaaatg atgtatgtca tataaaagac 28860 tgagattacc cgcagaatca
aataggtgta atcttcatcc ccagtgcagt catcttgaat 28920 ctgttccatc
aggtaattag caatgtccag cagctcctgg gtccctgtag ggtttgtctt 28980
atgatagcta cagaataaga gaagagagtc aggacttggt aaccccagtt aggtttgtct
29040 taaaacccaa acttgtgaat tagaaaaaat aattataaaa ttcatatgga
atccaaaaag 29100 agcctgaata gctgaagcaa ttttaagcaa aaagaacata
gctggaggca tcacattacc 29160 tgacttcaga ttatacaaca aggctatagt
aatctaaaca gcatggtact ggcataaaaa 29220 tagacacata gatcaatgga
acagaatact gaacccagaa ataaagtcac atacttacag 29280 tcaacggatc
tttgacaaaa ttgacaaaaa catacactag ggaaaggaca cccttttcaa 29340
taagtggtgc tgggaaagat ggattgccat atgcagaaga ataaaactgg actcctatct
29400 gtcaccatat aaaaaaatca actcaagatg gatcaaagac ttaaatataa
gacctgagac 29460 tataaaaatg caagaagaaa acctagggaa aacttctctg
gacattggcc tagacaaaga 29520 attcgtgaat aagacctcaa aggcaaagac
aacaaaaaca aaaaatagac aaatagaact 29580 taattaaagt aaaaagtttc
tgcacagtaa aacaaataat caacagggtg gtaacctgca 29640 gaatgggaga
aaatatttgc aaactattca tccaatgggg tactaatatc cggaatgtac 29700
aagcaattca aacaactcaa caaaaaaatc ccccaaataa tcccattaaa aagtaggcaa
29760 aggacatgaa tagacatttt tttcaaaaga agacatacaa atggcaatag
gcatatgaaa 29820 aaatgcataa cattactaat cattagagaa atgtaaatta
aaaccacaat gagataacat 29880 cttacaagag tcaggagggc cattattcaa
aagacaaaaa ataacagttg ttggtgagga 29940 tacagagaaa aggaaacagt
taatcactgt tgatgggatt gtaaataagt acaaccacta 30000 tggaaaacaa
tacagagatt tctcaaagaa ctaaaaatag aactaccaat aaatccaaca 30060
atcccattac tgggtatata cctaaaggaa aagaaatcat tatatccaaa ggaaacctgc
30120 acccatatgt ttatcacagc actattcaca atagcaaaga catggaatca
acctaagtgt 30180 ctatcaatgg acgattggat taaaaatgtg aaatgtatag
gcaataaaat actattcggc 30240 cataaaaaga ataaaatcat gtcatttgca
gcaacatgga tggaactgga ggtcattgtc 30300 ttaagtgaaa caggacagtc
acagaaagac aaatattgcg tgttgtcatt tataagtggg 30360 agtgaaataa
tgtgcacaca aggacatagt gtgtggaatg atagacaatt gagactcaga 30420
aggtgtggaa gtggagggag atggatggtg agaaattact tagtgggtac actatatgtt
30480 gtctgggtga tggataccct gaaagccctg acttaactac tgcacaatct
attcatgtaa 30540 caaaattaca cttgttccac ataaatttat ataaaaatgt
aaaaaagaaa aagccaaaaa 30600 atttcatcaa aggaaaaacc tgctagcctg
agcaacatgg tgaaaccctg tatctaccaa 30660 gaatacaaaa attagccagg
gcatggtagc atgcacctgt ggtcccagct actcaggagg 30720 ctgaggtggg
aggatcactt cagcccagga atttgaggct tcagtgagcc gtgactgcat 30780
cactgcactc cagcctgggt gacacagtga gaccatctca aaaaaacaac aacaacaaaa
30840 aaaaacaaaa caaacaaaca aaaaaattca gaggaaaaaa ctgctaacta
atattgaact 30900 gaagttaata atatgcaagc tgtaatactt aggggaaagt
gtgttgatgt ttgcaattta 30960 ctttgaaatg cttcagaaaa taagatgaat
taatggatgg atgtatatgt gataaagcaa 31020 atataataac acttaaatag
taggagatag ctggttttct aggggttttc cctgtgaaat 31080 tctgtcaact
ttgctatgtt caaaaacttt tataatataa tgtagagaaa atatttttaa 31140
aaattcaatt tgtgtttgct gatttcttat ttcaagtcat taccccaaaa atgatcaaga
31200 aaaaacacaa gagtaaggag cagagtttga aagtggaagg aggggttcag
ttttaataca 31260 gagatgcaca gaggtgcaag atgttcctct gctcctagga
ggagaaatac agtgtggaaa 31320 ctcacttgtt gaccgcgtgg ctcagcgcat
acaaggtggc tcggctgcgc tgatccctcg 31380 ccatgttgaa gatctctcgc
agctgctgtg ctgagggctc ggggatcagg gccaccaggt 31440 aggtgaccac
atctatcaga agggggttgg catgcacacg tttcagccac tggaggatgt 31500
gagtggagca ctgaggctgt ccacactgaa ccaaggcttg taaagtgatg gggctgagaa
31560 gaaagacatg gataaagtta tacagaccac cttcagggca cataaaatat
tgctcatggt 31620 gtgtcctctg ccaggagagc ccttaatcac ctcattcact
attcaaatct aacttcaaaa 31680 cccaacttgg agctcagaac
catgatgctt tccttaggaa tattacccac agaagaagcc 31740 caatcgagaa
aagttccaag aggggactgt ggatctgtag caacaagaag actgttatca 31800
cagtcctttg gccagcacac agaatatgcc tggccaacat tagctgggtc tcattcctgg
31860 cctcagtccc ttctctcacc ttcatagcac ttaaagccag tgccttgaag
ggtagagaaa 31920 tcatattata tttttcgtct gagttctctc tccaccagcc
tataagcttc tggagagcac 31980 ctgcaataga cctaggaatt aaaagttacc
catcaaaatg tgggaactat tctccttctg 32040 ttttaacaca caaatacata
gctgccttga acacagtatg cgcgtgtgtg tttcatggaa 32100 ctcagcgcag
cagctgatag ttctctatcc agcaatcatg aactgattga ctaattgatt 32160
aactggattg atatccaaat ggtccctgag attctccatt tggccaaggt ttgaaagttc
32220 agtcagttac catcagtttt ataaaaagtt gagctgtaac cattagatac
ctggacacct 32280 caatcagctg tggcaagaga gatgtgactg cttcatcact
gaggcctctc agctcagtaa 32340 ccagcttatt gaagagatta gctctctgga
tattttgctc agagatggtt agttttttca 32400 gttcctggag agtcttcaaa
acagcttcgg cctgctttgg aggtgatgtg gatttggtgc 32460 tctcaaatgc
gaggcccatc ttcttagtac ctggaagatg gaaagtgtca aaggaactct 32520
agctttcttc atctcaacca tatctttgtc tactggaagc tggaaattgt ggagtatcag
32580 cacaggggaa aagggaaaga aactatcctg tattcaatgc ctgctagatc
tggctctgcc 32640 ataggtgatt gacttgtttt cactgactca tcatcctatc
cctgtggggt ggcattattg 32700 ttcaatttaa tgataaattc agccctgctt
ctgagaccca cagagttttt gggccatgta 32760 aattgctcat ccctggatct
cagctctgag tcccagagat gagacaaaga gatgagaccc 32820 agagatgagg
