U.S. patent application number 14/163581 was filed with the patent office on 2014-07-24 for modulation of apolipoprotein (a) expression.
This patent application is currently assigned to ISIS PHARMACEUTICALS, INC.. The applicant listed for this patent is Isis Pharmaceuticals, Inc.. Invention is credited to Rosanne M. Crooke, Mark J. Graham.
Application Number | 20140206750 14/163581 |
Document ID | / |
Family ID | 35883353 |
Filed Date | 2014-07-24 |
United States Patent
Application |
20140206750 |
Kind Code |
A1 |
Crooke; Rosanne M. ; et
al. |
July 24, 2014 |
MODULATION OF APOLIPOPROTEIN (a) EXPRESSION
Abstract
Compounds, compositions and methods are provided for modulating
the expression of apolipoprotein(a). The compositions comprise
oligonucleotides, targeted to nucleic acid encoding
apolipoprotein(a). Methods of using these compounds for modulation
of apolipoprotein(a) expression and for diagnosis and treatment of
disease associated with expression of apolipoprotein(a) are
provided.
Inventors: |
Crooke; Rosanne M.;
(Carlsbad, CA) ; Graham; Mark J.; (San Clemente,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Isis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
ISIS PHARMACEUTICALS, INC.
Carlsbad
CA
|
Family ID: |
35883353 |
Appl. No.: |
14/163581 |
Filed: |
January 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12726286 |
Mar 17, 2010 |
8673632 |
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14163581 |
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10559647 |
Jul 31, 2006 |
7723508 |
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PCT/US2004/014540 |
Jun 2, 2004 |
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12726286 |
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10684440 |
Oct 15, 2003 |
7259150 |
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10559647 |
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60475402 |
Jun 2, 2003 |
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Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
C12N 2310/321 20130101;
A61P 9/10 20180101; C12N 2310/3341 20130101; C12N 2320/30 20130101;
A61P 3/00 20180101; C12N 2310/346 20130101; C12N 15/113 20130101;
A61K 38/00 20130101; C12N 2310/341 20130101; C12N 2310/315
20130101; A61P 9/00 20180101; C12N 2310/11 20130101; A61P 3/06
20180101; C12N 2310/321 20130101; C12N 2310/3525 20130101 |
Class at
Publication: |
514/44.A ;
536/24.5 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. A compound comprising a modified oligonucleotide 12 to 30 linked
nucleobases in length targeted to a nucleic acid molecule encoding
apolipoprotein (a), wherein the compound comprises at least 8
consecutive nucleobases of any of the nucleobase sequences of SEQ
ID NOs: 85, 89-93, 95-96 and is at least 80% complementary to SEQ
ID NO: 4.
2. The compound of claim 1, wherein the modified oligonucleotide is
at least 85%, at least 90%, at least 95%, or 100% complementary to
SEQ ID NO: 1.
3. The compound of claim 1, wherein the modified oligonucleotide is
single-stranded.
4. The compound of claim 1, wherein at least one internucleoside
linkage is a modified internucleoside linkage.
5. The compound of claim 4, wherein each internucleoside linkage is
a phosphorothioate internucleoside linkage.
6. The compound of claim 1, wherein the modified oligonucleotide
comprises at least one modified sugar.
7. The compound of claim 6, wherein at least one modified sugar is
a bicyclic sugar.
8. The compound of claim 6, wherein at least one modified sugar
comprises a 2'-.beta.-methoxyethyl, or a 4'-CH.sub.2--O-2'.
9. The compound of claim 1, wherein at least one nucleoside
comprises a modified nucleobase.
10. The compound of claim 9, wherein the modified nucleobase is a
5-methylcytosine.
11. The compound of claim 1, wherein the modified oligonucleotide
consists of 12 to 30 linked nucleosides and comprises: a gap
segment consisting of linked deoxynucleosides; a 5' wing segment
consisting of linked nucleosides; a 3' wing segment consisting of
linked nucleosides; wherein the gap segment is positioned between
the 5' wing segment and the 3' wing segment and wherein each
nucleoside of each wing segment comprises a modified sugar.
12. The compound of claim 11, wherein the modified oligonucleotide
consists of 20 linked nucleosides and comprises: a gap segment
consisting of ten linked deoxynucleosides; a 5' wing segment
consisting of five linked nucleosides; a 3' wing segment consisting
of five linked nucleosides; wherein the gap segment is positioned
between the 5' wing segment and the 3' wing segment, wherein each
nucleoside of each wing segment comprises a 2'-O-methoxyethyl
sugar, wherein each internucleoside linkage is a phosphorothioate
linkage and wherein each cytosine residue is a
5-methylcytosine.
13. The compound of claim 11, wherein the modified oligonucleotide
consists of 20 linked nucleosides.
14. A composition comprising the compound of claim 1, or a salt
thereof, and a pharmaceutically acceptable carrier or diluent.
15. A composition comprising the compound of claim 1, for use in
therapy.
16. The compound of claim 15, for use in treating, preventing, or
slowing progression of a disease related to elevated apo(a) and/or
elevated Lp(a).
17. The compound of claim 16, wherein the disease is an
inflammatory, cardiovascular or metabolic disease, disorder or
condition.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 12/726,286, filed Mar. 17, 2010, is a
Continuation of U.S. patent application Ser. No. 10/559,647, filed
Jul. 31, 2006, which is a US National Phase Application under 35
USC 371 of PCT/US2004/014540 filed on Jun. 2, 2004, which claims
priority to U.S. provisional patent application No. 60/475,402,
filed Jun. 2, 2003 and U.S. patent application Ser. No. 10/684,440,
filed Oct. 15, 2003, each of which is incorporated by reference in
its entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled ISPH0595USC5_ST25.txt, created on Jan. 21, 2014 which
is 168 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] The present invention provides compositions and methods for
modulating the expression of apolipoprotein(a).
[0004] Lipoproteins are globular, micelle-like particles that
consist of a non-polar core of acylglycerols and cholesteryl
esters, surrounded by an amphiphilic coating consisting of protein,
phospholipid and cholesterol. Lipoproteins have been classified
into five broad categories on the basis of their functional and
physical properties: chylomicrons (which transport dietary lipids
from intestine to tissues), very low density lipoproteins (VLDL),
intermediate density lipoproteins (IDL). Low density lipoproteins
(LDL), (all of which transport triacylglycerols and cholesterol
from the liver to tissues), and high density lipoproteins (HDL)
(which transport endogenous cholesterol from tissues to the liver).
Lipoprotein particles undergo continuous metabolic processing and
have variable properties and compositions. Lipoprotein densities
increase without decreasing particle diameter because the density
of their outer coatings is less than that of the inner core. The
protein components of lipoproteins are known as apolipoproteins. At
least nine apolipoproteins are distributed in significant amounts
among the various human lipoproteins.
[0005] Lipoprotein (a) (also known as Lp(a)) is a cholesterol rich
particle of the proatherogenic LDL class. Since Lp(a) is found only
in Old World primates and European hedgehogs, it has been suggested
that it
does not play an essential role in lipid and lipoprotein
metabolism. Most studies have shown that high concentrations of
Lp(a) are strongly associated with increased risk of cardiovascular
disease (Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61).
These observations have stimulated numerous studies in humans and
other primates to investigate the factors that control Lp(a)
concentrations and physiological properties (Rainwater and
Kammerer, J. Exp. Zool., 1998, 282, 54-61).
[0006] Lp(a) contains two disulfide-linked distinct proteins,
apolipoprotein(a) (or ApoA) and apolipoprotein B (or ApoB)
(Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61).
Apolipoprotein(a) is a unique apolipoprotein encoded by the LPA
gene which has been shown to exclusively control the physiological
concentrations of Lp(a) (Rainwater and Kammerer, J. Exp. Zool.,
1998, 282, 54-61). It varies in size due to interallelic
differences in the number of tandemly repeated Kringle-4-encoding
5.5 kb sequences in the LPA gene (Rainwater and Kammerer, J. Exp.
Zool., 1998, 282, 54-61).
[0007] Cloning of human apolipoprotein(a) in 1987 revealed homology
to human plasminogen (McLean et al., Nature, 1987, 330, 132-137).
The gene locus LPA encoding apolipoprotein(a) was localized to
chromosome 6q26-27, in close proximity to the homologous gene for
plasminogen (Frank et al., Hum. Genet., 1988, 79, 352-356).
[0008] Transgenic mice expressing human apolipoprotein(a) were
found to be more susceptible than control mice to the development
of lipid-staining lesions in the aorta. Consequently,
apolipoprotein(a) is co-localized with lipid deposition in the
artery walls (Lawn et al., Nature, 1992, 360, 670-672). As an
extension of these studies, it was established that the major in
vivo action of apolipoprotein(a) is inhibition of the conversion of
plasminogen to plasmin which causes decreased activation of latent
transforming growth factor-beta. Since transforming growth
factor-beta is a negative regulator of smooth muscle cell migration
and proliferation, inhibition of plasminogen activation indicates a
possible mechanism for apolipoprotein(a) induction of
atherosclerotic lesions (Grainger et al., Nature, 1994, 370,
460-462).
[0009] Elevated plasma levels of Lp(a), caused by increased
expression of apolipoprotein(a), are associated with increased risk
for atherosclerosis and its manifestations, which include
hypercholesterolemia (Seed et al., N. Engl. J. Med., 1990, 322,
1494-1499), myocardial infarction (Sandkamp et al., Clin. Chem.,
1990, 36, 20-23), and thrombosis (Nowak-Gottl et al., Pediatrics,
1997, 99, E11).
[0010] Moreover, the plasma concentration of Lp(a) is strongly
influenced by heritable factors and is refractory to most drug and
dietary manipulation (Katan and Beynen, Am. J. Epidemiol., 1987,
125, 387-399; Vessby et al., Atherosclerosis, 1982, 44, 61-71.).
Pharmacologic therapy of elevated Lp(a) levels has been only
moderately successful and apheresis remains the most effective
therapeutic modality (Hajjar and Nachman, Annu. Rev. Med., 1996,
47, 423-442).
[0011] Morishita et al. reported the use of three ribozyme
oligonucleotides against apolipoprotein(a) for inhibition of
apolipoprotein(a) expression in HepG2 cells (Morishita et al.,
Circulation, 1998, 98, 1898-1904).
[0012] U.S. Pat. No. 5,721,138 refers to nucleotide sequences
encoding the human apolipoprotein(a) gene 5'-regulatory region and
isolated nucleotide sequences comprising at least thirty
consecutive complementary nucleotides from human apolipoprotein(a)
from nucleotide positions 208 to 1448 (Lawn, 1998).
[0013] To date, investigative and therapeutic strategies aimed at
inhibiting apolipoprotein(a) function have involved the previously
cited use of Lp(a) apheresis and ribozyme oligonucleotides. No
existing drugs are available to specifically lower lipoprotein(a)
levels in humans, and only limited models exist in which to perform
drug discovery. Consequently, there remains a long-felt need for
additional agents and methods capable of effectively modulating,
e.g., inhibiting, apolipoprotein(a) function, and particularly a
need for agents capable of safe and efficacious administration to
lower alipoprotein(a) levels in patients at risk for the
development of coronary artery disease.
SUMMARY OF THE INVENTION
[0014] The present invention provides compositions and methods for
modulating the expression of apolipoprotein(a). Such novel
compositions and methods enable research into the pathways of
plasminogen and apolipoprotein(a), as well as other lipid metabolic
processes. Such novel compositions and methods are useful in
assessing the toxicity of chemical and pharmaceutical compounds on
apolipoprotein(a) function, plasminogen or other lipid metabolic
processes. Such novel compositions and methods are useful for drug
discovery and for the treatment of cardiovascular conditions,
including myocardial infarction and atherosclerosis, among
others.
[0015] Antisense technology is emerging as an effective means for
reducing the expression of specific gene products, and is uniquely
useful in a number of therapeutic, diagnostic, and research
applications for the modulation of apolipoprotein(a)
expression.
[0016] In particular, this invention relates to compounds,
particularly oligonucleotide compounds, which, in preferred
embodiments, hybridize with nucleic acid molecules or sequences
encoding apolipoprotein(a). Such compounds are shown herein to
modulate the expression of apolipoprotein(a). Additionally
disclosed are embodiments of oligonucleotide compounds that
hybridize with nucleic acid molecules encoding apolipoprotein(a) in
preference to nucleic acid molecules or sequences encoding
plasminogen.
[0017] The present invention is directed to compounds, especially
nucleic acid and nucleic acid-like oligomers, which are targeted to
a nucleic acid encoding apolipoprotein(a), and which modulate the
expression of apolipoprotein(a). Pharmaceutical and other
compositions comprising the compounds of the invention are also
provided.
[0018] Further provided are methods of screening for modulators of
apolipoprotein(a) and methods of modulating the expression of
apolipoprotein(a) in cells, tissues or animals comprising
contacting said cells, tissues or animals with one or more of the
compounds or compositions of the invention. In these methods, the
cells or tissues may be contacted in vivo. Alternatively, the cells
or tissues may be contacted ex vivo.
[0019] Methods of treating an animal, particularly a human, having,
suspected of having, or being prone to a disease or condition
associated with expression of apolipoprotein(a) 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.
[0020] In one aspect, the invention provides the use of a compound
or composition of the invention in the manufacture of a medicament
for the treatment of any and all conditions disclosed herein.
DETAILED DESCRIPTION OF THE INVENTION
A. Overview of the Invention
[0021] The present invention employs compounds, preferably
oligonucleotides and similar species, for use in modulating the
function or effect of nucleic acid molecules encoding
apolipoprotein(a). This is accomplished by providing
oligonucleotides that specifically hybridize with one or more
nucleic acid molecules encoding apolipoprotein(a). As used herein,
the terms "target nucleic acid" and "nucleic acid molecule encoding
apolipoprotein(a)" have been used for convenience to encompass DNA
encoding apolipoprotein(a), 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". Antisense technology is emerging as an effective means
of reducing the expression of specific gene products and is
uniquely useful in a number of therapeutic, diagnostic and research
applications involving modulation of apolipoprotein(a)
expression.
[0022] 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.
[0023] 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(a). 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.
[0024] 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 that pair through the formation of hydrogen bonds.
Hybridization can occur under varying circumstances.
[0025] 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. Such conditions include, e.g., physiological conditions
in the case of in vivo assays or therapeutic treatment, and
conditions in which assays are performed in the case of in vitro
assays.
[0026] 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. 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.
[0027] "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 that 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.
[0028] The sequence of an antisense compound can be, but need not
necessarily 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. In
one embodiment of this invention, 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. In other embodiments, the
antisense compounds of the present invention comprise at least 90%
sequence complementarity and even 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).
[0029] 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 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 other embodiments, homology, sequence identity, or
complementarity is between about 70% and about 80%. In still other
embodiments, homology, sequence identity, or complementarity is
between about 80% and about 90%. In yet other embodiments,
homology, sequence identity, or complementarity is about 90%, about
92%, about 94%, about 95%, about 96%, about 97%, about 98%, about
99%, or about 100%.