aagtgtgacc cagagatgag caacttaggt ttcccaagag ctctcagggt 32880
ctaagaggta gagctaaagt ggaatccaga cttgtctgag tctatcatca ggttaccctt
32940 agcttgaaat aattggtgag gaggtgctac tgttgacatg gttacaactg
ggaccaggca 33000 catgggtgct tatcaccatt tcttctaatt gggtctacca
gcagcttgtg gggaggggag 33060 acacttttgt atcctctcca ccacctactg
tgacctatgc caggaagaga ggtaaaagtc 33120 atcatggaaa tgtggtgtaa
tgagaatagt cctggcttag attacaacct ggcacttatt 33180 ttcccccctc
taatattctg cggccttgac tacatcaaac tctaagcctc tctgtgtcac 33240
tgtctcctct gtagaatagg tataaaatca catctctacc acctgacatc acaggttgct
33300 atgagcaaaa aggacaatga ggtaatgtac aagaaggaat ttggacagat
ataaatacaa 33360 tacaatactg ggtaagggtt ggacttgaga ttgttagagt
tccttctacc tctaaagact 33420 gactcctgag gttactatct ggccagactc
tgaaggttct ctcccacttg gtccacctcg 33480 tctgggacag ctctggatct
tatcacagga tgggtgatgt cagttctatc aaatatgtca 33540 tgttaaactt
aggattccag ttttctattt acaaatgcct aagtagcttt gggaacttct 33600
tttcctttat gccattgcaa cttgacatca tgaaatgagt ttgctttctc actagaggta
33660 gccagaacac cagtgtctgt atcacttgtt agtcagcaga catttaacag
ggctcagcaa 33720 gtggctaagc catgataggc acatcttgag tagattttcc
agcaactatg tggacagaaa 33780 ctcttacctt caccaaagaa gcggctgttg
atctttggtg tgtcttcaag tttcaaagtc 33840 tgtgtcactt gtgctaccat
cccatactta ttcctggtaa ccaaggaagc acaccatgtc 33900 acggatggcc
agaacatact attctttcca tgttgaaggt tttccctgtc tctgcatcta 33960
cccaagtcct gcccatcttt caaagcatgg gtaaatccag ccattgttgg ccctgaatga
34020 ataacatcca cctcttcctg gtcacgctag ttatcctccc atcaaaaaga
catgcgtgcc 34080 ttatactctc cactagtctg gagtggccag aggtaggaac
tagtgcaatg tctcaaaatt 34140 ctttcgtatc ttccacaaca tagagccaag
aatatggcag ttactcaata tatattcatt 34200 gattatcagt tttcaaagtg
gatttggata cacaagtttc cagtcagtat cccaaatctt 34260 ccatgaaacc
ttttctaacc agggtaccct gcagtgatcc tcaggttttc tggatctaca 34320
gcccttaaag aatgggtctt agatatcttt tgttggtctt ggattgtttt gcatgcctag
34380 gacttttctg ccaaatggcc tgttcgctta aaaaggggga cagggagggg
cggcatttta 34440 tccctctcca ttcctccctc ccaggcacag gtttgcctgg
aacagagcac ttgagaagtg 34500 ttcagttcac actgacccat taaatgacaa
atcaggggtg catcacatga cctacttgta 34560 ggagaaaggc aggaagaggt
gttgctcctt gcagatggct tctgccacat gcttcctctt 34620 agcgtccagt
gtgtactgac aggactggct gctgctgatc agagttgaca aggggcgggt 34680
ctatgaaaga gattggagac gagcattttg atcagtccct gtatcttctg ttaaaagcat
34740 ttaccttcaa ggtagaaggg agcaggagtc ctgtaccact agataaactc
agagaaagaa 34800 gataactggg tacttgacat ttagaccagg gggtgcaaca
agtcggcatc cccaaagctg 34860 agtcaagctg tagactcctc tttttccacc
ctcactggac aatgtctaaa ctttgtatcc 34920 tccacatgcc catcccacca
catagcccct cacacacctg ctcagtaact tttctttgcc 34980 caaaagccca
ttcttacatg tctctttcat ttacatattc tttgcatttt ttgggttcct 35040
gctccacatc ttccatgaaa tgttgcccgc tgagctacat gaataattct ttctccttcc
35100 tggtcaggcc caggacttct tgtctgcact gttcagctta tcaccttatt
gagtatacca 35160 cgtatacttt attgacctgg ctcctgcatt agactggaag
catcctgagg ggaaggtcca 35220 tggttcatct gccactgtcc cttccttggt
gctgcccaga gtggggttga gcaaacagca 35280 gatgctcaga aaagacccct
aagccagtgc attatcctct tctgactgtg gcactcatgg 35340 tcatcagact
ccctgttttc aaccctaagg acaatgacca ggaacagcgc ccagcatgac 35400
agtgattgcc caagttattc atgttcactg agacaggctg gggctcatat cagggaagat
35460 gttgcagggg tcaggcaggc tcaggtaata tggtgctcca tggccccagt
taacagagag 35520 gagagggtgc tccaggccgg tggcagtggg gccatgtttg
gtcagggtgg tactgggggt 35580 ggaaggacaa gaagctggag cactctaagt
cactccactt catgggatga gggagagggg 35640 gagaacacag gtcttgggac
ttcttcaaag gccagaggct tggagctggc acaaggagtc 35700 cttgtgcaga
aaagaacaga aggtggtttt gctgccctac atccatcccc ggaaccttct 35760
cagaggtgag tagtcattac tctcaagtga tgagattaga ggcagtttgg tgccagtgac
35820 agaaggacag gaggaaagac agagctctgg gctgggaggg aggagcctga
ctcaagcctt 35880 ggctcaactg atcccaggcc gtgaagcctg ggcagacctt
cccgtgcctg ctcagggtcc 35940 tatctctagt ctgtataaaa aacagccagg
gcaggctgtc ctcaaaggtg cccactagct 36000 caaaagttct ggaattgtat
taataagagg atgctccttg ctgtgcacga cagtgctgac 36060 atgggactta
ccatgccttt gatgagagca agtgggctga tgcctgtgcg gatgggcttg 36120
aagcgatcac actgccccag gtctctttca gtggatattt ctgttgccac attgcccttc
36180 ctcgtcttga cggtaaagtg agtggagcag tttccataca cggtatccta
tggaggaaga 36240 agatgcaacc acatgtattc aacacgggca acatccttgg
gtacttggga gggatggggt 36300 ggggatcgtg aaagaaaaag cagaaggaat
ctagctttgg gagggacttt ctttttcttc 36360 ctatgcagag tgtggtcttg
ctagtgcctg gccagcccaa gttgggagag agaaaaccag 36420 ttaaaggtgg
gtctgcagaa aggcctccgc aggttgcatc ggtgtcttct cccattatgg 36480
tgtcgtagag aaattagaac acacaaaagt gctcatggga ctgaggtgaa atgaattctg
36540 tctcacagtg aaacctgaaa ttcgtaaaag agaccagcaa cagatcccat
tatttttggt 36600 gtttaaaata cacacaggat ttactgtgta caccataacc
cagcaaacac aggtgaagca 36660 tcaaacaaaa gcaggggtgg ggtggccagg
actcctcaat gactgtttta aaattagacc 36720 caacctgata agcctgcttg
ggatgatctg tcagtgagcc taaggggcag gaggaacctc 36780 gggcaccagt
atttcacgcc aatccagggc ttcctatgta actagtcatg gagctgactc 36840
agtgatctgc tttgtatatg gtaggactgg tctctaacac atgaagatga gtttcaaggg
36900 ccactgctat cagctttcta aatcctcacc agaaacaaca cttgcttggc
ttcttctgtc 36960 tctgggggaa ccaggagggc agaaatgatg cccctcttga
tgttcaggat gtaagtaggt 37020 tcatctttct ccgggtaaag gaaaacctgc
ttcccttctg gaatggccag cttgagctca 37080 tacctgtccc agagagagga
tggtcacgga aatgtccttc tccattacaa cttgctggaa 37140 gtaagctggg
tggcactgaa gtttcttttc tcatattttt ttaaccaatt gttcttctga 37200
catcatttat ttgtaaatat agaatatagc tacacataga catacatttt caaaaaagta
37260 tgcacatata cataagtttt ggtgattctt tttttttctt tttttctttt
ttctttttct 37320 ttttttgaaa tggagtctcg ctcggtcgcc cgggctggag
tgcaatggcg caacctcagc 37380 tcactgcaac ctccacctcc tgggatcaag
tgattctcct gcctcagcct ccggagtagc 37440 taggactact ggcatgtgcc
atcacaccca gctaattttt gtatttttag tagagacagt 37500 tttcaccatg
ttggccaggc tggtctcaaa ctcctgacct caggtgattc tccctcctcg 37560
gcctcccaaa gtgctgggac tacaggcatg agccaccagg tccagctgtg ttttaacttt
37620 ttttgcttgg ctcatccagc cctgctctgt tctccaccca tatcagtctc
tttgcccatc 37680 ctccccatac ggacacccga gtggttgttc catttgtttc
tgcacactca tacaatcctc 37740 tgtaaacatg ggtgagtgca aacagagtca
tacaaagatt ttgccattgt ttgttttata 37800 caagtggcat tgtattatgc
atacttttat cttgtttttc tcacttaatt atacttcaca 37860 aaaatccttc
tgagtcactt ggtacagctc taacagattt ttttaaaagc tgcatgcaga 37920
acattccatt ccatggctaa atataattta ttcacacatt gccccatttg atggcatttg
37980 ccctctctct ttctggattt tttttaaacc acacaaacaa taccaaaaca
aacatcctta 38040 tgtgcctatc ttcacacatt actgctttta ttttcatggg
ctagagtccc aaagactaaa 38100 atatctgtgt caaagggtat taagtatttt
aaattacagt agatattgcc tgatggctct 38160 cgtttccacc agcagctcag
gacagcacac ttttcctcac tggcctgcca gcaacaggtg 38220 ctatcactgt
tttccagttt tgcagtatga tgggtaaaag cagataaata tcttgttgtt 38280
acattaattc gcatttcttg actaccagtg aattggagct tattttcata cacttgttgg
38340 acagtcattc ttttgtatgg tgtcaggcca tggctgaagc ccatgctgac
tttggtctta 38400 gggatttccc actgggtact ctctgagttt ctgaggacct
ggctgcagcc actctggtta 38460 catcaggagg aaaaggtgac ggacttatct
cccacagcct acactctctc agaaagttcc 38520 caacacgaag aaatgataaa
tgtttgagat gatgggtatg ctaattactc tgatctgatc 38580 accatacatt
acatgtatca aaatatcact atgttctcaa taaatatgta taattattgt 38640
atgtcaattt ttaaaaaaga cagttaaagt catgaagatg caaacatgga aagccctcct
38700 tttcaagaac aggagtctct tctgtatttg gagaagacca aggaccaaaa
aataaaccac 38760 tctttcctcc tgccaccata cccctctccc tcatatacag
ttaagtcttg gagaaaataa 38820 agcataggaa agaaagaagc ctcacagcct
attttggcaa gaaaggactg gcatctgaat 38880 ctaatgttta gcaaaactga
aaaatgggca aggatggtga tatggtttgg ctctgcgtcc 38940 ccacccaaat
ttcgtctcgt agctctcata attcccacgt gttgtgggag ggacccgggg 39000
ggaggtgatt gaatcatggg ggcaggtctt tcccgtgctg ttctcgtgat tgcagatggg
39060 tgtcgggaga tctgatggtt ctaaaaacag gagtttctct gcacaagctc
tctttgccag 39120 ccaccatcta cataagatgt gacttgctgt ccctcacctt
ccgccatgat tgtgagacct 39180 ccccagccac gtggaactgt gagtccaata
aacctttctt ttgtaaattg cccagtctca 39240 ggtatgtctt tatcagcagc
atgaaaacag actaatacag acagagtttt ctgatgctac 39300 caagttaaca
actctatcct gcctgtattc atacacacct aggaccctac aaactgtgat 39360
tgaggatgag gcaggggtga tgttgaaaat atttacaatg ggtgcgacat gggccctgac
39420 cagtcagcag agatgcagct gcagtggccg atcagcgtgc agtggctgaa
tgccagccta 39480 ggagggggag ccaccgaagc cttggtgctc ctctgccctg
cggtgaacag accctgcccc 39540 gccatgtgcc ggccacagca gccagtgcct
ctgggacccc acaccaaaga ccaccaagca 39600 ctagtcttga ctagttcttt
aaataagaat tcacttgttt aaacaaagca tctcagtttt 39660 ataatctcaa
gaagtttaaa gcatgagatt tttagaaaca catgaaaatg ataattaaag 39720
gaacctaact agggaaggtt aactaatgct tctctctttt tgatgattgt gaaaactcag
39780 taattccctg atccacgatg gatacggaat acaaatactt acagtcacat
ccgtgcctgg 39840 tgcaaacaca caagttcata cctcagcgga cacacacaca
tgcgtgtgct catgtacaac 39900 atgacttacc tggacatggc tgcagcaaac
tcctcagagt tcttggtttt cttcagcaag 39960 gctttgccct cagggttgaa
gccatacacc tctttcaggg tgcactggct ggtcttcagg 40020 atgaagctgc
agagctgggg aacctccagc tcaacctgag aattcagggt agcagagcat 40080
tgaggttgtc tatcaagaat gagaggtggc ccctgaagcc cagggcttaa tctctaatca
40140 gagcaccaaa gggaatggtg ctgggaacac cactgcctgc gcctcaacac
cacatgcctt 40200 atcaacatgc ctcctgggtt ctctgtgcac caacctagac
ttagtcctat tgctgacgtt 40260 ttccccctcc cgggtaacac atttcttaag
tttgccccta ggcatgggaa ggaggatgtc 40320 cttttattgg ttctaaagtt
actcacttta attataaaga gctgtggtta aatagaagcg 40380 ctgcagacta
ggagtgaaag tgaagaagaa aaacagaaag caggaagagg gccatccttc 40440
gtttccacag caaatgtctc cttaatgtca cccaaaaaac tggttatatc tattagggcc
40500 tatttagagc tttgcatata gctggagttt caacggattc ctacttatta
tccagcacta 40560 ttcaaataac tttatagaaa tgttgtacat gtgtgctgtt
caatatggga gccagtagcc 40620 agggtggctg ttgagcagtt gaaatgcagc
taatgtgaca acgaatatga atttttaatt 40680 tcattccatt taaatagccc
aatgtggctc atgtctacca tattggacag cacatacaag 40740 agctacggta
ctctctaaaa aaaggtgact gctcagcttg acttctcttc cccacccaaa 40800
ccccaatagc agcctgtaga tctgctccac atgtatgtaa catgagtaca accagtctca
40860 ataataagca aagttttatt ttagaacaaa ctgtatgttt atattttttt