B. Compounds of the Invention
[0030] According to the present invention, "compounds" include
antisense oligomeric compounds, antisense oligonucleotides, siRNAs,
external guide sequence (EGS) oligonucleotides, alternate splicers,
and other oligomeric compounds that 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,
partially single-stranded, or circular oligomeric compounds.
Specifically excluded from the definition of "compounds" herein are
ribozymes that contain internal or external "bulges" that do not
hybridize to the target sequence. 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.
[0031] 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 that 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.
[0032] While one 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.
[0033] The first evidence that dsRNA could lead to gene silencing
in animals came in 1995 from work in the nematode, Caenorhabditis
elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). 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, the single-stranded RNA oligomers of antisense
polarity of the dsRNAs have been reported to be the potent inducers
of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).
[0034] 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.
[0035] The oligonucleotides of the present invention also include
modified oligonucleotides in which a different base is present at
one or more of the nucleotide positions in the oligonucleotide. For
example, if the first nucleotide is an adenosine, modified
oligonucleotides may be produced that contain thymidine, guanosine
or cytidine at this position. This may be done at any of the
positions of the oligonucleotide. These oligonucleotides are then
tested using the methods described herein to determine their
ability to inhibit expression of apolipoprotein(a) mRNA.
[0036] While oligonucleotides are a preferred form of the compounds
of this invention, the present invention comprehends other families
of compounds as well, including but not limited to, oligonucleotide
analogs and mimetics such as those described herein.
[0037] The compounds in accordance with this invention 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.
[0038] In one embodiment, the 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.
[0039] In another embodiment, the 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.
[0040] In another embodiment, compounds of this invention are
oligonucleotides from about 12 to about 50 nucleobases. In another
embodiment, compounds of this invention comprise from about 15 to
about 30 nucleobases.
[0041] In another embodiment, the antisense compounds comprise at
least 8 contiguous nucleobases of an antisense compound disclosed
herein.
[0042] 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.
[0043] Exemplary 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 that is specifically
hybridizable to the target nucleic acid, and continuing until the
oligonucleotide contains about 8 to about 80 nucleobases).
Similarly, exemplary 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
that is specifically hybridizable to the target nucleic acid and
continuing until the oligonucleotide contains about 8 to about 80
nucleobases).
[0044] Exemplary compounds of this invention may be found
identified in the Examples and listed in Tables 1 and 7. In
addition to oligonucleotide compounds that bind to target sequences
of apolipoprotein(a) in general, there are also exemplified
oligonucleotide compounds of this invention that bind to target
nucleotide sequences of apolipoprotein(a), but do not bind to, or
do not bind preferentially to, sequences of plasminogen due to lack
of homology between the two nucleic acid molecules or a sufficient
number of mismatches in the target sequences. These latter
compounds are also useful in various therapeutic methods of this
invention. Examples of antisense compounds to such `mismatched`
target sequences as described above include SEQ ID NO: 12 and SEQ
ID NO: 23 of Table 1 below. See, also, the discussion of target
regions below.
[0045] One having skill in the art armed with the exemplary
antisense compounds illustrated herein will be able, without undue
experimentation, to identify further useful antisense
compounds.
C. Targets of the Invention
[0046] "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(a).
[0047] 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.
[0048] 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 having translation
initiation codons with 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). 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(a), regardless of the
sequence(s) of such codons. 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).
[0049] 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 that
may be targeted effectively with the antisense compounds of the
present invention.
[0050] 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.
[0051] Another target region includes the 5' untranslated region
(5'UTR), known in the art to refer to the portion of an mRNA in the
5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA (or corresponding nucleotides on the
gene). Still another target region includes the 3' untranslated
region (3'UTR), known in the art to refer to the portion of an mRNA
in the 3' direction from the translation termination codon, and
thus including nucleotides between the translation termination
codon and 3' end of an mRNA (or corresponding nucleotides on the
gene). The 5' cap site of an mRNA comprises an N7-methylated
guanosine residue joined to the 5'-most residue of the mRNA via a
5'-5' triphosphate linkage. The 5' cap region of an mRNA is
considered to include the 5' cap structure itself as well as the
first 50 nucleotides adjacent to the cap site. Another target
region for this invention is the 5' cap region.
[0052] Accordingly, the present invention provides antisense
compounds that target a portion of nucleotides 1-2480 as set forth
in SEQ ID NO: 4. In another embodiment, the antisense compounds
target at least an 8-nucleobase portion of nucleotides 1-45,
comprising the 5'UTR as set forth in SEQ ID NO: 4. In another
embodiment, the antisense compounds target at least an 8-nucleobase
portion of nucleotides 13593-13938, comprising the 3'UTR as set
forth in SEQ ID NO: 4. In another embodiment, the antisense
compounds target at least an 8-nucleobase portion of nucleotides
46-13592, comprising the coding region as set forth in SEQ ID NO:
4. In still other embodiments, the antisense compounds target at
least an 8-nucleobase portion of a "preferred target segment" (as
defined herein) as set forth in Table 2.
[0053] 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. In one embodiment, targeting
splice sites, i.e., intron-exon junctions or exon-intron junctions,
is particularly useful in situations where aberrant splicing is
implicated in disease, or where an overproduction of a particular
splice product is implicated in disease. An aberrant fusion
junction due to rearrangement or deletion is another embodiment of
a target site. mRNA transcripts produced via the process of
splicing of two (or more) mRNAs from different gene sources known
as "fusion transcripts" are also suitable target sites. Introns can
be effectively targeted using antisense compounds targeted to, for
example, DNA or pre-mRNA.
[0054] 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.
[0055] 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.
[0056] Variants can be produced through the use of alternative
signals to start or stop transcription. 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 embodiments of target nucleic
acids.
[0057] 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 that are accessible for hybridization.
[0058] While the specific sequences of certain exemplary 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
target segments are readily identifiable by one having ordinary
skill in the art in view of this disclosure.
[0059] 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.
[0060] 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). One having skill in the art armed with the target
segments illustrated herein will be able, without undue
experimentation, to identify further preferred target segments.
[0061] 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.
[0062] In various embodiments of this invention, the oligomeric
compounds are targeted to regions of a target apolipoprotein(a)
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:
[0063] Let R(m, 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.
[0064] This set of regions can be represented using the following
mathematical notation:
A = m S ( m ) ##EQU00001## where m .di-elect cons. N | 8 .ltoreq. m
.ltoreq. 80 ##EQU00001.2## and ##EQU00001.3## S ( m ) = { R n , n +
m - 1 | n .di-elect cons. { 1 , 2 , 3 , , L - m + 1 } }
##EQU00001.4##
[0065] where the mathematical operator | indicates "such that",
[0066] 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),
[0067] where x is a variable,
[0068] where N indicates all natural numbers, defined as positive
integers,
[0069] and where the mathematical operator .orgate. indicates "the
union of sets".
[0070] For example, the set of regions for m equal to 8, 9 and 80
can be constructed in the following manner. The set of regions,
each 8 nucleobases in length, S(m=8), in a target nucleobase
sequence 100 nucleobases in length (L=100), beginning at position 1
(n=1) of the target nucleobase sequence, can be created using the
following expression:
S(8)={R.sub.1,8|n.epsilon.{1,2,3, . . . ,93}}
and describes the set of regions comprising nucleobases 1-8, 2-9,
3-10, 4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19,
13-20, 14-21, 15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28,
22-29, 23-30, 24-31, 25-32, 26-33, 27-34, 28-35, 29-36, 30-37,
31-38, 32-39, 33-40, 34-41, 35-42, 36-43, 37-44, 38-45, 39-46,
40-47, 41-48, 42-49, 43-50, 44-51, 45-52, 46-53, 47-54, 48-55,
49-56, 50-57, 51-58, 52-59, 53-60, 54-61, 55-62, 56-63, 57-64,
58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71, 65-72, 66-73,
67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81, 75-82,
76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91,
85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.
[0071] An additional set for regions 20 nucleobases in length, in a
target sequence 100 nucleobases in length, beginning at position 1
of the target nucleobase sequence, can be described using the
following expression:
S(20)={R.sub.1,20|n.epsilon.{1,2,3, . . . ,81}}
and describes the set of regions comprising nucleobases 1-20, 2-21,
3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31,
13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40,
22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49,
31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58,
40-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67,
49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76,
58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85,
67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94,
76-95, 77-96, 78-97, 79-98, 80-99, 81-100.
[0072] 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|n.epsilon.{1,2,3, . . . ,21}}
and describes the set of regions comprising nucleobases 1-80, 2-81,
3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91,
13-92, 14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.
[0073] 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.
[0074] The union of these aforementioned example sets and other
sets for lengths from 10 to 19 and 21 to 79 can be described using
the mathematical expression:
A = m S ( m ) ##EQU00002##
[0075] where .orgate. represents the union of the sets obtained by
combining all members of all sets.
[0076] The mathematical expressions described herein define 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
where m is less than L, and where n is less than L-m+1.
[0077] In one embodiment, the oligonucleotide compounds of this
invention are 100% complementary to these sequences or to small
sequences found within each of the above listed sequences. In
another embodiment the oligonucleotide compounds have from at least
3 or 5 mismatches per 20 consecutive nucleobases in individual
nucleobase positions to these target regions. Still other compounds
of the invention are targeted to overlapping regions of the
above-identified portions of the apolipoprotein(a) sequence.
[0078] In still another embodiment, target regions include those
portions of the apolipoprotion(a) sequence that do not overlap with
plasminogen sequences. For example, among such apolipoprotein(a)
target sequences are included those found within the following
nucleobase sequences: 10624-10702, 10963-11036, 11325-11354,
11615-11716, 11985-12038, 12319-12379, 13487-13491, and
13833-13871. As a further example, target sequences of
apolipoprotein(a) that have at least 6 mismatches with the sequence
of plasminogen over at least 20 consecutive nucleotides are
desirable targets for antisense compounds that bind preferentially
to apolipoprotein(a) rather than to plasminogen. Such target
sequences can readily be identified by a BLAST comparison of the
two GENBANK.RTM. sequences of plasminogen (e.g., GENBANK.RTM.
Accession No. NM.sub.--000301) and apolipoprotein(a) (e.g.,
GENBANK.RTM. Accession No. NM.sub.--005577.1).
[0079] In still another embodiment, the target regions include
portions of the apolipoprotein (a) sequence that overlap with
portions of the plasminogen or apolipoprotein B sequence, but to
which antisense compounds bind to inhibit apolipoprotein (a) but do
not inhibit, to any appreciable degree, plasminogen and/or
apolipoprotein B. Such targets may be obtained from the target
regions of SEQ ID NOs: 46, 54, 56, 57, 59, 60, 61, 62, 64, 67, 68
and 69 of Table 2. These target regions are bound by antisense
oligonucleotides of SEQ ID Nos: 11, 23, 28, 30, 31, 33, 34, 35, 36,
39, 42, 43, and 45, for example, which inhibit apolipoprotein(a)
but not a second protein, which is plasminogen (see Example 22) or
apolipoprotein B (see Example 23).
D. Screening and Target Validation
[0080] 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(a).
"Modulators" are those compounds that decrease or increase the
expression of a nucleic acid molecule encoding apolipoprotein(a)
and which comprise at least an 8-nucleobase portion that 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(a) 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(a). 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(a), the modulator may then be
employed in further investigative studies of the function of
apolipoprotein(a), or for use as a research, diagnostic, or
therapeutic agent in accordance with the present invention.
[0081] 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.
[0082] 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 the antisense
strand of the duplex to the target, thereby triggering enzymatic
degradation of the target (Tijsterman et al., Science, 2002, 295,
694-697).
[0083] The 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(a) and a disease
state, phenotype, or condition. These methods include detecting or
modulating apolipoprotein(a) comprising contacting a sample,
tissue, cell, or organism with the compounds of the present
invention, measuring the nucleic acid or protein level of
apolipoprotein(a) and/or a related phenotypic or chemical endpoint
at some time after treatment, and optionally comparing the measured
value to a non-treated sample or sample treated with a further
compound of the invention. These methods can also be performed in
parallel or in combination with other experiments to determine the
function of unknown genes for the process of target validation or
to determine the validity of a particular gene product as a target
for treatment or prevention of a particular disease, condition, or
phenotype.
E. Kits, Research Reagents, Diagnostics, and Therapeutics
[0084] The compounds of the present invention are utilized for
diagnostics, therapeutics, and prophylaxis, and as research
reagents and components of 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.
[0085] For use in kits and diagnostics and in various biological
systems, the compounds of the present invention, either alone or in
combination with other compounds or therapeutics, are 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.
[0086] As used herein the term "biological system" or "system" is
defined as any organism, cell, cell culture or tissue that
expresses, or is made competent to express products of the LPA
gene. These include, but are not limited to, humans, transgenic
animals, cells, cell cultures, tissues, xenografts, transplants and
combinations thereof.
[0087] 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 that affect
expression patterns.
[0088] 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).
[0089] The compounds of the invention are useful for research and
diagnostics, because these compounds hybridize to nucleic acids
encoding apolipoprotein(a). Primers and probes disclosed herein are
useful in methods requiring the specific detection of nucleic acid
molecules encoding apolipoprotein(a) and in the amplification of
said nucleic acid molecules for detection or for use in further
studies of apolipoprotein(a). Hybridization of the primers and
probes with a nucleic acid encoding apolipoprotein(a) can be
detected by means known in the art. Such means may include
conjugation of an enzyme to the primers and probes, radiolabelling
of the primers and probes, or any other suitable detection means.
Kits using such detection means for detecting the level of
apolipoprotein(a) in a sample may also be prepared.
[0090] The invention further provides for the use of a compound or
composition of the invention in the manufacture of a medicament for
the treatment of any and all conditions disclosed herein.
[0091] The specificity and sensitivity of antisense are 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 have been safely and effectively
administered to humans and numerous clinical trials are 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.
[0092] For therapeutics, an animal, preferably a human, suspected
of having a disease or disorder which can be treated by modulating
the expression of apolipoprotein(a) is treated by administering
antisense compounds in accordance with this invention. For example,
in one non-limiting embodiment, the methods comprise the step of
administering to the animal in need of treatment, a therapeutically
effective amount of a apolipoprotein(a) inhibitor. The
apolipoprotein(a) inhibitors of the present invention effectively
inhibit the activity of the apolipoprotein(a) protein or inhibit
the expression of the apolipoprotein(a) protein. In one embodiment,
the activity or expression of apolipoprotein(a) in an animal is
inhibited by about 10%. Preferably, the activity or expression of
apolipoprotein(a) in an animal is inhibited by about 30%. More
preferably, the activity or expression of apolipoprotein(a) in an
animal is inhibited by 50% or more. Thus, the oligomeric compounds
modulate expression of apolipoprotein(a) mRNA by at least 10%, by
at least 20%, by at least 25%, by at least 30%, by at least 40%, by
at least 50%, by at least 60%, by at least 70%, by at least 75%, by
at least 80%, by at least 85%, by at least 90%, by at least 95%, by
at least 98%, by at least 99%, or by 100%.