cttctttcca 40920 ttcatttttc agcaacagat tctgtttgac ttaaattata
aaaactgcat ttcacagtgc 40980 gattccgagt tgcctgcctc ccatagctca
cctactgggc ctctctcacg ctgaaatcta 41040 cagacccaca ctgctgctaa
tctagatcat ggattcctat tgcatctggg aagttaacgg 41100 gaaaatactt
ctgacttgcg aaatttggtg ggggcagacc acatctcagc aatgtggctg 41160
acttacaaac agaggcaaag tcccagtgct ttgatcagat tacaacagtt ctgggatgtt
41220 ctgcgtttgc tcagtacaca ccctccggga aggtcgcgtg ttgggcgccc
gctggaacag 41280 ggctggggga aagctgtggg ctctaggtcc ctcctgcctg
catcctccat accttgcagt 41340 tgatcctggt ggcacttctt gaatcagcag
tcccagggac tccactggaa ctctcagcct 41400 catagttgta tgtgtacttc
cggaggtgct tgaatcgggt cgcatcttct aacgtgggga 41460 gaaatacgtc
agccacatag cagaaatagc tctcccaagg acagccaatt ctgggcagag 41520
aggggcagtg gcatagagaa ttaaaaatgg taattcaact caaattattt tttaattgac
41580 tttatactta attttccttt atagatttga cttctccatt atgtttttgc
tgaggctaat 41640 gtttaaattg gctggcatgt cttgaatatc tcatctgagg
cacagggaag ggagtgacaa 41700 gtgtcaagta atggggctca gaaaatctgc
atgagagacc ctctggcaaa ttaagagatg 41760 acaacattac gcaaatagta
ctatctctaa tgctttttta gcttgaacac gccgtaattc 41820 cgtgactcta
agactaccaa atacttaagg caaagttcat taagcattaa gcgtaactaa 41880
cttaacaaac tcctagaaaa gaatttctgg aggtttccac tgtagttgag aggaattttc
41940 tgaacaattg aagaaaaaga acatctgggt cctgagtcca gctgcagtga
tgacagacgg 42000 ccactagatg gcggtgctgc cccatcagaa gtccgccccg
cggctccagc acacagggtc 42060 gcgcttggag gccagttcct ttaaccccca
ggggtcaggt aaataggaag ggggtggagg 42120 ttcgatttct tcacaaaggt
taaagtcagt atttcctcac cctcacatgc ttcgaatcaa 42180 tgactagtca
ctgataaaca ggacagtgat gtttcttcaa agtgtgcaca ggccagcgga 42240
gagacaatcc cttccctccc ccgcccccag gctgaacttt tgggacagaa cagaaggcca
42300 ggtagaagag agttggcatc ccttgggtgt gggcagaggc tcagggaact
tgcttcctgg 42360 ggtcagagca aggcacacca cgatgccatc tcagccctgt
agagtgggag gccctcaggg 42420 acccgggtgt aggagagtgc acggggctgg
gcgcccttcc acgccccatg cgcagatgcc 42480 ttacttggac agaccaggct
gacattttcc agcatttcct cttctgtaag acaggagaaa 42540 gaaatctgtg
agcttcccac gtcttcccag cgggtgctag ggcccgacag ggggaccacc 42600
ggcacaggtt tcacctttca ggagggaggc aggctccgga gaccccctcc tcagcccctc
42660 catcccgcgc cccccatcct gagcctgcag gggccgccag ctggtccaat
ccccccactc 42720 gccctggacc ctgtggctgc cctccctctg gcctaggccc
aggctgcccc ggccaacctc 42780 gtgccgccgg ctccctcccg ctccctctgc
gcccgcagag cggccgcgca ctcaccggcc 42840 ctggcgcccg ccagcagcag
cagcagcagc gcaggcagcg ccagcagcgc cagcagcgcg 42900 ggcctcggcg
ggtccatcgc cagctgcggt ggggcggctc ctgggctgcg gcctggcctc 42960
ggcctcgcgg ccctggctgg ctgggcgggc tcctcagcgg cagcaaccga gaagggcact
43020 cagccccgca ggtcccggtg ggaatgcgcg gccggcgccc gcaccccatt
tataggaagc 43080 ccaggctgca agagcgccag gattgcaaaa ggtccaaagg
gcgcctcccg ggcctgacct 43140 gtttgctttt ctacactggc ttctctttga
gccttgaaga gcctcgggga gggggcccac 43200 ctgggatgca gccgcagcca
ccaggggctg ggtcccaggt gggttccctt ccccaagcgt 43260 cttcagtgct
ctggcgcggc ccttcctgtg tctcagtggg gccatggcca gcgcctcagg 43320
gtctgagaag cctgccctga ccaggggtgc cttcttcaga tgacccacca tggggacaaa
43380 atctctgctt ctcctggact gaattgggag ccacgaggag aaccagtcct
gagcgctgtc 43440 ttggt 43445 3 19 RNA Artificial Sequence Antisense
Oligonucleotide 3 cgagaggcgg acgggaccg 19 4 19 RNA Artificial
Sequence Antisense Oligonucleotide 4 gcucuccgcc ugcccuggc 19 5 21
DNA Artificial Sequence Antisense Oligonucleotide 5 cgagaggcgg
acgggaccgt t 21 6 21 DNA Artificial Sequence Antisense
Oligonucleotide 6 ttgcucuccg ccugcccugg c 21 7 20 DNA Artificial
Sequence Antisense Oligonucleotide 7 tccgtcatcg ctcctcaggg 20 8 20
DNA Artificial Sequence Antisense Oligonucleotide 8 gtgcgcgcga
gcccgaaatc 20 9 20 DNA Artificial Sequence Antisense
Oligonucleotide 9 atgcattctg cccccaagga 20 10 14121 DNA H. sapiens
10 attcccaccg ggacctgcgg ggctgagtgc ccttctcggt tgctgccgct
gaggagcccg 60 cccagccagc cagggccgcg aggccgaggc caggccgcag
cccaggagcc gccccaccgc 120 agctggcgat ggacccgccg aggcccgcgc
tgctggcgct gctggcgctg cctgcgctgc 180 tgctgctgct gctggcgggc
gccagggccg aagaggaaat gctggaaaat gtcagcctgg 240 tctgtccaaa
agatgcgacc cgattcaagc acctccggaa gtacacatac aactatgagg 300
ctgagagttc cagtggagtc cctgggactg ctgattcaag aagtgccacc aggatcaact
360 gcaaggttga gctggaggtt ccccagctct gcagcttcat cctgaagacc
agccagtgca 420 ccctgaaaga ggtgtatggc ttcaaccctg agggcaaagc
cttgctgaag aaaaccaaga 480 actctgagga gtttgctgca gccatgtcca
ggtatgagct caagctggcc attccagaag 540 ggaagcaggt tttcctttac
ccggagaaag atgaacctac ttacatcctg aacatcaaga 600 ggggcatcat
ttctgccctc ctggttcccc cagagacaga agaagccaag caagtgttgt 660
ttctggatac cgtgtatgga aactgctcca ctcactttac cgtcaagacg aggaagggca
720 atgtggcaac agaaatatcc actgaaagag acctggggca gtgtgatcgc
ttcaagccca 780 tccgcacagg catcagccca cttgctctca tcaaaggcat
gacccgcccc ttgtcaactc 840 tgatcagcag cagccagtcc tgtcagtaca
cactggacgc taagaggaag catgtggcag 900 aagccatctg caaggagcaa
cacctcttcc tgcctttctc ctacaacaat aagtatggga 960 tggtagcaca
agtgacacag actttgaaac ttgaagacac accaaagatc aacagccgct 1020
tctttggtga aggtactaag aagatgggcc tcgcatttga gagcaccaaa tccacatcac
1080 ctccaaagca ggccgaagct gttttgaaga ctctccagga actgaaaaaa
ctaaccatct 1140 ctgagcaaaa tatccagaga gctaatctct tcaataagct
ggttactgag ctgagaggcc 1200 tcagtgatga agcagtcaca tctctcttgc
cacagctgat tgaggtgtcc agccccatca 1260 ctttacaagc cttggttcag
tgtggacagc ctcagtgctc cactcacatc ctccagtggc 1320 tgaaacgtgt
gcatgccaac ccccttctga tagatgtggt cacctacctg gtggccctga 1380
tccccgagcc ctcagcacag cagctgcgag agatcttcaa catggcgagg gatcagcgca
1440 gccgagccac cttgtatgcg ctgagccacg cggtcaacaa ctatcataag
acaaacccta 1500 cagggaccca ggagctgctg gacattgcta attacctgat
ggaacagatt caagatgact 1560 gcactgggga tgaagattac acctatttga
ttctgcgggt cattggaaat atgggccaaa 1620 ccatggagca gttaactcca
gaactcaagt cttcaatcct caaatgtgtc caaagtacaa 1680 agccatcact
gatgatccag aaagctgcca tccaggctct gcggaaaatg gagcctaaag 1740
acaaggacca ggaggttctt cttcagactt tccttgatga tgcttctccg ggagataagc
1800 gactggctgc ctatcttatg ttgatgagga gtccttcaca ggcagatatt
aacaaaattg 1860 tccaaattct accatgggaa cagaatgagc aagtgaagaa
ctttgtggct tcccatattg 1920 ccaatatctt gaactcagaa gaattggata
tccaagatct gaaaaagtta gtgaaagaag 1980 ctctgaaaga atctcaactt
ccaactgtca tggacttcag aaaattctct cggaactatc 2040 aactctacaa
atctgtttct cttccatcac ttgacccagc ctcagccaaa atagaaggga 2100
atcttatatt tgatccaaat aactaccttc ctaaagaaag catgctgaaa actaccctca
2160 ctgcctttgg atttgcttca gctgacctca tcgagattgg cttggaagga
aaaggctttg 2220 agccaacatt ggaagctctt tttgggaagc aaggattttt
cccagacagt gtcaacaaag 2280 ctttgtactg ggttaatggt caagttcctg
atggtgtctc taaggtctta gtggaccact 2340 ttggctatac caaagatgat
aaacatgagc aggatatggt aaatggaata atgctcagtg 2400 ttgagaagct
gattaaagat ttgaaatcca aagaagtccc ggaagccaga gcctacctcc 2460
gcatcttggg agaggagctt ggttttgcca gtctccatga cctccagctc ctgggaaagc
2520 tgcttctgat gggtgcccgc actctgcagg ggatccccca gatgattgga
gaggtcatca 2580 ggaagggctc aaagaatgac ttttttcttc actacatctt
catggagaat gcctttgaac 2640 tccccactgg agctggatta cagttgcaaa
tatcttcatc tggagtcatt gctcccggag 2700 ccaaggctgg agtaaaactg
gaagtagcca acatgcaggc tgaactggtg gcaaaaccct 2760 ccgtgtctgt
ggagtttgtg acaaatatgg gcatcatcat tccggacttc gctaggagtg 2820
gggtccagat gaacaccaac ttcttccacg agtcgggtct ggaggctcat gttgccctaa
2880 aagctgggaa gctgaagttt atcattcctt ccccaaagag accagtcaag
ctgctcagtg 2940 gaggcaacac attacatttg gtctctacca ccaaaacgga
ggtgatccca cctctcattg 3000 agaacaggca gtcctggtca gtttgcaagc
aagtctttcc tggcctgaat tactgcacct 3060 caggcgctta ctccaacgcc
agctccacag actccgcctc ctactatccg ctgaccgggg 3120 acaccagatt
agagctggaa ctgaggccta caggagagat tgagcagtat tctgtcagcg 3180
caacctatga gctccagaga gaggacagag ccttggtgga taccctgaag tttgtaactc
3240 aagcagaagg tgcgaagcag actgaggcta ccatgacatt caaatataat
cggcagagta 3300 tgaccttgtc cagtgaagtc caaattccgg attttgatgt
tgacctcgga acaatcctca 3360 gagttaatga tgaatctact gagggcaaaa
cgtcttacag actcaccctg gacattcaga 3420 acaagaaaat tactgaggtc
gccctcatgg gccacctaag ttgtgacaca aaggaagaaa 3480 gaaaaatcaa
gggtgttatt tccatacccc gtttgcaagc agaagccaga agtgagatcc 3540
tcgcccactg gtcgcctgcc aaactgcttc tccaaatgga ctcatctgct acagcttatg
3600 gctccacagt ttccaagagg gtggcatggc attatgatga agagaagatt
gaatttgaat 3660 ggaacacagg caccaatgta gataccaaaa aaatgacttc
caatttccct gtggatctct 3720 ccgattatcc taagagcttg catatgtatg
ctaatagact cctggatcac agagtccctg 3780 aaacagacat gactttccgg
cacgtgggtt ccaaattaat agttgcaatg agctcatggc 3840 ttcagaaggc
atctgggagt cttccttata cccagacttt gcaagaccac ctcaatagcc 3900
tgaaggagtt caacctccag aacatgggat tgccagactt ccacatccca gaaaacctct
3960 tcttaaaaag cgatggccgg gtcaaatata ccttgaacaa gaacagtttg
aaaattgaga 4020 ttcctttgcc ttttggtggc aaatcctcca gagatctaaa
gatgttagag actgttagga 4080 caccagccct ccacttcaag tctgtgggat
tccatctgcc atctcgagag ttccaagtcc 4140 ctacttttac cattcccaag
ttgtatcaac tgcaagtgcc tctcctgggt gttctagacc 4200 tctccacgaa
tgtctacagc aacttgtaca actggtccgc ctcctacagt ggtggcaaca 4260
ccagcacaga ccatttcagc cttcgggctc gttaccacat gaaggctgac tctgtggttg
4320 acctgctttc ctacaatgtg caaggatctg gagaaacaac atatgaccac
aagaatacgt 4380 tcacactatc atgtgatggg tctctacgcc acaaatttct
agattcgaat atcaaattca 4440 gtcatgtaga aaaacttgga aacaacccag
tctcaaaagg tttactaata ttcgatgcat 4500 ctagttcctg gggaccacag
atgtctgctt cagttcattt ggactccaaa aagaaacagc 4560 atttgtttgt
caaagaagtc aagattgatg ggcagttcag agtctcttcg ttctatgcta 4620
aaggcacata tggcctgtct tgtcagaggg atcctaacac tggccggctc aatggagagt
4680 ccaacctgag gtttaactcc tcctacctcc aaggcaccaa ccagataaca
ggaagatatg 4740 aagatggaac cctctccctc acctccacct ctgatctgca
aagtggcatc attaaaaata 4800 ctgcttccct aaagtatgag aactacgagc
tgactttaaa atctgacacc aatgggaagt 4860 ataagaactt tgccacttct
aacaagatgg atatgacctt ctctaagcaa aatgcactgc 4920 tgcgttctga
atatcaggct gattacgagt cattgaggtt cttcagcctg ctttctggat 4980
cactaaattc ccatggtctt gagttaaatg ctgacatctt aggcactgac aaaattaata