[0093] For example, the reduction of the expression of
apolipoprotein(a) 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(a)
protein and/or the apolipoprotein(a) protein itself. For example,
apolipoprotein(a) is produced in the liver, and can be found in
normal and atherosclerotic vessel walls.
[0094] The compounds of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of a
compound to a suitable pharmaceutically acceptable diluent or
carrier. Use of the compounds and methods of the invention may also
be useful prophylactically.
F. Modifications
[0095] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. In turn, the respective ends of this
linear polymeric compound can be further joined to form a circular
compound, however, linear compounds are generally preferred. In
addition, linear compounds may have internal nucleobase
complementarity and may therefore fold in a manner as to produce a
fully or partially double-stranded compound. Within
oligonucleotides, the phosphate groups are commonly referred to as
forming the internucleoside backbone of the oligonucleotide. The
normal linkage or backbone of RNA and DNA is a 3' to 5'
phosphodiester linkage.
Modified Internucleoside Linkages (Backbones)
[0096] 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.
[0097] Preferred modified oligonucleotide backbones containing a
phosphorus atom therein include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates, 5'-alkylene phosphonates and
chiral phosphonates, phosphinates, phosphoramidates including
3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, selenophosphates and boranophosphates
having normal 3'-5' linkages, 2'-5' linked analogs of these, and
those having inverted polarity wherein one or more internucleotide
linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred
oligonucleotides having inverted polarity comprise a single 3' to
3' linkage at the 3'-most internucleotide linkage i.e. a single
inverted nucleoside residue which may be abasic (the nucleobase is
missing or has a hydroxyl group in place thereof). Various salts,
mixed salts and free acid forms are also included.
[0098] 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, certain of which are
commonly owned with this application, and each of which is herein
incorporated by reference.
[0099] 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.
[0100] 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, certain of which are commonly owned with
this application, and each of which is herein incorporated by
reference.
Modified Sugar and Internucleoside Linkages--Mimetics
[0101] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage (i.e. the backbone) of the
nucleotide units are replaced with novel groups. The 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, each of which is herein
incorporated by reference. Further teaching of PNA compounds can be
found in Nielsen et al., Science, 1991, 254, 1497-1500.
[0102] Further embodiments of the invention are oligonucleotides
with phosphorothioate backbones and oligonucleosides with
heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-[wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified Sugars
[0103] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-,
S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein
the alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are O[(CH.sub.2).sub.mO]CH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[CH.sub.2).sub.nCH.sub.3].sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-.beta.-methoxyethyl
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-methoxyethoxy 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 herein below.
[0104] Other modifications include 2'-methoxy (2'-O--CH.sub.3),
2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified 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; certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0105] A further 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
International Patent Publication Nos. WO 98/39352 and WO
99/14226.
Natural and Modified Nucleobases
[0106] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
(--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further modified nucleobases include tricyclic
pyrimidines such as phenoxazine
cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the compounds of the
invention. These include 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C. and
are presently preferred base substitutions, even more particularly
when combined with 2'-O-methoxyethyl sugar modifications.
[0107] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653;
5,763,588; 6,005,096; and 5,681,941; certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference, and U.S. Pat. No. 5,750,692, which is
commonly owned with the instant application and also herein
incorporated by reference.
Conjugates
[0108] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates that 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 No. 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-5-tritylthiol, a
thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl
residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a
polyamine or a polyethylene glycol chain, or adamantane acetic
acid, a palmityl moiety, or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the
invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999), which is incorporated herein by reference in its
entirety.
[0109] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928; and 5,688,941; certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference.
[0110] Oligomeric compounds used in the compositions of the present
invention can also be modified to have one or more stabilizing
groups that are generally attached to one or both termini of
oligomeric compounds to enhance properties such as for example
nuclease stability. Included in stabilizing groups are cap
structures. By "cap structure or terminal cap moiety" is meant
chemical modifications, which have been incorporated at either
terminus of oligonucleotides (see for example Wincott et al.,
International Patent Publication No. WO 97/26270, incorporated by
reference herein). These terminal modifications protect the
oligomeric compounds having terminal nucleic acid molecules from
exonuclease degradation, and can help in delivery and/or
localization within a cell. The cap can be present at the
5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or at both
termini. In non-limiting examples, the 5'-cap includes inverted
abasic residue (moiety), 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide,
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide;
L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted
nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate;
or bridging or non-bridging methylphosphonate moiety (for more
details see Wincott et al., International Patent Publication No. WO
97/26270, incorporated by reference herein).
[0111] Particularly preferred 3'-cap structures of the present
invention include, for example 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide,
carbocyclic nucleotide; 5'-amino-alkyl phosphate;
1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate; bridging or non
bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0112] Further 3' and 5'-stabilizing groups that can be used to cap
one or both ends of an oligomeric compound to impart nuclease
stability include those disclosed in International Patent
Publication No. WO 03/004602, published Jan. 16, 2003.
Chimeric Compounds
[0113] 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.
[0114] The present invention also includes antisense compounds that
are chimeric compounds. "Chimeric" antisense compounds, or
"chimeras," in the context of this invention, are antisense
compounds, particularly oligonucleotides, which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of an oligonucleotide
compound. These oligonucleotides typically contain at least one
region wherein the oligonucleotide is modified so as to confer upon
the oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, 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.
[0115] Preferred chimeric oligonucleotides are those disclosed in
the Examples herein. Particularly preferred chimeric
oligonucleotides are those referred to as ISIS 144367, ISIS 144368,
ISIS 144379, ISIS 144381, and ISIS 144396.
[0116] 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. 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. 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".
[0117] 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').
[0118] 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.
[0119] 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; certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference in its entirety.
G. Formulations
[0120] The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor-targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption-assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756; each of which is herein incorporated by
reference.
[0121] 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.
[0122] 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.
[0123] The present invention also includes pharmaceutical
compositions and formulations that 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.
[0124] 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.
[0125] 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 that increase the viscosity of the suspension including,
for example, sodium carboxymethylcellulose, sorbitol and/or
dextran. The suspension may also contain stabilizers.
[0126] 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.
[0127] 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 that 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.
[0128] 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 that 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.
[0129] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids. When incorporated into liposomes, these
specialized lipids result in liposomes with 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.
[0130] 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.
[0131] In one embodiment, the present invention employs various
penetration enhancers to affect 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.
[0132] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e., route of
administration.
[0133] 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.
dioleoyl-phosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl
choline DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidyl
ethanolamine DOTMA).
[0134] 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 May
20, 1999, which is incorporated herein by reference in its
entirety.
[0135] 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. Published Patent
Application No. 2003/0040497 (Feb. 27, 2003) and its parent
applications; U.S. Published Patent Application No. 2003/0027780
(Feb. 6, 2003) and its parent applications; and U.S. patent
application Ser. No. 10/071,822, filed Feb. 8, 2002, each of which
is incorporated herein by reference in their entirety.
[0136] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions that 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.
[0137] 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.
[0138] 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, rat, mouse, a rat, a dog, a guinea pig, a monkey,
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 monkey, a
non-human primate, a cat or a pig.
[0139] 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
apolipoprotein(a) in order to obtain therapeutic indications
discussed in more detail herein.
[0140] 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).
[0141] 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).
[0142] 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.
[0143] 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.
[0144] 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 such a manner the oligonucleotide
may be introduced into the body of an animal without unacceptable
side-effects such as toxicity, irritation or allergic response.
[0145] 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.
[0146] 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 another
biological property.
[0147] 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.
[0148] 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'-.beta.-substituted
alkyl, e.g. 2'-O-methoxyethyl, 2'-.beta.-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.
[0149] 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.
[0150] 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).
[0151] 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.
[0152] 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).
[0153] 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.
[0154] 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).
[0155] Fatty acids and their derivatives which act as penetration
enhancers and may be used in compositions of the present invention
include, for example, oleic acid, lauric acid, capric acid
(n-decanoic acid), myristic acid, palmitic acid, stearic acid,
linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein
(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic
acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one,
acylcarnitines, acylcholines and mono- and di-glycerides thereof
and/or physiologically acceptable salts thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1; El-Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651).
[0156] 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 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. 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, and 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.
[0157] 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).
[0158] 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. Another 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.
[0159] Other excipients include chelating agents, i.e. compounds
that remove metallic ions from solution by forming complexes
therewith, with the result that absorption of oligonucleotides
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).
[0160] 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).
[0161] 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.
[0162] 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-tisothiocyano-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).
[0163] 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.TM. disintegrating agent); and wetting agents (e.g.,
sodium lauryl sulphate, etc.).
[0164] 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.
[0165] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other chemotherapeutic agents that 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-chloroethyl-nitrosurea, busulfan, mitomycin C,
actinomycin D, mithramycin, prednisone, hydroxyprogesterone,
testosterone, tamoxifen, dacarbazine, procarbazine,
hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,
chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards,
melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine,
cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclo-phosphoramide, 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.
[0166] 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. For example, the first target may be an apolipoprotein(a)
target, and the second target may be a region from another
nucleotide sequence. Alternatively, compositions of the invention
may contain two or more antisense compounds targeted to different
regions of the same apolipoprotein(a) nucleic acid target. Numerous
examples of antisense compounds are illustrated herein, and others
may be selected from among suitable compounds known in the art. Two
or more combined compounds may be used together or
sequentially.
H. Dosing
[0167] 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 .mu.g to 100 g per kg of body weight, from 0.1 .mu.g
to 10 g per kg of body weight, from 1.0 .mu.g to 1 g per kg of body
weight, from 10.0 .mu.g to 100 mg per kg of body weight, from 100
.mu.g to 10 mg per kg of body weight, or from 1 mg to 5 mg per kg
of body weight, and may be given once or more daily, weekly,
monthly or yearly, or even once every 2 to 20 years. Persons of
ordinary skill in the art can easily estimate repetition rates for
dosing based on measured residence times and concentrations of the
drug in bodily fluids or tissues. Following successful treatment,
it may be desirable to have the patient undergo maintenance therapy
to prevent the recurrence of the disease state, wherein the
oligonucleotide is administered in maintenance doses, ranging from
0.01 .mu.g to 100 g per kg of body weight, once or more daily, to
once every 20 years.
[0168] 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. Additionally, the mRNA or protein
expression levels of other genes known or suspected to be
associated with the specific disease state, condition or phenotype
can be assessed. mRNA levels can be measured or evaluated by
real-time PCR, Northern blot, in situ hybridization or DNA array
analysis. Protein levels can be measured or evaluated by ELISA,
immunoblotting, quantitative protein assays, protein activity
assays (for example, caspase activity assays) immunohistochemistry
or immunocytochemistry. Furthermore, the effects of treatment can
be assessed by measuring biomarkers associated with the disease or
condition in the aforementioned tissues and fluids, collected from
a patient or subject receiving treatment, by routine clinical
methods known in the art. These biomarkers include but are not
limited to: glucose, cholesterol, lipoproteins, triglycerides, free
fatty acids and other markers of glucose and lipid metabolism;
liver transaminases, bilirubin, albumin, blood urea nitrogen,
creatine and other markers of kidney and liver function;
interleukins, tumor necrosis factors, intracellular adhesion
molecules, C-reactive protein and other markers of inflammation;
testosterone, estrogen and other hormones; tumor markers; vitamins,
minerals and electrolytes.
[0169] 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, as well as each
application from which the present application claims priority, and
the like recited in the present application is incorporated herein
by reference in its entirety.
EXAMPLES
Example 1
Synthesis of Nucleoside Phosphoramidites
[0170] The following compounds, including amidites and their
intermediates were prepared as described in U.S. Pat. No. 6,426,220
and International Patent Publication No. WO 02/36743;
5'-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC
amidite, 51-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine
intermediate for 5-methyl-dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcytidine
penultimate intermediate for 5-methyl dC amidite,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N.sup.4-benzoyl-5-methylcy-
tidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(5-methyl dC amidite), 2'-Fluorodeoxyadenosine,
2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine,
2'-O-(2-Methoxyethyl) modified amidites,
2'-O-(2-methoxyethyl)-5-methyluridine intermediate,
5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate
intermediate,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi-
n-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T
amidite),
5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine
intermediate,
5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N.sup.4-benzoyl-5-methyl-cytid-
ine penultimate intermediate,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-benzo-
yl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE 5-Me-C amidite),
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.6-benzo-
yladenosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE A amdite),
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su-
p.4-isobutyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidit-
e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites,
2'-(Dimethylaminooxyethoxy) nucleoside amidites,
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine,
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine,
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine,
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri-
dine, 5'-O-tert-Butyldiphenylsilyl-2'-O--[N,N
dimethylaminooxyethyl]-5-methyluridine,
2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite], 2'-(Aminooxyethoxy)
nucleoside amidites,
N2-isobutyryl-6-.beta.-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5-
'-O-(4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite], 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
Oligonucleotide and Oligonucleoside Synthesis
[0171] 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.
[0172] 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.
[0173] Phosphorothioates (P.dbd.S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions:
thiation was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time was increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligonucleotides were recovered by precipitating with >3 volumes
of ethanol from a 1M NH.sub.4OAc solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270, herein incorporated by reference.
[0174] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0175] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050,
herein incorporated by reference.
[0176] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or 5,366,878, herein incorporated by
reference.
[0177] Alkylphosphonothioate oligonucleotides are prepared as
described in International Patent Application Nos. PCT/US94/00902
and PCT/US93/06976 (published as International Patent Publication
Nos. WO 94/17093 and WO 94/02499, respectively), herein
incorporated by reference.
[0178] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0179] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0180] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated
by reference.
[0181] Oligonucleosides: Methylenemethylimino linked
oligonucleosides, also identified as MMI linked oligonucleosides,
methylenedimethylhydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825; 5,386,023;
5,489,677; 5,602,240; and 5,610,289; all of which are herein
incorporated by reference.
[0182] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564,
herein incorporated by reference.
[0183] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 3
RNA Synthesis
[0184] 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.
[0185] 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.
[0186] 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.
[0187] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate
(S.sub.2Na.sub.2) in DMF. The deprotection solution is washed from
the solid support-bound oligonucleotide using water. The support is
then treated with 40% methylamine in water for 10 minutes at
55.degree. C. This releases the RNA oligonucleotides into solution,
deprotects the exocyclic amines, and modifies the 2'-groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this
stage.
[0188] 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 that 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.
[0189] Additionally, methods of RNA synthesis are well known in the
art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996;
Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821;
Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103,
3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett.,
1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990,
44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25,
4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23,
2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2315-2331).
[0190] 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 .mu.M 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.
Example 4
Synthesis of Chimeric Oligonucleotides
[0191] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0192] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spectrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry).
[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric
Phosphorothioate Oligonucleotides
[0193] [2'-O-(2-methoxyethyl)]-[2'-deoxy]-[-2'-O-(methoxyethyl)]
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
[2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy
Phosphorothioate]-[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0194] [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.
[0195] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/5 oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 5
Design and Screening of Duplexed Antisense Compounds Targeting
Apolipoprotein(a)
[0196] 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(a). 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. The antisense and sense strands
of the duplex comprise from about 17 to 25 nucleotides, or from
about 19 to 23 nucleotides. Alternatively, the antisense and sense
strands comprise 20, 21 or 22 nucleotides.
[0197] For example, a duplex comprising an antisense strand having
the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 97) and having a
two-nucleobase overhang of deoxythymidine(dT) has the following
structure (Antisense SEQ ID NO: 98, Complement SEQ ID NO: 99):
##STR00001##
[0198] 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 may have an overhang on
only one terminus.
[0199] In another embodiment, a duplex comprising an antisense
strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 97)
is prepared with blunt ends (no single stranded overhang) as shown
(Antisense SEQ ID NO: 97, Complement SEQ ID NO: 100):
##STR00002##
[0200] 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.
[0201] 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 .mu.M. Once diluted, 30 .mu.L of each strand is
combined with 15 .mu.L 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 .mu.L. 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 .mu.M. This solution can be stored frozen
(-20.degree. C.) and freeze-thawed up to 5 times.
[0202] Once prepared, the duplexed antisense compounds are
evaluated for their ability to modulate apolipoprotein(a)
expression.
[0203] When cells reached 80% confluency, they are treated with
duplexed antisense compounds of the invention. For cells grown in
96-well plates, wells are washed once with 200 .mu.L OPTI-MEM-1
reduced-serum medium (Gibco BRL) and then treated with 130 .mu.L of
OPTI-MEM-1 containing 12 .mu.g/mL LIPOFECTIN.TM. reagent
(Invitrogen Life Technologies, Carlsbad, Calif.) and the desired
duplex antisense compound at a final concentration of 200 nM. After
5 hours of treatment, the medium is replaced with fresh medium.
Cells are harvested 16 hours after treatment, at which time RNA is
isolated and target reduction measured by RT-PCR.
Example 6
Oligonucleotide Isolation
[0204] 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 were determined
by the ratio of correct molecular weight relative to the -16 amu
product (+/-32+/-48). For some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7
Oligonucleotide Synthesis--96 Well Plate Format
[0205] 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.
[0206] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
Example 8
Oligonucleotide Analysis-96-Well Plate Format
[0207] 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 apparatus) or, for individually prepared
samples, on a commercial CE apparatus (e.g., Beckman P/ACE.TM.
5000, ABI 270 apparatus). Base and backbone composition was
confirmed by mass analysis of the compounds utilizing
electrospray-mass spectroscopy. All assay test plates were diluted
from the master plate using single and multi-channel robotic
pipettors. Plates were judged to be acceptable if at least 85% of
the compounds on the plate were at least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
[0208] The effects of antisense compounds on target nucleic acid
expression are 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.
T-24 Cells:
[0209] The human transitional cell bladder carcinoma cell line T-24
was obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). T-24 cells were routinely cultured in complete
McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.)
supplemented with 10% fetal calf serum (Invitrogen Corporation,
Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin
100 .mu.g/mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were
routinely passaged by trypsinization and dilution when they reached
90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #353872) at a density of 7000 cells/well for use
in RT-PCR analysis.
[0210] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
A549 Cells:
[0211] The human lung carcinoma cell line A549 was obtained from
the American Type Culture Collection (ATCC) (Manassas, Va.). A549
cells were routinely cultured in DMEM basal media (Invitrogen
Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf
serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100
units per mL, and streptomycin 100 .mu.g/mL (Invitrogen
Corporation, Carlsbad, Calif.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
NHDF Cells:
[0212] Human neonatal dermal fibroblasts (NHDFs) were obtained from
the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville, Md.) supplemented as recommended by the supplier.
Cells were maintained for up to 10 passages as recommended by the
supplier.
HEK Cells:
[0213] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville, Md.). HEKs were routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville, Md.) formulated as recommended by the supplier. Cells
were routinely maintained for up to 10 passages as recommended by
the supplier.
Treatment with Antisense Compounds:
[0214] When cells reached 65-75% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 100 .mu.L OPTI-MEM.TM.-1 reduced-serum medium
(Invitrogen Corporation, Carlsbad, Calif.) and then treated with
130 .mu.L of OPTI-MEM.TM.-1 medium containing 3.75 .mu.g/mL
LIPOFECTIN.TM. reagent (Invitrogen Corporation, Carlsbad, Calif.)
and the desired concentration of oligonucleotide. Cells are treated
and data are obtained in triplicate. After 4-7 hours of treatment
at 37.degree. C., the medium was replaced with fresh medium. Cells
were harvested 16-24 hours after oligonucleotide treatment.
[0215] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is selected from either ISIS 13920
(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human
H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is
targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are
2'-O-methoxyethyl gapmers (2'-O-methoxyethyls shown in bold) with a
phosphorothioate backbone. For mouse or rat cells the positive
control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID
NO: 3, a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in
bold) with a phosphorothioate backbone which is targeted to both
mouse and rat c-raf. The concentration of positive control
oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS
13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is
then utilized as the screening concentration for new
oligonucleotides in subsequent experiments for that cell line. If
80% inhibition is not achieved, the lowest concentration of
positive control oligonucleotide that results in 60% inhibition of
c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% inhibition is not achieved, that particular cell line
is deemed as unsuitable for oligonucleotide transfection
experiments. The concentrations of antisense oligonucleotides used
herein are from 50 nM to 300 nM.
Example 10
Analysis of Oligonucleotide Inhibition of Apolipoprotein(a)
Expression
[0216] Antisense modulation of apolipoprotein(a) expression can be
assayed in a variety of ways known in the art. For example,
apolipoprotein(a) mRNA levels can be quantitated by, e.g., Northern
blot analysis, competitive polymerase chain reaction (PCR), or
real-time PCR (RT-PCR). Real-time quantitative PCR is presently
preferred. RNA analysis can be performed on total cellular RNA or
poly(A)+ mRNA. The preferred method of RNA analysis of the present
invention is the use of total cellular RNA as described in other
examples herein. Methods of RNA isolation are well known in the
art. Northern blot analysis is also routine in the art. Real-time
quantitative (PCR) can be conveniently accomplished using the
commercially available ABI PRISM.TM. 7600, 7700, or 7900 Sequence
Detection System, available from PE-Applied Biosystems, Foster
City, Calif. and used according to manufacturer's instructions.
[0217] Protein levels of apolipoprotein(a) can be quantitated in a
variety of ways well known in the art, such as immunoprecipitation,
Western blot analysis (immunoblotting), enzyme-linked immunosorbent
assay (ELISA) or fluorescence-activated cell sorting (FACS).
Antibodies directed to apolipoprotein(a) can be identified and
obtained from a variety of sources, such as the MSRS catalog of
antibodies (Aerie Corporation, Birmingham, Mich.), or can be
prepared via conventional monoclonal or polyclonal antibody
generation methods well known in the art.
Example 11
Design of Phenotypic Assays and In Vivo Studies for the Use of
Apolipoprotein(a) Inhibitors
Phenotypic Assays
[0218] Once apolipoprotein(a) 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(a) 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.).
[0219] 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(a) 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.
[0220] 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.
[0221] Analysis of the genotype of the cell (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(a) 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.
[0222] The cells subjected to the phenotypic assays described
herein derive from in vitro cultures or from tissues or fluids
isolated from living organisms, both human and non-human. 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, brain, stem cells, blood vessel,
liver, lung, bone, breast, cartilage, cervix, colon, cornea,
embryonic, endometrium, endothelial, epithelial, esophagus, facia,
fibroblast, follicular, ganglion cells, glial cells, goblet cells,
kidney, lymph node, muscle, neuron, ovaries, pancreas, peripheral
blood, prostate, skin, skin, small intestine, spleen, stomach,
testes and fetal tissue. In other embodiments, a fluid and its
constituent cells comprise, but is not limited to, blood, urine,
synovial fluid, lymphatic fluid and cerebro-spinal fluid. The
phenotypic assays may also be performed on tissues treated with
apolipoprotein(a) inhibitors ex vivo.
In Vivo Studies
[0223] The individual subjects of the in vivo studies described
herein are warm-blooded vertebrate animals, including humans.
[0224] The clinical trial is subjected to rigorous controls to
ensure that individuals are not unnecessarily put at risk and that
they are fully informed about their role in the study.
[0225] To account for the psychological effects of receiving
treatments, volunteers are randomly given placebo or
apolipoprotein(a) inhibitor. Furthermore, to prevent the doctors
from being biased in treatments, they are not informed as to
whether the medication they are administering is a
apolipoprotein(a) inhibitor or a placebo. Using this randomization
approach, each volunteer has the same chance of being given either
the new treatment or the placebo.
[0226] Volunteers receive either the apolipoprotein(a) inhibitor or
placebo for eight week period with biological parameters associated
with the indicated disease state or condition being measured at the
beginning (baseline measurements before any treatment), end (after
the final treatment), and at regular intervals during the study
period. Such measurements include the levels of nucleic acid
molecules encoding apolipoprotein(a) or apolipoprotein(a) protein
levels in body fluids, tissues or organs compared to pre-treatment
levels. Other measurements include, but are not limited to, indices
of the disease state or condition being treated, body weight, blood
pressure, serum titers of pharmacologic indicators of disease or
toxicity as well as ADME (absorption, distribution, metabolism and
excretion) measurements.
[0227] Information recorded for each patient includes age (years),
gender, height (cm), family history of disease state or condition
(yes/no), motivation rating (some/moderate/great) and number and
type of previous treatment regimens for the indicated disease or
condition.
[0228] Volunteers taking part in this study are healthy adults (age
18 to 65 years) and roughly an equal number of males and females
participate in the study. Volunteers with certain characteristics
are equally distributed for placebo and apolipoprotein(a) inhibitor
treatment. In general, the volunteers treated with placebo have
little or no response to treatment, whereas the volunteers treated
with the apolipoprotein(a) inhibitor show positive trends in their
disease state or condition index at the conclusion of the
study.
Example 12
RNA Isolation
[0229] Poly(A)+ mRNA Isolation
[0230] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.
Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA
isolation are routine in the art. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C., was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0231] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Total RNA Isolation
[0232] Total RNA was isolated using an RNEASY.TM. 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. 150 .mu.L Buffer RLT
was added to each well and the plate vigorously agitated for 20
seconds. 150 .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.TM. 96 well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum was applied for 1
minute. 500 .mu.L of Buffer RW1 was added to each well of the
RNEASY.TM. 96 plate and incubated for 15 minutes and the vacuum was
again applied for 1 minute. An additional 500 .mu.L of Buffer RW1
was added to each well of the RNEASY.TM. 96 plate and the vacuum
was applied for 2 minutes. 1 mL of Buffer RPE was then added to
each well of the RNEASY.TM. 96 plate and the vacuum applied for a
period of 90 seconds. The Buffer RPE wash was then repeated and the
vacuum was applied for an additional 3 minutes. The plate was then
removed from the QIAVAC.TM. manifold and blotted dry on paper
towels. The plate was then re-attached to the QIAVAC.TM. manifold
fitted with a collection tube rack containing 1.2 mL collection
tubes. RNA was then eluted by pipetting 140 .mu.L of RNase free
water into each well, incubating 1 minute, and then applying the
vacuum for 3 minutes.
[0233] The repetitive pipetting and elution steps may be automated
using a QIAGEN.RTM. Bio-Robot.TM. 9604 (Qiagen, Inc., Valencia
Calif.). Essentially, after lysing of the cells on the culture
plate, the plate is transferred to the robot deck where the
pipetting, DNase treatment and elution steps are carried out.
Example 13
Real-Time Quantitative PCR Analysis of Apolipoprotein(a) mRNA
Levels
[0234] Quantitation of apolipoprotein(a) mRNA levels was
accomplished by real-time quantitative PCR using the ABI PRISM.TM.
7600, 7700, or 7900 Sequence Detection System (PE-Applied
Biosystems, Foster City, Calif.) according to manufacturer's
instructions. This is a closed-tube, non-gel-based, fluorescence
detection system which allows high-throughput quantitation of
polymerase chain reaction (PCR) products in real-time. As opposed
to standard PCR in which amplification products are quantitated
after the PCR is completed, products in real-time quantitative PCR
are quantitated as they accumulate. This is accomplished by
including in the PCR reaction an oligonucleotide probe that anneals
specifically between the forward and reverse PCR primers, and
contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,
obtained from either PE-Applied Biosystems, Foster City, Calif.,
Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 5' end of
the probe and a quencher dye (e.g., TAMRA, obtained from either
PE-Applied Biosystems, Foster City, Calif., Operon Technologies
Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,
Coralville, Iowa) is attached to the 3' end of the probe. When the
probe and dyes are intact, reporter dye emission is quenched by the
proximity of the 3' quencher dye. During amplification, annealing
of the probe to the target sequence creates a substrate that can be
cleaved by the 5'-exonuclease activity of Taq polymerase. During
the extension phase of the PCR amplification cycle, cleavage of the
probe by Taq polymerase releases the reporter dye from the
remainder of the probe (and hence from the quencher moiety) and a
sequence-specific fluorescent signal is generated. With each cycle,
additional reporter dye molecules are cleaved from their respective
probes, and the fluorescence intensity is monitored at regular
intervals by laser optics built into the ABI PRISM.TM. Sequence
Detection System. In each assay, a series of parallel reactions
containing serial dilutions of mRNA from untreated control samples
generates a standard curve that is used to quantitate the percent
inhibition after antisense oligonucleotide treatment of test
samples.
[0235] 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.
[0236] Prior to the real-time PCR, isolated RNA is subjected to a
reverse transcriptase (RT) reaction, for the purpose of generating
complementary DNA (cDNA), from which the real-time PCR product is
amplified. Reverse transcriptase and PCR reagents were obtained
from Invitrogen Corporation, (Carlsbad, Calif.). RT, real-time PCR
reactions carried out by adding 20 .mu.L PCR cocktail
(2.5.times.PCR buffer minus MgCl.sub.2, 6.6 mM MgCl.sub.2, 375
.mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward
primer and reverse primer, 125 nM of probe, 4 Units RNase
inhibitor, 1.25 Units PLATINUM.RTM. Taq polymerase, 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 polymerase, 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).
The method of obtaining gene target quantities by RT, real-time PCR
is herein referred to as real-time PCR.
[0237] Gene target quantities obtained by RT, 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.TM. reagent (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.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RIBOGREEN.TM. reagent are taught in Jones, L. J.,
et al, (Analytical Biochemistry, 1998, 265, 368-374).