5040 gtggtgctca caaggcgaca ctaaggattg gccaagatgg aatatctacc
agtgcaacga 5100 ccaacttgaa gtgtagtctc ctggtgctgg agaatgagct
gaatgcagag cttggcctct 5160 ctggggcatc tatgaaatta acaacaaatg
gccgcttcag ggaacacaat gcaaaattca 5220 gtctggatgg gaaagccgcc
ctcacagagc tatcactggg aagtgcttat caggccatga 5280 ttctgggtgt
cgacagcaaa aacattttca acttcaaggt cagtcaagaa ggacttaagc 5340
tctcaaatga catgatgggc tcatatgctg aaatgaaatt tgaccacaca aacagtctga
5400 acattgcagg cttatcactg gacttctctt caaaacttga caacatttac
agctctgaca 5460 agttttataa gcaaactgtt aatttacagc tacagcccta
ttctctggta actactttaa 5520 acagtgacct gaaatacaat gctctggatc
tcaccaacaa tgggaaacta cggctagaac 5580 ccctgaagct gcatgtggct
ggtaacctaa aaggagccta ccaaaataat gaaataaaac 5640 acatctatgc
catctcttct gctgccttat cagcaagcta taaagcagac actgttgcta 5700
aggttcaggg tgtggagttt agccatcggc tcaacacaga catcgctggg ctggcttcag
5760 ccattgacat gagcacaaac tataattcag actcactgca tttcagcaat
gtcttccgtt 5820 ctgtaatggc cccgtttacc atgaccatcg atgcacatac
aaatggcaat gggaaactcg 5880 ctctctgggg agaacatact gggcagctgt
atagcaaatt cctgttgaaa gcagaacctc 5940 tggcatttac tttctctcat
gattacaaag gctccacaag tcatcatctc gtgtctagga 6000 aaagcatcag
tgcagctctt gaacacaaag tcagtgccct gcttactcca gctgagcaga 6060
caggcacctg gaaactcaag acccaattta acaacaatga atacagccag gacttggatg
6120 cttacaacac taaagataaa attggcgtgg agcttactgg acgaactctg
gctgacctaa 6180 ctctactaga ctccccaatt aaagtgccac ttttactcag
tgagcccatc aatatcattg 6240 atgctttaga gatgagagat gccgttgaga
agccccaaga atttacaatt gttgcttttg 6300 taaagtatga taaaaaccaa
gatgttcact ccattaacct cccatttttt gagaccttgc 6360 aagaatattt
tgagaggaat cgacaaacca ttatagttgt agtggaaaac gtacagagaa 6420
acctgaagca catcaatatt gatcaatttg taagaaaata cagagcagcc ctgggaaaac
6480 tcccacagca agctaatgat tatctgaatt cattcaattg ggagagacaa
gtttcacatg 6540 ccaaggagaa actgactgct ctcacaaaaa agtatagaat
tacagaaaat gatatacaaa 6600 ttgcattaga tgatgccaaa atcaacttta
atgaaaaact atctcaactg cagacatata 6660 tgatacaatt tgatcagtat
attaaagata gttatgattt acatgatttg aaaatagcta 6720 ttgctaatat
tattgatgaa atcattgaaa aattaaaaag tcttgatgag cactatcata 6780
tccgtgtaaa tttagtaaaa acaatccatg atctacattt gtttattgaa aatattgatt
6840 ttaacaaaag tggaagtagt actgcatcct ggattcaaaa tgtggatact
aagtaccaaa 6900 tcagaatcca gatacaagaa aaactgcagc agcttaagag
acacatacag aatatagaca 6960 tccagcacct agctggaaag ttaaaacaac
acattgaggc tattgatgtt agagtgcttt 7020 tagatcaatt gggaactaca
atttcatttg aaagaataaa tgatgttctt gagcatgtca 7080 aacactttgt
tataaatctt attggggatt ttgaagtagc tgagaaaatc aatgccttca 7140
gagccaaagt ccatgagtta atcgagaggt atgaagtaga ccaacaaatc caggttttaa
7200 tggataaatt agtagagttg acccaccaat acaagttgaa ggagactatt
cagaagctaa 7260 gcaatgtcct acaacaagtt aagataaaag attactttga
gaaattggtt ggatttattg 7320 atgatgctgt gaagaagctt aatgaattat
cttttaaaac attcattgaa gatgttaaca 7380 aattccttga catgttgata
aagaaattaa agtcatttga ttaccaccag tttgtagatg 7440 aaaccaatga
caaaatccgt gaggtgactc agagactcaa tggtgaaatt caggctctgg 7500
aactaccaca aaaagctgaa gcattaaaac tgtttttaga ggaaaccaag gccacagttg
7560 cagtgtatct ggaaagccta caggacacca aaataacctt aatcatcaat
tggttacagg 7620 aggctttaag ttcagcatct ttggctcaca tgaaggccaa
attccgagag actctagaag 7680 atacacgaga ccgaatgtat caaatggaca
ttcagcagga acttcaacga tacctgtctc 7740 tggtaggcca ggtttatagc
acacttgtca cctacatttc tgattggtgg actcttgctg 7800 ctaagaacct
tactgacttt gcagagcaat attctatcca agattgggct aaacgtatga 7860
aagcattggt agagcaaggg ttcactgttc ctgaaatcaa gaccatcctt gggaccatgc
7920 ctgcctttga agtcagtctt caggctcttc agaaagctac cttccagaca
cctgatttta 7980 tagtccccct aacagatttg aggattccat cagttcagat
aaacttcaaa gacttaaaaa 8040 atataaaaat cccatccagg ttttccacac
cagaatttac catccttaac accttccaca 8100 ttccttcctt tacaattgac
tttgtcgaaa tgaaagtaaa gatcatcaga accattgacc 8160 agatgcagaa
cagtgagctg cagtggcccg ttccagatat atatctcagg gatctgaagg 8220
tggaggacat tcctctagcg agaatcaccc tgccagactt ccgtttacca gaaatcgcaa
8280 ttccagaatt cataatccca actctcaacc ttaatgattt tcaagttcct
gaccttcaca 8340 taccagaatt ccagcttccc cacatctcac acacaattga
agtacctact tttggcaagc 8400 tatacagtat tctgaaaatc caatctcctc
ttttcacatt agatgcaaat gctgacatag 8460 ggaatggaac cacctcagca
aacgaagcag gtatcgcagc ttccatcact gccaaaggag 8520 agtccaaatt
agaagttctc aattttgatt ttcaagcaaa tgcacaactc tcaaacccta 8580
agattaatcc gctggctctg aaggagtcag tgaagttctc cagcaagtac ctgagaacgg
8640 agcatgggag tgaaatgctg ttttttggaa atgctattga gggaaaatca
aacacagtgg 8700 caagtttaca cacagaaaaa aatacactgg agcttagtaa
tggagtgatt gtcaagataa 8760 acaatcagct taccctggat agcaacacta
aatacttcca caaattgaac atccccaaac 8820 tggacttctc tagtcaggct
gacctgcgca acgagatcaa gacactgttg aaagctggcc 8880 acatagcatg
gacttcttct ggaaaagggt catggaaatg ggcctgcccc agattctcag 8940
atgagggaac acatgaatca caaattagtt tcaccataga aggacccctc acttcctttg
9000 gactgtccaa taagatcaat agcaaacacc taagagtaaa ccaaaacttg
gtttatgaat 9060 ctggctccct caacttttct aaacttgaaa ttcaatcaca
agtcgattcc cagcatgtgg 9120 gccacagtgt tctaactgct aaaggcatgg