[0238] In this assay, 170 .mu.L of RIBOGREEN.TM. working reagent
(RIBOGREEN.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000
apparatus (PE Applied Biosystems) with excitation at 485 nm and
emission at 530 nm.
[0239] Probes and primers to human apolipoprotein(a) were designed
to hybridize to a human apolipoprotein(a) sequence, using published
sequence information (GENBANK.RTM. accession number
NM.sub.--005577.1, incorporated herein as SEQ ID NO: 4). For human
apolipoprotein(a) the PCR primers were:
forward primer: CAGCTCCTTATTGTTATACGAGGGA (SEQ ID NO: 5) reverse
primer: TGCGTCTGAGCATTGCGT (SEQ ID NO: 6) and the PCR probe was:
FAM-CCCGGTGTCAGGTGGGAGTACTGC-TAMRA (SEQ ID NO: 7) where FAM is the
fluorescent dye and TAMRA is the quencher dye.
[0240] Gene target quantities in mouse cells are tissues are
normalized using mouse GAPDH expression. For mouse GAPDH the PCR
primers were:
forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 8) reverse primer:
GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 9) and the PCR probe was: 5'
JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3' (SEQ ID NO: 10) where JOE
is the fluorescent reporter dye and TAMRA is the quencher dye.
Example 14
Northern Blot Analysis of Apolipoprotein(a) mRNA Levels
[0241] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM.
reagent (TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was
prepared following manufacturer's recommended protocols. Twenty
micrograms of total RNA was fractionated by electrophoresis through
1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer
system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the
gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.TM.
UV Crosslinker 2400 apparatus (Stratagene, Inc, La Jolla, Calif.)
and then probed using QUICKHYB.TM. hybridization solution
(Stratagene, La Jolla, Calif.) using manufacturer's recommendations
for stringent conditions.
[0242] To detect human apolipoprotein(a), a human apolipoprotein(a)
specific probe was prepared by PCR using the forward primer
CAGCTCCTTATTGTTATACGAGGGA (SEQ ID NO: 5) and the reverse primer
TGCGTCTGAGCATTGCGT (SEQ ID NO: 6). To normalize for variations in
loading and transfer efficiency membranes were stripped and probed
for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA
(Clontech, Palo Alto, Calif.).
[0243] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. apparatus and IMAGEQUANT.TM. Software V3.3
(Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to
GAPDH levels in untreated controls.
Example 15
Antisense Inhibition of Human Apolipoprotein(a) Expression by
Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and
a Deoxy Gap
[0244] In accordance with the present invention, a series of
antisense compounds was designed to target different regions of the
human apolipoprotein(a) RNA, using published sequences
(GENBANK.RTM. accession number NM.sub.--005577.1, incorporated
herein as SEQ ID NO: 4). The compounds are shown in Table 1.
"Target site" indicates the first (5'-most) nucleotide number on
the particular target sequence to which the compound binds. All
compounds in Table 1 are chimeric oligonucleotides ("gapmers") 20
nucleotides in length, composed of a central "gap" region
consisting of ten 2'-deoxynucleotides, which is flanked on both
sides (5' and 3' directions) by five-nucleotide "wings". The wings
are composed of 2'-O-methoxyethyl (2'-MOE) nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
throughout the oligonucleotide. All cytidine residues are
5-methylcytidines.
[0245] Apolipoprotein(a) is found in humans, nonhuman primates and
the European hedgehog, but not in common laboratory animals such as
rats and mice. Transgenic mice which express human
apolipoprotein(a) have been engineered (Chiesa et al., J. Biol.
Chem., 1992, 267, 24369-24374). The use of primary hepatocytes
prepared from human apolipoprotein(a) transgenic mice circumvents
the issue of variability when testing antisense oligonucleotide
activity in primary cells. Accordingly, primary mouse hepatocytes
prepared from the human apolipoprotein(a) transgenic mice were used
to investigate the effects of antisense oligonucleotides on human
apolipoprotein(a) expression. The human apolipoprotein(a)
transgenic mice were obtained from Dr. Robert Pitas and Dr.
Matthias Schneider in the Gladstone Institute at the University of
California, San Francisco. Primary hepatocytes were isolated from
these mice and were cultured in DMEM, high glucose (Invitrogen
Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine
serum, (Invitrogen Corporation, Carlsbad, Calif.), 100 units per mL
penicillin and 100 .mu.g/mL streptomycin (Invitrogen Corporation,
Carlsbad, Calif.). For treatment with oligonucleotide, cells were
washed once with serum-free DMEM and subsequently transfected with
a dose of 150 nM of antisense oligonucleotide using LIPOFECTIN.TM.
reagent (Invitrogen Corporation, Carlsbad, Calif.) as described in
other examples herein. The compounds were analyzed for their effect
on human apolipoprotein(a) mRNA levels by quantitative real-time
PCR as described in other examples herein. Gene target quantities
obtained by real time RT-PCR were normalized using mouse GAPDH.
[0246] Data are averages from three experiments in which primary
transgenic mouse hepatocytes were treated with 150 nM of antisense
oligonucleotides targeted to human apolipoprotein(a).
TABLE-US-00001 TABLE 1 Inhibition of human apolipoprotein(a) mRNA
levels by chimeric phosphorothioate oligonucleotides having 2'-MOE
wings and a deoxy gap TAR- GET SEQ TAR- SEQ ID GET % ID ISIS #
REGION NO SITE SEQUENCE INHIB NO 144367 Coding 4 174
ggcaggtccttcctgtgaca 53 11 144368 Coding 4 352 tctgcgtctgagcattgcgt
87 12 144369 Coding 4 522 aagcttggcaggttcttcct 0 13 144370 Coding 4
1743 tcggaggcgcgacggcagtc 40 14 144371 Coding 4 2768
cggaggcgcgacggcagtcc 0 15 144372 Coding 4 2910 ggcaggttcttcctgtgaca
65 16 144373 Coding 4 3371 ataacaataaggagctgcca 50 17 144374 Coding
4 4972 gaccaagcttggcaggttct 62 18 144375 Coding 4 5080
taacaataaggagctgccac 36 19 144376 Coding 4 5315
tgaccaagcttggcaggttc 25 20 144377 Coding 4 5825
ttctgcgtctgagcattgcg 38 21 144378 Coding 4 6447
aacaataaggagctgccaca 29 22 144379 Coding 4 7155
acctgacaccgggatccctc 79 23 144380 Coding 4 7185
ctgagcattgcgtcaggttg 16 24 144381 Coding 4 8463
agtagttcatgatcaagcca 71 25 144382 Coding 4 8915
gacggcagtcccttctgcgt 34 26 144383 Coding 4 9066
ggcaggttcttccagtgaca 5 27 144384 Coding 4 10787
tgaccaagcttggcaagttc 31 28 144385 Coding 4 11238
tataacaccaaggactaatc 9 29 144386 Coding 4 11261
ccatctgacattgggatcca 66 30 144387 Coding 4 11461
tgtggtgtcatagaggacca 36 31 144388 Coding 4 11823
atgggatcctccgatgccaa 55 32 144389 Coding 4 11894
acaccaagggcgaatctcag 58 33 144390 Coding 4 11957
ttctgtcactggacatcgtg 59 34 144391 Coding 4 12255
cacacggatcggttgtgtaa 58 35 144392 Coding 4 12461
acatgtccttcctgtgacag 51 36 144393 Coding 4 12699
cagaaggaggccctaggctt 33 37 144394 Coding 4 13354
ctggcggtgaccatgtagtc 52 38 144395 3'UTR 4 13711
tctaagtaggttgatgcttc 68 39 144396 3'UTR 4 13731
tccttacccacgtttcagct 70 40 144397 3'UTR 4 13780
ggaacagtgtcttcgtttga 63 41 144398 3'UTR 4 13801
gtttggcatagctggtagct 44 42 144399 3'UTR 4 13841
accttaaaagcttatacaca 57 43 144400 3'UTR 4 13861
atacagaatttgtcagtcag 21 44 144401 3'UTR 4 13881
gtcatagctatgacacctta 46 45
[0247] As shown in Table 1, SEQ ID NOs 11, 12, 14, 16, 17, 18, 19,
21, 23, 25, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43 and
45 demonstrated at least 35% inhibition of human apolipoprotein(a)
expression in this assay and are therefore preferred. More
preferred are SEQ ID NOs 23, 12 and 40. The target regions to which
these preferred sequences are complementary are herein referred to
as "preferred target segments" and are therefore preferred for
targeting by compounds of the present invention. These preferred
target segments are shown in Table 2. These sequences are shown to
contain thymine (T) but one of skill in the art will appreciate
that thymine (T) is generally replaced by uracil (U) in RNA
sequences. The sequences represent the reverse complement of the
preferred antisense compounds shown in Table 1. "Target site"
indicates the first (5'-most) nucleotide number on the particular
target nucleic acid to which the oligonucleotide binds. Also shown
in Table 2 is the species in which each of the preferred target
segments was found.
TABLE-US-00002 TABLE 2 Sequence and position of preferred target
segments identified in apolipoprotein(a). TARGET REV SITE SEQ ID
TARGET COMP OF SEQ ID ID NO SITE SEQUENCE SEQ ID ACTIVE IN NO 57364
4 174 tgtcacaggaaggacctgcc 11 H. sapiens 46 57365 4 352
acgcaatgctcagacgcaga 12 H. sapiens 47 57367 4 1743
gactgccgtcgcgcctccga 14 H. sapiens 48 57369 4 2910
tgtcacaggaagaacctgcc 16 H. sapiens 49 57370 4 3371
tggcagctccttattgttat 17 H. sapiens 50 57371 4 4972
agaacctgccaagcttggtc 18 H. sapiens 51 57372 4 5080
gtggcagctccttattgtta 19 H. sapiens 52 57374 4 5825
cgcaatgctcagacgcagaa 21 H. sapiens 53 57376 4 7155
gagggatcccggtgtcaggt 23 H. sapiens 54 57378 4 8463
tggcttgatcatgaactact 25 H. sapiens 55 57383 4 11261
tggatcccaatgtcagatgg 30 H. sapiens 56 57384 4 11461
tggtcctctatgacaccaca 31 H. sapiens 57 57385 4 11823
ttggcatcggaggatcccat 32 H. sapiens 58 57386 4 11894
ctgagattcgcccttggtgt 33 H. sapiens 59 57387 4 11957
cacgatgtccagtgacagaa 34 H. sapiens 60 57388 4 12255
ttacacaaccgatccgtgtg 35 H. sapiens 61 57389 4 12461
ctgtcacaggaaggacatgt 36 H. sapiens 62 57391 4 13354
gactacatggtcaccgccag 38 H. sapiens 63 57392 4 13711
gaagcatcaacctacttaga 39 H. sapiens 64 57393 4 13731
agctgaaacgtgggtaagga 40 H. sapiens 65 57394 4 13780
tcaaacgaagacactgttcc 41 H. sapiens 66 57395 4 13801
agctaccagctatgccaaac 42 H. sapiens 67 57396 4 13841
tgtgtataagcttttaaggt 43 H. sapiens 68 57398 4 13881
taaggtgtcatagctatgac 45 H. sapiens 69
[0248] 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(a).
[0249] According to the present invention, antisense compounds
include antisense oligomeric compounds, antisense oligonucleotides,
siRNAs, external guide sequence (EGS) oligonucleotides, alternate
splicers, and other short oligomeric compounds that hybridize to at
least a portion of the target nucleic acid.
Example 16
Western Blot Analysis of Apolipoprotein(a) Protein Levels
[0250] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 .mu.l/well), boiled for 5 minutes and loaded on
a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to apolipoprotein(a) is used, with a radiolabeled
or fluorescently labeled secondary antibody directed against the
primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. apparatus (Molecular Dynamics, Sunnyvale
Calif.).
Example 17
Antisense Inhibition of Human Apolipoprotein(a) in Transgenic
Primary Mouse Hepatocytes: Dose Response
[0251] In accordance with the present invention, antisense
oligonucleotides identified as having good activity based on the
results in Example 15 were further investigated in dose-response
studies. Primary hepatocytes from human apolipoprotein(a)
transgenic mice were treated with 10, 50, 150 or 300 nM of ISIS
144396 (SEQ ID NO: 40), ISIS 144368 (SEQ ID NO: 12), ISIS 144379
(SEQ ID NO: 23) or ISIS 113529 (CTCTTACTGTGCTGTGGACA, SEQ ID NO:
70). ISIS 113529, which does not target apolipoprotein(a), was used
as a control oligonucleotide and is a chimeric oligonucleotides
("gapmers") 20 nucleotides in length, composed of a central "gap"
region consisting of ten 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by five-nucleotide "wings". The
wings are composed of 2'-O-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
throughout the oligonucleotide. All cytidine residues are
5-methylcytidines.
[0252] Following 24 hours of exposure to antisense
oligonucleotides, target mRNA expression levels were evaluated by
quantitative real-time PCR as described in other examples herein.
The results are the average of 4 experiments for apolipoprotein(a)
antisense oligonucleotides and the average of 12 experiments for
the control oligonucleotide. The data are expressed as percent
inhibition of apolipoprotein(a) expression relative to untreated
controls and are shown in Table 3.
TABLE-US-00003 TABLE 3 Antisense inhibition of human
apolipoprotein(a) in transgenic primary mouse hepatocytes: dose
response % Inhibition of transgenic human lipoprotein(a) ISIS #
Oligonucleotide dose 144396 144368 144379 113529 10 nM 0 11 55 N.D.
50 nM 0 26 73 N.D. 150 nM 0 58 85 N.D. 300 nM 9 62 89 0
[0253] These data demonstrate that ISIS 144368 and ISIS 144379
inhibited the expression of human apolipoprotein(a) in a
dose-dependent fashion.
Example 18
Oil Red O Stain
[0254] Hepatic steatosis, or accumulation of lipids in the liver,
is assessed by routine histological analysis of frozen liver tissue
sections stained with oil red 0 stain, which is commonly used to
visualize lipid deposits, and counterstained with hematoxylin and
eosin, to visualize nuclei and cytoplasm, respectively. Tissue is
preserved in 10% neutral-buffered formalin, embedded in paraffin,
sectioned and stained.
Example 19
Animal Models
[0255] In addition to human systems, which express
apolipoprotein(a), biological systems of other mammals are also
available for studies of expression products of the LPA gene as
well as for studies of the Lp(a) particles and their role in
physiologic processes.
[0256] Transgenic mice which express human apolipoprotein(a) have
been engineered (Chiesa et al., J. Biol. Chem., 1992, 267,
24369-24374) and are used as an animal model for the investigation
of the in vivo activity of the oligonucleotides of this invention.
Although transgenic mice expressing human apolipoprotein(a) exist,
they fail to assemble Lp(a) particles because of the inability of
human apolipoprotein(a) to associate with mouse apolipoprotein B.
When mice expressing human apolipoprotein(a) are bred to mice
expressing human apolipoprotein B, the Lp(a) particle is
efficiently assembled (Callow et al., Proc. Natl. Acad. Sci. USA,
1994, 91, 2130-2134). Accordingly mice expressing both human
apolipoprotein(a) and human apolipoprotein B transgenes are used
for animal model studies in which the secretion of the Lp(a)
particle is evaluated.
[0257] Where additional genetic alterations are necessary, mice
with either a single human transgene (human apolipoprotein(a) or
human apolipoprotein B) or both human transgenes (human
apolipoprotein(a) and human apolipoprotein B) are bred to mice with
a desired genetic mutation. The offspring with the desired
combination of transgene(s) and genetic mutation(s) is selected for
use as an animal model. In one nonlimiting example, mice expressing
both human apolipoprotein(a) and human apolipoprotein B are bred to
mice with a mutation in the leptin gene, yielding offspring
producing human Lp(a) particles in an ob/ob model of obesity and
diabetes.
ob/ob Mice
[0258] Leptin is a hormone produced by fat that regulates appetite.
Deficiencies in this hormone in both humans and non-human animals
leads to obesity. ob/ob mice have a mutation in the leptin gene
which results in obesity and hyperglycemia. As such, these mice are
a useful model for the investigation of obesity and treatments
designed to reduce obesity.
[0259] Seven-week old male C57Bl/6J-Lep ob/ob mice (Jackson
Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of
10-15% and are subcutaneously injected with oligonucleotides of the
present invention or a control oligonucleotide at a dose of 5, 10
or 25 mg/kg two times per week for 4 weeks. Saline-injected animals
and leptin wildtype littermates (i.e. lean littermates) serve as
controls. After the treatment period, mice are sacrificed and
target levels are evaluated in liver, brown adipose tissue (BAT)
and white adipose tissue (WAT). RNA isolation and target mRNA
expression level quantitation are performed as described by other
examples herein.
[0260] To assess the physiological effects resulting from antisense
inhibition of target apolipoprotein(a) mRNA, the ob/ob mice that
receive antisense oligonucleotide treatment are further evaluated
at the end of the treatment period for serum lipids, serum
apolipoproteins, serum free fatty acids, serum cholesterol (CHOL),
liver triglycerides, and fat tissue triglycerides. Serum components
are measured on routine clinical diagnostic instruments. Tissue
triglycerides are extracted using an acetone extraction technique
known in the art, and subsequently measured by ELISA. The presence
of the Lp(a) particle in the serum is measured using a commercially
available ELISA kit (ALerCHEK Inc., Portland, Me.). Hepatic
steatosis, or accumulation of lipids in the liver, is assessed by
measuring the liver triglyceride content. Hepatic steatosis is also
assessed by routine histological analysis of frozen liver tissue
sections stained with oil red O stain, which is commonly used to
visualize lipid deposits, and counterstained with hematoxylin and
eosin, to visualize nuclei and cytoplasm, respectively.
[0261] The effects of apolipoprotein(a) inhibition on glucose and
insulin metabolism are also evaluated in the ob/ob mice treated
with antisense oligonucleotides of this invention. Plasma glucose
is measured at the start of the antisense oligonucleotide treatment
and after 2 weeks and 4 weeks of treatment. Plasma insulin is
similarly at the beginning to of the treatment, and following 2
weeks and 4 weeks of treatment. Glucose and insulin tolerance tests
are also administered in fed and fasted mice. Mice receive
intraperitoneal injections of either glucose or insulin, and the
blood glucose and insulin levels are measured before the insulin or
glucose challenge and at 15, 20 or 30 minute intervals for up to 3
hours.
[0262] To assess the metabolic rate of ob/ob mice treated with
antisense oligonucleotides of this invention, the respiratory
quotient and oxygen consumption of the mice are also measured.
[0263] The ob/ob mice that received antisense oligonucleotide
treatment are further evaluated at the end of the treatment period
for the effects of apolipoprotein(a) inhibition on the expression
of genes that participate in lipid metabolism, cholesterol
biosynthesis, fatty acid oxidation, fatty acid storage,
gluconeogenesis and glucose metabolism. These genes include, but
are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and
acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and
glycogen phosphorylase, glucose-6-phosphatase and
phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormone
sensitive lipase. mRNA levels in liver and white and brown adipose
tissue are quantitated by real-time PCR as described in other
examples herein, employing primer-probe sets that were generated
using published sequences of each gene of interest.
db/db Mice
[0264] A deficiency in the leptin hormone receptor mouse also
results in obesity and hyperglycemia. These mice are referred to as
db/db mice and, like the ob/ob mice, are used as a mouse model of
obesity.
[0265] Seven-week old male C57Bl/6J-Lepr db/db mice (Jackson
Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of
15-20% and are subcutaneously injected with oligonucleotides of
this invention or a control oligonucleotide at a dose of 5, 10 or
25 mg/kg two times per week for 4 weeks. Saline-injected animals
and leptin receptor wildtype littermates (i.e. lean littermates)
serve as controls. After the treatment period, mice are sacrificed
and apolipoprotein(a) levels are evaluated in liver, brown adipose
tissue (BAT) and white adipose tissue (WAT). RNA isolation and
apolipoprotein(a) mRNA expression level quantitation are performed
as described by other examples herein.
[0266] After the treatment period, mice are sacrificed and
apolipoprotein(a) levels are evaluated in liver, brown adipose
tissue (BAT) and white adipose tissue (WAT). RNA isolation and
apolipoprotein(a) mRNA expression level quantitation are performed
as described by other examples herein.
[0267] To assess the physiological effects resulting from antisense
inhibition of apolipoprotein(a) mRNA, the db/db mice that receive
antisense oligonucleotide treatment are further evaluated at the
end of the treatment period for serum lipids, serum apolipoproeins,
serum free fatty acids, serum cholesterol (CHOL), liver
triglycerides, and fat tissue triglycerides. Serum components are
measured on routine clinical diagnostic instruments. Tissue
triglycerides are extracted using an acetone extraction technique
known in the art, and subsequently measured by ELISA. The presence
of the Lp(a) particle in the serum is measured using a commercially
available ELISA kit (ALerCHEK Inc., Portland, Me.). Hepatic
steatosis, or accumulation of lipids in the liver, is assessed by
measuring the liver triglyceride content. Hepatic steatosis is also
assessed by routine histological analysis of frozen liver tissue
sections stained with oil red O stain, which is commonly used to
visualize lipid deposits, and counterstained with hematoxylin and
eosin, to visualize nuclei and cytoplasm, respectively.
[0268] The effects of apolipoprotein(a) inhibition on glucose and
insulin metabolism are also evaluated in the db/db mice treated
with antisense oligonucleotides. Plasma glucose is measured at the
start of the antisense oligonucleotide treatment and after 2 weeks
and 4 weeks of treatment. Plasma insulin is similarly at the
beginning to of the treatment, and following 2 weeks and 4 weeks of
treatment. Glucose and insulin tolerance tests are also
administered in fed and fasted mice. Mice receive intraperitoneal
injections of either glucose or insulin, and the blood glucose
levels are measured before the insulin or glucose challenge and 15,
30, 60, 90 and 120 minutes following the injection.
[0269] To assess the metabolic rates of db/db mice treated with
antisense oligonucleotides, the respiratory quotients and oxygen
consumptions of the mice are also measured.
[0270] The db/db mice that received antisense oligonucleotide
treatment are further evaluated at the end of the treatment period
for the effects of apolipoprotein(a) inhibition on the expression
of genes that participate in lipid metabolism, cholesterol
biosynthesis, fatty acid oxidation, fatty acid storage,
gluconeogenesis and glucose metabolism. These genes include, but
are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and
acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and
glycogen phosphorylase, glucose-6-phosphatase and
phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormone
sensitive lipase. mRNA levels in liver and white and brown adipose
tissue are quantitated by real-time PCR as described in other
examples herein, employing primer-probe sets that were generated
using published sequences of each gene of interest.
Lean Mice
[0271] C57Bl/6 mice are maintained on a standard rodent diet and
are used as control (lean) animals. Seven-week old male C57Bl/6
mice are fed a diet with a fat content of 4% and are subcutaneously
injected with oligonucleotides of this invention or control
oligonucleotide at a dose of 5, 10 or 25 mg/kg two times per week
for 4 weeks. Saline-injected animals serve as a control. After the
treatment period, mice are sacrificed and apolipoprotein(a) levels
are evaluated in liver, brown adipose tissue (BAT) and white
adipose tissue (WAT). RNA isolation and apolipoprotein(a) mRNA
expression level quantitation are performed as described by other
examples herein.
[0272] To assess the physiological effects resulting from antisense
inhibition of apolipoprotein(a) mRNA, the lean mice that receive
antisense oligonucleotide treatment are further evaluated at the
end of the treatment period for serum lipids, serum free fatty
acids, serum cholesterol (CHOL), liver triglycerides, and fat
tissue triglycerides. Serum components are measured on routine
clinical diagnostic instruments. Tissue triglycerides are extracted
using an acetone extraction technique known in the art, and
subsequently measured by ELISA. The presence of the Lp(a) particle
in the serum is measured using a commercially available ELISA kit
(ALerCHEK Inc., Portland, Me.). Hepatic steatosis, i.e.
accumulation of lipids in the liver, is assessed by measuring the
liver triglyceride content. Hepatic steatosis is also assessed by
routine histological analysis of frozen liver tissue sections
stained with oil red O stain, which is commonly used to visualize
lipid deposits, and counterstained with hematoxylin and eosin, to
visualize nuclei and cytoplasm, respectively.
[0273] The effects of apolipoprotein(a) inhibition on glucose and
insulin metabolism are also evaluated in the lean mice treated with
antisense oligonucleotides of this invention. Plasma glucose is
measured at the start of the antisense oligonucleotide treatment
and after 2 weeks and 4 weeks of treatment. Plasma insulin is
similarly at the beginning to of the treatment, and following 2
weeks and 4 weeks of treatment. Glucose and insulin tolerance tests
are also administered in fed and fasted mice. Mice receive
intraperitoneal injections of either glucose or insulin, and the
blood glucose levels are measured before the insulin or glucose
challenge and 15, 30, 60, 90 and 120 minutes following the
injection.
[0274] To assess the metabolic rates of lean mice treated with
antisense oligonucleotides of this invention, the respiratory
quotients and oxygen consumptions of the mice can also be
measured.
[0275] The lean mice that received antisense oligonucleotide
treatment are further evaluated at the end of the treatment period
for the effects of apolipoprotein(a) inhibition on the expression
of genes that participate in lipid metabolism, cholesoterol
biosynthesis, fatty acid oxidation, fatty acid storage,
gluconeogenesis and glucose metabolism. These genes include, but
are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and
acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and
glycogen expressing both human apolipoprotein(a) and human
apolipoprotein B are bred to mice with a mutation in the leptin
gene, yielding offspring producing human Lp(a) particles in an
ob/ob model of obesity and diabetes.
ob/ob Mice
[0276] Leptin is a hormone produced by fat that regulates appetite.
Deficiencies in this hormone in both humans and non-human animals
leads to obesity. ob/ob mice have a mutation in the leptin gene
which results in obesity and hyperglycemia. As such, these mice are
a useful model for the investigation of obesity and treatments
designed to reduce obesity.
[0277] Seven-week old male C57Bl/6J-Lep ob/ob mice (Jackson
Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of
10-15% and are subcutaneously injected with oligonucleotides of the
present invention or a control oligonucleotide at a dose of 5, 10
or 25 mg/kg two times per week for 4 weeks. Saline-injected animals
and leptin wildtype littermates (i.e. lean littermates) serve as
controls. After the treatment period, mice are sacrificed and
target levels are evaluated in liver, brown adipose tissue (BAT)
and white adipose tissue (WAT). RNA isolation and target mRNA
expression level quantitation are performed as described by other
examples herein.
[0278] To assess the physiological effects resulting from antisense
inhibition of target apolipoprotein(a) mRNA, the ob/ob mice that
receive antisense oligonucleotide treatment are further evaluated
at the end of the treatment period for serum lipids, serum
apolipoproteins, serum free fatty acids, serum cholesterol (CHOL),
liver reverse primer: ACAGGGCTTTTCTCAGGTGGT (SEQ ID NO: 72) and the
additional PCR probe was: FAM-CCAAGCACAGAGGCTCCTTCTGAACAAG-TAMRA
(SEQ ID NO: 73). Gene target quantities were normalized using GAPDH
expression levels. For human GAPDH the PCR primers were:
forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 74) reverse primer:
GAAGATGGTGATGGGATTTC (SEQ ID NO: 75) and the PCR probe was: 5'
JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 76) where JOE is the
fluorescent reporter dye and TAMRA is the quencher dye.
[0279] Primary human hepatocytes were treated with 150 nM of the
compounds shown in Table 4. Untreated cells served as the control
to which all data were normalized. Following 24 hours of treatment,
apolipoprotein(a) expression levels were measured by real-time PCR
as described herein, using the primers and probe described by SEQ
ID NOs 71, 72 and 73. The data, shown in Table 4, represent the
average of three experiments and are normalized to untreated
control cells.
TABLE-US-00004 TABLE 4 Antisense inhibition of human
apolipoprotein(a) using chimeric phosphorothioate oligonucleotides
having 2'-MOE wings and a deoxy gap: primary human hepatocytes
TARGET ISIS SEQ TARGET % SEQ # REGION ID NO SITE INHIB ID NO 144367
Coding 4 174 77 11 144368 Coding 4 352 59 12 144369 Coding 4 522 69
13 144370 Coding 4 1743 75 14 144371 Coding 4 2768 57 15 144372
Coding 4 2910 54 16 144373 Coding 4 3371 49 17 144374 Coding 4 4972
80 18 144375 Coding 4 5080 11 19 144376 Coding 4 5315 82 20 144377
Coding 4 5825 72 21 144378 Coding 4 6447 72 22 144379 Coding 4 7155
46 23 144380 Coding 4 7185 78 24 144381 Coding 4 8463 64 25 144382
Coding 4 8915 58 26 144383 Coding 4 9066 79 27 144384 Coding 4
10787 0 28 144385 Coding 4 11238 94 29 144386 Coding 4 11261 61 30
144387 Coding 4 11461 60 31 144388 Coding 4 11823 57 32 144389
Coding 4 11894 39 33 144390 Coding 4 11957 0 34 144391 Coding 4
12255 57 35 144392 Coding 4 12461 50 36 144393 Coding 4 12699 82 37
144394 Coding 4 13354 76 38 144395 3'UTR 4 13711 84 39 144396 3'UTR
4 13731 72 40 144397 3'UTR 4 13780 64 41 144398 3'UTR 4 13801 33 42
144399 3'UTR 4 13841 44 43 144400 3'UTR 4 13861 75 44 144401 3'UTR
4 13881 72 45
Example 21
Effects of Antisense Oligonucleotides Targeted to Human
Apolipoprotein(a) on Human Plasminogen Expression
[0280] Human apolipoprotein(a) sequence shares a high degree of
homology with the human plasminogen sequence. Thus it was of
interest to determine if antisense oligonucleotides targeting
apolipoprotein(a) would exhibit an inhibitory effect on human
plasminogen.