cactgtttgg agaagggaag gcagagttta 9180 ctgggaggca tgatgctcat
ttaaatggaa aggttattgg aactttgaaa aattctcttt 9240 tcttttcagc
ccagccattt gagatcacgg catccacaaa caatgaaggg aatttgaaag 9300
ttcgttttcc attaaggtta acagggaaga tagacttcct gaataactat gcactgtttc
9360 tgagtcccag tgcccagcaa gcaagttggc aagtaagtgc taggttcaat
cagtataagt 9420 acaaccaaaa tttctctgct ggaaacaacg agaacattat
ggaggcccat gtaggaataa 9480 atggagaagc aaatctggat ttcttaaaca
ttcctttaac aattcctgaa atgcgtctac 9540 cttacacaat aatcacaact
cctccactga aagatttctc tctatgggaa aaaacaggct 9600 tgaaggaatt
cttgaaaacg acaaagcaat catttgattt aagtgtaaaa gctcagtata 9660
agaaaaacaa acacaggcat tccatcacaa atcctttggc tgtgctttgt gagtttatca
9720 gtcagagcat caaatccttt gacaggcatt ttgaaaaaaa cagaaacaat
gcattagatt 9780 ttgtcaccaa atcctataat gaaacaaaaa ttaagtttga
taagtacaaa gctgaaaaat 9840 ctcacgacga gctccccagg acctttcaaa
ttcctggata cactgttcca gttgtcaatg 9900 ttgaagtgtc tccattcacc
atagagatgt cggcattcgg ctatgtgttc ccaaaagcag 9960 tcagcatgcc
tagtttctcc atcctaggtt ctgacgtccg tgtgccttca tacacattaa 10020
tcctgccatc attagagctg ccagtccttc atgtccctag aaatctcaag ctttctcttc
10080 cacatttcaa ggaattgtgt accataagcc atatttttat tcctgccatg
ggcaatatta 10140 cctatgattt ctcctttaaa tcaagtgtca tcacactgaa
taccaatgct gaacttttta 10200 accagtcaga tattgttgct catctccttt
cttcatcttc atctgtcatt gatgcactgc 10260 agtacaaatt agagggcacc
acaagattga caagaaaaag gggattgaag ttagccacag 10320 ctctgtctct
gagcaacaaa tttgtggagg gtagtcataa cagtactgtg agcttaacca 10380
cgaaaaatat ggaagtgtca gtggcaaaaa ccacaaaagc cgaaattcca attttgagaa
10440 tgaatttcaa gcaagaactt aatggaaata ccaagtcaaa acctactgtc
tcttcctcca 10500 tggaatttaa gtatgatttc aattcttcaa tgctgtactc
taccgctaaa ggagcagttg 10560 accacaagct tagcttggaa agcctcacct
cttacttttc cattgagtca tctaccaaag 10620 gagatgtcaa gggttcggtt
ctttctcggg aatattcagg aactattgct agtgaggcca 10680 acacttactt
gaattccaag agcacacggt cttcagtgaa gctgcagggc acttccaaaa 10740
ttgatgatat ctggaacctt gaagtaaaag aaaattttgc tggagaagcc acactccaac
10800 gcatatattc cctctgggag cacagtacga aaaaccactt acagctagag
ggcctctttt 10860 tcaccaacgg agaacataca agcaaagcca ccctggaact
ctctccatgg caaatgtcag 10920 ctcttgttca ggtccatgca agtcagccca
gttccttcca tgatttccct gaccttggcc 10980 aggaagtggc cctgaatgct
aacactaaga accagaagat cagatggaaa aatgaagtcc 11040 ggattcattc
tgggtctttc cagagccagg tcgagctttc caatgaccaa gaaaaggcac 11100
accttgacat tgcaggatcc ttagaaggac acctaaggtt cctcaaaaat atcatcctac
11160 cagtctatga caagagctta tgggatttcc taaagctgga tgtaaccacc
agcattggta 11220 ggagacagca tcttcgtgtt tcaactgcct ttgtgtacac
caaaaacccc aatggctatt 11280 cattctccat ccctgtaaaa gttttggctg
ataaattcat tactcctggg ctgaaactaa 11340 atgatctaaa ttcagttctt
gtcatgccta cgttccatgt cccatttaca gatcttcagg 11400 ttccatcgtg
caaacttgac ttcagagaaa tacaaatcta taagaagctg agaacttcat 11460
catttgccct caacctacca acactccccg aggtaaaatt ccctgaagtt gatgtgttaa
11520 caaaatattc tcaaccagaa gactccttga ttcccttttt tgagataacc
gtgcctgaat 11580 ctcagttaac tgtgtcccag ttcacgcttc caaaaagtgt
ttcagatggc attgctgctt 11640 tggatctaaa tgcagtagcc aacaagatcg
cagactttga gttgcccacc atcatcgtgc 11700 ctgagcagac cattgagatt
ccctccatta agttctctgt acctgctgga attgtcattc 11760 cttcctttca
agcactgact gcacgctttg aggtagactc tcccgtgtat aatgccactt 11820
ggagtgccag tttgaaaaac aaagcagatt atgttgaaac agtcctggat tccacatgca
11880 gctcaaccgt acagttccta gaatatgaac taaatgtttt gggaacacac
aaaatcgaag 11940 atggtacgtt agcctctaag actaaaggaa cacttgcaca
ccgtgacttc agtgcagaat 12000 atgaagaaga tggcaaattt gaaggacttc
aggaatggga aggaaaagcg cacctcaata 12060 tcaaaagccc agcgttcacc
gatctccatc tgcgctacca gaaagacaag aaaggcatct 12120 ccacctcagc
agcctcccca gccgtaggca ccgtgggcat ggatatggat gaagatgacg 12180
acttttctaa atggaacttc tactacagcc ctcagtcctc tccagataaa aaactcacca
12240 tattcaaaac tgagttgagg gtccgggaat ctgatgagga aactcagatc
aaagttaatt 12300 gggaagaaga ggcagcttct ggcttgctaa cctctctgaa
agacaacgtg cccaaggcca 12360 caggggtcct ttatgattat gtcaacaagt
accactggga acacacaggg ctcaccctga 12420 gagaagtgtc ttcaaagctg
agaagaaatc tgcagaacaa tgctgagtgg gtttatcaag 12480 gggccattag
gcaaattgat gatatcgacg tgaggttcca gaaagcagcc agtggcacca 12540
ctgggaccta ccaagagtgg aaggacaagg cccagaatct gtaccaggaa ctgttgactc
12600 aggaaggcca agccagtttc cagggactca aggataacgt gtttgatggc
ttggtacgag 12660 ttactcaaaa attccatatg aaagtcaagc atctgattga
ctcactcatt gattttctga 12720 acttccccag attccagttt ccggggaaac
ctgggatata cactagggag gaactttgca 12780 ctatgttcat aagggaggta
gggacggtac tgtcccaggt atattcgaaa gtccataatg 12840 gttcagaaat
actgttttcc tatttccaag acctagtgat tacacttcct ttcgagttaa 12900
ggaaacataa actaatagat gtaatctcga tgtataggga actgttgaaa gatttatcaa
12960 aagaagccca agaggtattt aaagccattc agtctctcaa gaccacagag
gtgctacgta 13020 atcttcagga ccttttacaa ttcattttcc aactaataga
agataacatt aaacagctga 13080 aagagatgaa atttacttat cttattaatt
atatccaaga tgagatcaac acaatcttca 13140 atgattatat cccatatgtt