[0281] In a further embodiment, compounds designed to target human
apolipoprotein(a), shown in Table 1, were tested for their effects
on human plasminogen mRNA expression. Pre-plated primary human
hepatocytes were purchased from InVitro Technologies (Baltimore,
Md.). Cells were cultured in high-glucose DMEM (Invitrogen Life
Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine
serum, 100 units per mL penicillin, and 100 .mu.g/mL streptomycin
(all supplements from Invitrogen Life Technologies, Carlsbad,
Calif.). Immediately upon receipt from the vendor, cells were
transfected with a dose of 150 nM of antisense oligonucleotide as
described in other examples herein.
[0282] Following 24 hours of exposure to antisense
oligonucleotides, human plasminogen mRNA levels were measured by
quantitative real-time PCR as described in other examples herein.
Probes and primers to human plasminogen were designed to hybridize
to a human plasminogen sequence, using published sequence
information (GENBANK.RTM. accession number NM 000301.1,
incorporated herein as SEQ ID NO: 77). For human plasminogen, the
PCR primers were:
forward primer: CGCTGGGAACTTTGTGACATC (SEQ ID NO: 78) reverse
primer: CCCGCTGCACAACACCTCCACC (SEQ ID NO: 79) and the PCR probe
was: 5' JOE-CACTGGTAGGTGGGACCAGAA-TAMRA 3' (SEQ ID NO: 80) where
JOE is the fluorescent reporter dye and TAMRA is the quencher dye.
Gene target quantities were normalized using GAPDH expression
levels.
[0283] Data, shown in Table 5, are averages from three experiments
in which primary human hepatocytes were treated with antisense
oligonucleotides targeted to human apolipoprotein(a).
TABLE-US-00005 TABLE 5 Effects of chimeric phosphorothioate
oligonucleotides targeted to human apolipoprotein (a) on human
plamsinogen expression ISIS # % INHIB SEQ ID NO 144367 62 11 144368
49 12 144369 8 13 144370 44 14 144371 0 15 144372 11 16 144373 33
17 144374 60 18 144375 9 19 144376 32 20 144377 43 21 144378 8 22
144379 0 23 144380 31 24 144381 13 25 144382 45 26 144383 47 27
144384 0 28 144385 0 29 144386 0 30 144387 0 31 144388 36 32 144389
0 33 144390 0 34 144391 0 35 144392 0 36 144393 58 37 144394 24 38
144395 35 39 144396 62 40 144397 25 41 144398 0 42 144399 0 43
144400 60 44 144401 0 45
[0284] These data illustrate that ISIS 144371, 144379, 144384,
144385, 144386, 144387, 144389, 144390, 144391, 144392, 144398,
144399 and 144401 do not inhibit plasminogen expression. Thus, in
this assay, these compounds selectively inhibit apolipoprotein(a)
expression. ISIS 144369, 144378 and 144375 demonstrated less than
10% inhibition of plasminogen. The target sites in human
apolipoprotein(a) to which ISIS 144379, ISIS 144368 and ISIS 144376
bind share 70%, 70% and 80% nucleotide identity with human
plasminogen, respectively.
Example 22
Antisense Inhibition of Human Apolipoprotein(a) In Vivo: Transgenic
Mouse Study
[0285] Apolipoprotein(a) is found in humans, nonhuman primates and
the European hedgehog, but not in common laboratory animals such as
rats and mice. Accordingly, mice harboring a human
apolipoprotein(a) transgene are required to investigate the effects
of antisense oligonucleotides on human apolipoprotein(a)
expression.
[0286] In a further embodiment, antisense oligonucleotides targeted
to human apolipoprotein(a) were tested for their effects in mice
transgenic for both human apolipoprotein(a) and human
apolipoprotein B, as well as in mice transgenic for human
apolipoprotein B alone. The transgenic mice were provided by Dr.
Robert Pitas and Dr. Matthias Schneider in the Gladstone Institute
at the University of California, San Francisco.
[0287] Mice were treated with 25 mg/kg of ISIS 144379 (SEQ ID NO:
23), twice weekly, for a period of 4 weeks. A control group
consisting of mice transgenic for both human genes was treated with
saline. Each treatment group consisted of 4 animals. At the end of
the 4 week treatment period, animals were sacrificed, and
apolipoprotein(a) mRNA levels in liver tissue were measured by
real-time PCR, as described herein. Apolipoprotein B mRNA was also
measured by real-time PCR with probes and primers designed using
published sequence information (GENBANK.RTM. accession number
NM.sub.--000384.1, incorporated herein as SEQ ID NO: 81). For human
apolipoprotein B the PCR primers were:
forward primer: TGCTAAAGGCACATATGGCCT (SEQ ID NO: 82) reverse
primer: CTCAGGTTGGACTCTCCATTGAG (SEQ ID NO: 83) and the PCR probe
was: FAM-CTTGTCAGAGGGATCCTAACACTGGCCG-TAMRA (SEQ ID NO: 84) where
FAM is the fluorescent reporter dye and TAMRA is the quencher dye.
Gene target quantities were normalized using mouse GAPDH expression
levels, as described herein.
[0288] The data, shown in Table 6, represent the average of all
animals in each treatment group and are normalized to
saline-treated control animals.
TABLE-US-00006 TABLE 6 Antisense inhibition of human
apolipoprotein(a) in transgenic mice mRNA expression % control
Transgene apoB apo(a) apolipoprotein B 101 0 apolipoprotein B 133
61 apolipoprotein(a)
[0289] These data illustrate that treatment of mice transgenic for
human apolipoprotein(a) and human apolipoprotein B with ISIS 144379
resulted in a decrease in apolipoprotein(a), but not apolipoprotein
B, mRNA expression.
Example 23
Antisense Oligonucleotides Targeted to Apolipoprotein(a) Having
2'-MOE Wings and Deoxy Gaps
[0290] In a further embodiment, and additional series of
oligonucleotides was designed to target the human apolipoprotein(a)
sequence, using public sequence information (GENBANK.RTM. accession
#NM.sub.--005577.1, incorporated herein as SEQ ID NO: 4). The
compounds are shown in Table 7. "Target site" indicates the first
(5'-most) nucleotide number on the particular target sequence to
which the compound binds. All compounds in Table 7 are chimeric
oligonucleotides ("gapmers") 20 nucleotides in length, composed of
a central "gap" region consisting of ten 2'-deoxynucleotides, which
is flanked on both sides (5' and 3' directions) by five-nucleotide
"wings". The wings are composed of 2'-O-methoxyethyl (2'-MOE)
nucleotides. The internucleoside (backbone) linkages are
phosphorothioate (P.dbd.S) throughout the oligonucleotide. All
cytidine residues are 5-methylcytidines.
TABLE-US-00007 TABLE 7 Antisense oligonucleotides targeted to
apolipo- protein(a) having 2'-MOE wings and a deoxy gap TARGET SEQ
SEQ ID TARGET ID ISIS # REGION NO SITE SEQUENCE NO 359474 5' UTR 4
11 cagtgtccagaaagtgtgtc 85 359475 Coding 4 12380
ggtttgctcagttggtgctg 86 359476 Coding 4 12409 ttaccatggtagcactgccg
87 359477 Coding 4 12419 actctggccattaccatggt 88 359478 Coding 4
12449 tgtgacagtggtggagaatg 89 359479 Coding 4 12669
tgacagtcggaggagcgacc 90 359480 Coding 4 12839 tgcccatttatttgtccctg
91 359481 Coding 4 12919 agttttcttggattcattgt 92 359482 Coding 4
12944 gagagggatatcacagtagt 93 359483 Coding 4 13359
cagtcctggcggtgaccatg 94 359484 Coding 4 13466 cttatagtgattgcacactt
95 359485 Coding 4 13493 tctggccaaatgctcagcac 96
Example 24
Antisense Inhibition of Apolipoprotein(a) in Human Primary
Hepatocytes: Dose Response
[0291] In a further embodiment, antisense oligonucleotides targeted
to human apolipoprotein(a) were selected for dose response studies.
Human primary hepatocytes were treated with 25, 50, 150 and 300 nM
of ISIS 144367, ISIS 144370, ISIS 144385, ISIS 144393 and ISIS
144395. ISIS 133529 was used as a control oligonucleotide.
Untreated cells served as the control to which data were
normalized. Following 24 hours of exposure to antisense
oligonucleotides, target mRNA expression levels were measured by
real-time PCR as described by other examples herein. The results,
shown in Table 8, are the average of 3 experiments and are
expressed as percent inhibition of apolipoprotein(a) expression
relative to untreated control cells. "N.D." indicates not
determined.
TABLE-US-00008 TABLE 8 Antisense inhibition of apolipoprotein(a) in
human primary hepatocytes: dose response % Inhibition relative to
untreated control cells Dose of oligonucleotide ISIS # 25 50 150
300 144367 57 76 88 87 144370 47 62 56 26 144385 33 36 59 39 144393
23 32 35 30 144395 34 35 35 35 113529 N.D. N.D. 8 21
[0292] These data demonstrate that ISIS 144367 inhibited
apolipoprotein(a) in a dose-dependent manner. The other
oligonucleotides tested were able to reduce apolipoprotein(a)
expression.
Example 25
Effects of Antisense Inhibition of Apolipoprotein(a) on Plasminogen
Expression: Dose Response in Primary Human Hepatocytes
[0293] In a further embodiment, antisense oligonucleotides targeted
to human apolipoprotein(a) were tested for their ability to inhibit
human plasminogen expression. Human primary hepatocytes were
treated with 25, 50, 150 and 300 nM of ISIS 144367, ISIS 144370,
ISIS 144385, ISIS 144393 and ISIS 144395. ISIS 113529 was used as a
control oligonucleotide. Untreated cells served as the control to
which data were normalized. Following 24 hours of exposure to
antisense oligonucleotides, target mRNA expression levels were
measured by real-time PCR as described by other examples herein.
The results, shown in Table 9, are the average of 3 experiments and
are expressed as percent inhibition of apolipoprotein(a) expression
relative to untreated control cells. "N.D." indicates not
determined.
TABLE-US-00009 TABLE 9 Effects of antisense inhibition, of
apolipoprotein(a) on plasminogen expression in human primary
hepatocytes: dose response % plasminogen expression relative to
untreated control cells Dose of oligonucleotide (nM) ISIS # 25 50
150 300 144367 0 0 0 0 144370 0 6 9 0 144385 10 5 12 0 144393 10 39
2 0 144395 0 0 0 0 113529 N.D. N.D. 76 89
[0294] These data demonstrate that ISIS 144367 and ISIS 144395 did
not inhibit the expression of plasminogen in this assay and are
therefore apolipoprotein(a)-specific antisense oligonucleotides.
ISIS 144370 and ISIS 144385 did not result in a considerable
reduction in plasminogen expression.
Example 26
Effects of Antisense Inhibition of Apolipoprotein(a) in
Cytokine-Induced Cells
[0295] Elevated plasma levels of Lp(a), caused by increased
expression of apolipoprotein(a), is an independent risk factor for
a variety of cardiovascular disorders, including atherosclerosis,
hypercholesterolemia, myocardial infarction and thrombosis (Seed et
al., N. Engl. J. Med., 1990, 322, 1494-1499; Sandkamp et al., Clin.
Chem., 1990, 36, 20-23; Nowak-Gottl et al., Pediatrics, 1997, 99,
E11). Furthermore, increases in plasma Lp(a) are associated with
elevations in several acute-phase proteins, which participate in
the acute-phase of the immune response and function to promote
inflammation, activate the complement cascade, and stimulate
chemotaxis of phagocytes. Thus, Lp(a) is proposed to be an
acute-phase reactant and, consequently, responsive to cytokines.
The apolipoprotein(a) promoter contains several functional
cis-acting elements that are responsive to interleukin-6 (Wade et
al., Proc. natl. Acad. Sci. USA, 1993, 90, 1369-1373), a major
mediator of the acute phase response, further suggesting a link
between Lp(a) and the acute phase response. An association between
cytokines and Lp(a) was observed in primary monkey hepatocytes,
where stimulation of the cells with interleukin-6 resulted in an
increase in Lp(a) protein, as well as in apolipoprotein(a) mRNA
(Ramharack et al., Arterioscler. Thromb. Vasc. Biol., 1998, 18,
984-990). To date, no direct association between cytokines and
apolipoprotein(a) expression has been demonstrated in humans. Thus,
it is of interest to determine whether the antisense inhibition of
apolipoprotein(a) is affected by cytokine induction.
[0296] In a further embodiment, the ability of ISIS 144367 (SEQ ID
NO: 11) to inhibit apolipoprotein(a) expression was investigated in
primary human hepatocytes which were induced with cytokines. For a
period of 24 hours, cells were induced using culture media
supplemented with a final concentration of 1 .mu.M dexamethasone,
400 U/ml interleukin-1B and 200 U/ml interleukin-6. At the end of
this induction period, cells were treated with oligonucleotide as
described herein, for a period of 48 hours. One group of cells was
cytokine-induced and treated with 12.5, 25, 50, 100 or 200 nM of
ISIS 144367; data from these cells was normalized to data from
cells receiving only cytokine treatment. A second group of cells
received no cytokine induction and were treated with 12.5, 25, 50,
100 and 200 nM of ISIS 144367; data from these cells was normalized
to cells that received neither cytokine nor oligonucleotide
treatment. After the 48 oligonucleotide treatment period, cells
were harvested and apolipoprotein(a) expression was measured by
real-time PCR as described herein. The data, presented in Table 10,
are the average of 3 experiments and are normalized to the
respective controls as described. Results are shown as percent
inhibition of apolipoprotein(a) expression.
TABLE-US-00010 TABLE 10 Antisense inhibition of apolipoprotein(a)
in cytokine-induced primary human hepatocytes % Inhibition relative
to control Dose of No Cytokine oligonucleotide (nM) induction
induction 12.5 37 42 25 37 37 50 42 62 100 75 87 200 65 89
[0297] These data demonstrate a dose-dependent reduction in
apolipoprotein(a) expression cytokine-induced cells following
treatment with ISIS 144367. In cells receiving no oligonucleotide
treatment, the expression of apolipoprotein(a) was similar in
cytokine-induced cells relative to cells that were not exposed to
cytokines. Furthermore, ISIS 144367 inhibited apolipoprotein(a)
expression to a greater extent in cytokine-induced cells relative
to cells not exposed to cytokines. Thus, ISIS 144367 is a more
effective inhibitor of apolipoprotein(a) expression in
cytokine-induced cells. These data demonstrate a link between
cytokine stimulation of primary human hepatocytes and the antisense
inhibition of apolipoprotein(a) expression.