tttaaattgt tgaaagaaaa cctatgcctt aatcttcata 13200 agttcaatga
atttattcaa aacgagcttc aggaagcttc tcaagagtta cagcagatcc 13260
atcaatacat tatggccctt cgtgaagaat attttgatcc aagtatagtt ggctggacag
13320 tgaaatatta tgaacttgaa gaaaagatag tcagtctgat caagaacctg
ttagttgctc 13380 ttaaggactt ccattctgaa tatattgtca gtgcctctaa
ctttacttcc caactctcaa 13440 gtcaagttga gcaatttctg cacagaaata
ttcaggaata tcttagcatc cttaccgatc 13500 cagatggaaa agggaaagag
aagattgcag agctttctgc cactgctcag gaaataatta 13560 aaagccaggc
cattgcgacg aagaaaataa tttctgatta ccaccagcag tttagatata 13620
aactgcaaga tttttcagac caactctctg attactatga aaaatttatt gctgaatcca
13680 aaagattgat tgacctgtcc attcaaaact accacacatt tctgatatac
atcacggagt 13740 tactgaaaaa gctgcaatca accacagtca tgaaccccta
catgaagctt gctccaggag 13800 aacttactat catcctctaa ttttttaaaa
gaaatcttca tttattcttc ttttccaatt 13860 gaactttcac atagcacaga
aaaaattcaa actgcctata ttgataaaac catacagtga 13920 gccagccttg
cagtaggcag tagactataa gcagaagcac atatgaactg gacctgcacc 13980
aaagctggca ccagggctcg gaaggtctct gaactcagaa ggatggcatt ttttgcaagt
14040 taaagaaaat caggatctga gttattttgc taaacttggg ggaggaggaa
caaataaatg 14100 gagtctttat tgtgtatcat a 14121 11 21 DNA Artificial
Sequence PCR Primer 11 tgctaaaggc acatatggcc t 21 12 23 DNA
Artificial Sequence PCR Primer 12 ctcaggttgg actctccatt gag 23 13
28 DNA Artificial Sequence PCR Probe 13 cttgtcagag ggatcctaac
actggccg 28 14 19 DNA Artificial Sequence PCR Primer 14 gaaggtgaag
gtcggagtc 19 15 20 DNA Artificial Sequence PCR Primer 15 gaagatggtg
atgggatttc 20 16 20 DNA Artificial Sequence PCR Probe 16 caagcttccc
gttctcagcc 20 17 2354 DNA M. musculus 17 gaattccaac ttcctcacct
ctcacataca attgaaatac ctgcttttgg caaactgcat 60 agcatcctta
agatccaatc tcctctcttt atattagatg ctaatgccaa catacagaat 120
gtaacaactt cagggaacaa agcagagatt gtggcttctg tcactgctaa aggagagtcc
180 caatttgaag ctctcaattt tgattttcaa gcacaagctc aattcctgga
gttaaatcct 240 catcctccag tcctgaagga atccatgaac ttctccagta
agcatgtgag aatggagcat 300 gagggtgaga tagtatttga tggaaaggcc
attgagggga aatcagacac agtcgcaagt 360 ttacacacag agaaaaatga
agtagagttt aataatggta tgactgtcaa agtaaacaat 420 cagctcaccc
ttgacagtca cacaaagtac ttccacaagt tgagtgttcc taggctggac 480
ttctccagta aggcttctct taataatgaa atcaagacac tattagaagc tggacatgtg
540 gcattgacat cttcagggac agggtcatgg aactgggcct gtcccaactt
ctcggatgaa 600 ggcatacatt cgtcccaaat tagctttact gtggatggtc
ccattgcttt tgttggacta 660 tccaataaca taaatggcaa acacttacgg
gtcatccaaa aactgactta tgaatctggc 720 ttcctcaact attctaagtt
tgaagttgag tcaaaagttg aatctcagca cgtgggctcc 780 agcattctaa
cagccaatgg tcgggcactg ctcaaggacg caaaggcaga aatgactggt 840
gagcacaatg ccaacttaaa tggaaaagtt attggaactt tgaaaaattc tctcttcttt
900 tcagcacaac catttgagat tactgcatcc acaaataatg aaggaaattt
gaaagtgggt 960 tttccactaa agctgactgg gaaaatagac ttcctgaata
actatgcatt gtttctgagt 1020 ccccgtgccc aacaagcaag ctggcaagcg
agtaccagat tcaatcagta caaatacaat 1080 caaaactttt ctgctataaa
caatgaacac aacatagaag ccagtatagg aatgaatgga 1140 gatgccaacc
tggatttctt aaacatacct ttaacaattc ctgaaattaa cttgccttac 1200
acggagttca aaactccctt actgaaggat ttctccatat gggaagaaac aggcttgaaa
1260 gaatttttga agacaacaaa gcaatcattt gatttgagtg taaaggctca
atataaaaag 1320 aacagtgaca agcattccat tgttgtccct ctgggtatgt
tttatgaatt tattctcaac 1380 aatgtcaatt cgtgggacag aaaatttgag
aaagtcagaa acaatgcttt acattttctt 1440 accacctcct ataatgaagc
aaaaattaag gttgataagt acaaaactga aaattccctt 1500 aatcagccct
ctgggacctt tcaaaatcat ggctacacta tcccagttgt caacattgaa 1560
gtatctccat ttgctgtaga gacactggct tccaggcatg tgatccccac agcaataagc
1620 accccaagtg tcacaatccc tggtcctaac atcatggtgc cttcatacaa
gttagtgctg 1680 ccacccctgg agttgccagt tttccatggt cctgggaatc
tattcaagtt tttcctccca 1740 gatttcaagg gattcaacac tattgacaat
atttatattc cagccatggg caactttacc 1800 tatgactttt cttttaaatc
aagtgtcatc acactgaata ccaatgctgg actttataac 1860 caatcagata
tcgttgccca tttcctttct tcctcttcat ttgtcactga cgccctgcag 1920
tacaaattag agggaacatc acgtctgatg cgaaaaaggg gattgaaact agccacagct
1980 gtctctctaa ctaacaaatt tgtaaagggc agtcatgaca gcaccattag
tttaaccaag 2040 aaaaacatgg aagcatcagt gagaacaact gccaacctcc
atgctcccat attctcaatg 2100 aacttcaagc aggaacttaa tggaaatacc
aagtcaaaac ccactgtttc atcatccatt 2160 gaactaaact atgacttcaa
ttcctcaaag ctgcactcta ctgcaacagg aggcattgat 2220 cacaagttca
gcttagaaag tctcacttcc tacttttcca ttgagtcatt caccaaagga 2280
aatatcaaga gttccttcct ttctcaggaa tattcaggaa gtgttgccaa tgaagccaat
2340 gtatatctga attc 2354 18 19 DNA Artificial Sequence PCR Primer
18 cgtgggctcc agcattcta 19 19 21 DNA Artificial Sequence PCR Primer
19 agtcatttct gcctttgcgt c 21 20 22 DNA Artificial Sequence PCR
Probe 20 ccaatggtcg ggcactgctc aa 22 21 20 DNA Artificial Sequence
PCR Primer 21 ggcaaattca acggcacagt
20 22 20 DNA Artificial Sequence PCR Primer 22 gggtctcgct
cctggaagat 20 23 27 DNA Artificial Sequence PCR Probe 23 aaggccgaga
atgggaagct tgtcatc 27
* * * * *