[0298] The expression of plasminogen was also tested in
cytokine-induced cells that received ISIS 144367 treatment. Cells
were induced and treated as described for the apolipoprotein(a)
mRNA expression experiment. Plasminogen mRNA was measured by
real-time PCR as described herein. The data, averaged from 3
experiments and normalized to the appropriate controls,
demonstrated that in this assay, in unstimulated cells as well as
cytokine-induced cells, ISIS 144367 did not inhibit plasminogen.
Thus, the effects of ISIS 144367 are specific to apolipoprotein(a)
expression both in the presence and absence of cytokines.
Sequence CWU 1
1
100120DNAArtificial SequenceAntisense Oligonucleotide 1tccgtcatcg
ctcctcaggg 20220DNAArtificial SequenceAntisense Oligonucleotide
2gtgcgcgcga gcccgaaatc 20320DNAArtificial SequenceAntisense
Oligonucleotide 3atgcattctg cccccaagga 20413938DNAHomo
sapiensCDS(46)..(13692) 4ctgggattgg gacacacttt ctggacactg
ctggccagtc ccaaa atg gaa cat aag 57 Met Glu His Lys 1 gaa gtg gtt
ctt cta ctt ctt tta ttt ctg aaa tca gca gca cct gag 105Glu Val Val
Leu Leu Leu Leu Leu Phe Leu Lys Ser Ala Ala Pro Glu 5 10 15 20 caa
agc cat gtg gtc cag gat tgc tac cat ggt gat gga cag agt tat 153Gln
Ser His Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr 25 30
35 cga ggc acg tac tcc acc act gtc aca gga agg acc tgc caa gct tgg
201Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp
40 45 50 tca tct atg aca cca cat caa cat aat agg acc aca gaa aac
tac cca 249Ser Ser Met Thr Pro His Gln His Asn Arg Thr Thr Glu Asn
Tyr Pro 55 60 65 aat gct ggc ttg atc atg aac tac tgc agg aat cca
gat gct gtg gca 297Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro
Asp Ala Val Ala 70 75 80 gct cct tat tgt tat acg agg gat ccc ggt
gtc agg tgg gag tac tgc 345Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly
Val Arg Trp Glu Tyr Cys 85 90 95 100 aac ctg acg caa tgc tca gac
gca gaa ggg act gcc gtc gcg cct ccg 393Asn Leu Thr Gln Cys Ser Asp
Ala Glu Gly Thr Ala Val Ala Pro Pro 105 110 115 act gtt acc ccg gtt
cca agc cta gag gct cct tcc gaa caa gca ccg 441Thr Val Thr Pro Val
Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro 120 125 130 act gag caa
agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag 489Thr Glu Gln
Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln 135 140 145 agt
tat cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa 537Ser
Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln 150 155
160 gct tgg tca tct atg aca cca cac tcg cat agt cgg acc cca gaa tac
585Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr
165 170 175 180 tac cca aat gct ggc ttg atc atg aac tac tgc agg aat
cca gat gct 633Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn
Pro Asp Ala 185 190 195 gtg gca gct cct tat tgt tat acg agg gat ccc
ggt gtc agg tgg gag 681Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro
Gly Val Arg Trp Glu 200 205 210 tac tgc aac ctg acg caa tgc tca gac
gca gaa ggg act gcc gtc gcg 729Tyr Cys Asn Leu Thr Gln Cys Ser Asp
Ala Glu Gly Thr Ala Val Ala 215 220 225 cct ccg act gtt acc ccg gtt
cca agc cta gag gct cct tcc gaa caa 777Pro Pro Thr Val Thr Pro Val
Pro Ser Leu Glu Ala Pro Ser Glu Gln 230 235 240 gca ccg act gag caa
agg cct ggg gtg cag gag tgc tac cat ggt aat 825Ala Pro Thr Glu Gln
Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn 245 250 255 260 gga cag
agt tat cga ggc aca tac tcc acc act gtc aca gga aga acc 873Gly Gln
Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr 265 270 275
tgc caa gct tgg tca tct atg aca cca cac tcg cat agt cgg acc cca
921Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro
280 285 290 gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg
aat cca 969Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg
Asn Pro 295 300 305 gat gct gtg gca gct cct tat tgt tat acg agg gat
ccc ggt gtc agg 1017Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp
Pro Gly Val Arg 310 315 320 tgg gag tac tgc aac ctg acg caa tgc tca
gac gca gaa ggg act gcc 1065Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser
Asp Ala Glu Gly Thr Ala 325 330 335 340 gtc gcg cct ccg act gtt acc
ccg gtt cca agc cta gag gct cct tcc 1113Val Ala Pro Pro Thr Val Thr
Pro Val Pro Ser Leu Glu Ala Pro Ser 345 350 355 gaa caa gca ccg act
gag caa agg cct ggg gtg cag gag tgc tac cat 1161Glu Gln Ala Pro Thr
Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His 360 365 370 ggt aat gga
cag agt tat cga ggc aca tac tcc acc act gtc aca gga 1209Gly Asn Gly
Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly 375 380 385 aga
acc tgc caa gct tgg tca tct atg aca cca cac tcg cat agt cgg 1257Arg
Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg 390 395
400 acc cca gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg
1305Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg
405 410 415 420 aat cca gat gct gtg gca gct cct tat tgt tat acg agg
gat ccc ggt 1353Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg
Asp Pro Gly 425 430 435 gtc agg tgg gag tac tgc aac ctg acg caa tgc
tca gac gca gaa ggg 1401Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys
Ser Asp Ala Glu Gly 440 445 450 act gcc gtc gcg cct ccg act gtt acc
ccg gtt cca agc cta gag gct 1449Thr Ala Val Ala Pro Pro Thr Val Thr
Pro Val Pro Ser Leu Glu Ala 455 460 465 cct tcc gaa caa gca ccg act
gag caa agg cct ggg gtg cag gag tgc 1497Pro Ser Glu Gln Ala Pro Thr
Glu Gln Arg Pro Gly Val Gln Glu Cys 470 475 480 tac cat ggt aat gga
cag agt tat cga ggc aca tac tcc acc act gtc 1545Tyr His Gly Asn Gly
Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val 485 490 495 500 aca gga
aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 1593Thr Gly
Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 505 510 515
agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac tac
1641Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr
520 525 530 tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acg
agg gat 1689Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr
Arg Asp 535 540 545 ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa
tgc tca gac gca 1737Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln
Cys Ser Asp Ala 550 555 560 gaa ggg act gcc gtc gcg cct ccg act gtt
acc ccg gtt cca agc cta 1785Glu Gly Thr Ala Val Ala Pro Pro Thr Val
Thr Pro Val Pro Ser Leu 565 570 575 580 gag gct cct tcc gaa caa gca
ccg act gag caa agg cct ggg gtg cag 1833Glu Ala Pro Ser Glu Gln Ala
Pro Thr Glu Gln Arg Pro Gly Val Gln 585 590 595 gag tgc tac cat ggt
aat gga cag agt tat cga ggc aca tac tcc acc 1881Glu Cys Tyr His Gly
Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 600 605 610 act gtc aca
gga aga acc tgc caa gct tgg tca tct atg aca cca cac 1929Thr Val Thr
Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His 615 620 625 tcg
cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg 1977Ser
His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met 630 635
640 aac tac tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acg
2025Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr
645 650 655 660 agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg
caa tgc tca 2073Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr
Gln Cys Ser 665 670 675 gac gca gaa ggg act gcc gtc gcg cct ccg act
gtt acc ccg gtt cca 2121Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr
Val Thr Pro Val Pro 680 685 690 agc cta gag gct cct tcc gaa caa gca
ccg act gag caa agg cct ggg 2169Ser Leu Glu Ala Pro Ser Glu Gln Ala
Pro Thr Glu Gln Arg Pro Gly 695 700 705 gtg cag gag tgc tac cat ggt
aat gga cag agt tat cga ggc aca tac 2217Val Gln Glu Cys Tyr His Gly
Asn Gly Gln Ser Tyr Arg Gly Thr Tyr 710 715 720 tcc acc act gtc aca
gga aga acc tgc caa gct tgg tca tct atg aca 2265Ser Thr Thr Val Thr
Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr 725 730 735 740 cca cac
tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg 2313Pro His
Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu 745 750 755
atc atg aac tac tgc agg aat cca gat gct gtg gca gct cct tat tgt
2361Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys
760 765 770 tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg
acg caa 2409Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu
Thr Gln 775 780 785 tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg
act gtt acc ccg 2457Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro
Thr Val Thr Pro 790 795 800 gtt cca agc cta gag gct cct tcc gaa caa
gca ccg act gag caa agg 2505Val Pro Ser Leu Glu Ala Pro Ser Glu Gln
Ala Pro Thr Glu Gln Arg 805 810 815 820 cct ggg gtg cag gag tgc tac
cat ggt aat gga cag agt tat cga ggc 2553Pro Gly Val Gln Glu Cys Tyr
His Gly Asn Gly Gln Ser Tyr Arg Gly 825 830 835 aca tac tcc acc act
gtc aca gga aga acc tgc caa gct tgg tca tct 2601Thr Tyr Ser Thr Thr
Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser 840 845 850 atg aca cca
cac tcg cat agt cgg acc cca gaa tac tac cca aat gct 2649Met Thr Pro
His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala 855 860 865 ggc
ttg atc atg aac tac tgc agg aat cca gat gct gtg gca gct cct 2697Gly
Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro 870 875
880 tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg
2745Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu
885 890 895 900 acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct
ccg act gtt 2793Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro
Pro Thr Val 905 910 915 acc ccg gtt cca agc cta gag gct cct tcc gaa
caa gca ccg act gag 2841Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu
Gln Ala Pro Thr Glu 920 925 930 caa agg cct ggg gtg cag gag tgc tac
cat ggt aat gga cag agt tat 2889Gln Arg Pro Gly Val Gln Glu Cys Tyr
His Gly Asn Gly Gln Ser Tyr 935 940 945 cga ggc aca tac tcc acc act
gtc aca gga aga acc tgc caa gct tgg 2937Arg Gly Thr Tyr Ser Thr Thr
Val Thr Gly Arg Thr Cys Gln Ala Trp 950 955 960 tca tct atg aca cca
cac tcg cat agt cgg acc cca gaa tac tac cca 2985Ser Ser Met Thr Pro
His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro 965 970 975 980 aat gct
ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 3033Asn Ala
Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 985 990 995
gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac 3078Ala
Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr 1000 1005
1010 tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg
3123Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala
1015 1020 1025 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct
tcc gaa 3168Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser
Glu 1030 1035 1040 caa gca ccg act gag caa agg cct ggg gtg cag gag
tgc tac cat 3213Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys
Tyr His 1045 1050 1055 ggt aat gga cag agt tat cga ggc aca tac tcc
acc act gtc aca 3258Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr
Thr Val Thr 1060 1065 1070 gga aga acc tgc caa gct tgg tca tct atg
aca cca cac tcg cat 3303Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr
Pro His Ser His 1075 1080 1085 agt cgg acc cca gaa tac tac cca aat
gct ggc ttg atc atg aac 3348Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala
Gly Leu Ile Met Asn 1090 1095 1100 tac tgc agg aat cca gat gct gtg
gca gct cct tat tgt tat acg 3393Tyr Cys Arg Asn Pro Asp Ala Val Ala
Ala Pro Tyr Cys Tyr Thr 1105 1110 1115 agg gat ccc ggt gtc agg tgg
gag tac tgc aac ctg acg caa tgc 3438Arg Asp Pro Gly Val Arg Trp Glu
Tyr Cys Asn Leu Thr Gln Cys 1120 1125 1130 tca gac gca gaa ggg act
gcc gtc gcg cct ccg act gtt acc ccg 3483Ser Asp Ala Glu Gly Thr Ala
Val Ala Pro Pro Thr Val Thr Pro 1135 1140 1145 gtt cca agc cta gag
gct cct tcc gaa caa gca ccg act gag caa 3528Val Pro Ser Leu Glu Ala
Pro Ser Glu Gln Ala Pro Thr Glu Gln 1150 1155 1160 agg cct ggg gtg
cag gag tgc tac cat ggt aat gga cag agt tat 3573Arg Pro Gly Val Gln
Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 1165 1170 1175 cga ggc aca
tac tcc acc act gtc aca gga aga acc tgc caa gct 3618Arg Gly Thr Tyr
Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala 1180 1185 1190 tgg tca
tct atg aca cca cac tcg cat agt cgg acc cca gaa tac 3663Trp Ser Ser
Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr 1195 1200 1205 tac
cca aat gct ggc ttg atc atg aac tac tgc agg aat cca gat 3708Tyr Pro
Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp 1210 1215 1220
gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 3753Ala
Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 1225 1230
1235 tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act
3798Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr
1240 1245 1250 gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta
gag gct 3843Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu
Ala 1255 1260 1265 cct tcc gaa caa gca ccg act gag caa agg cct ggg
gtg cag gag 3888Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val
Gln Glu
1270 1275 1280 tgc tac cat ggt aat gga cag agt tat cga ggc aca tac
tcc acc 3933Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser
Thr 1285 1290 1295 act gtc aca gga aga acc tgc caa gct tgg tca tct
atg aca cca 3978Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met
Thr Pro 1300 1305 1310 cac tcg cat agt cgg acc cca gaa tac tac cca
aat gct ggc ttg 4023His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn
Ala Gly Leu 1315 1320 1325 atc atg aac tac tgc agg aat cca gat gct
gtg gca gct cct tat 4068Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val
Ala Ala Pro Tyr 1330 1335 1340 tgt tat acg agg gat ccc ggt gtc agg
tgg gag tac tgc aac ctg 4113Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp
Glu Tyr Cys Asn Leu 1345 1350 1355 acg caa tgc tca gac gca gaa ggg
act gcc gtc gcg cct ccg act 4158Thr Gln Cys Ser Asp Ala Glu Gly Thr
Ala Val Ala Pro Pro Thr 1360 1365 1370 gtt acc ccg gtt cca agc cta
gag gct cct tcc gaa caa gca ccg 4203Val Thr Pro Val Pro Ser Leu Glu
Ala Pro Ser Glu Gln Ala Pro 1375 1380 1385 act gag caa agg cct ggg
gtg cag gag tgc tac cat ggt aat gga 4248Thr Glu Gln Arg Pro Gly Val
Gln Glu Cys Tyr His Gly Asn Gly 1390 1395 1400 cag agt tat cga ggc
aca tac tcc acc act gtc aca gga aga acc 4293Gln Ser Tyr Arg Gly Thr
Tyr Ser Thr Thr Val Thr Gly Arg Thr 1405 1410 1415 tgc caa gct tgg
tca tct atg aca cca cac tcg cat agt cgg acc 4338Cys Gln Ala Trp Ser
Ser Met Thr Pro His Ser His Ser Arg Thr 1420 1425 1430 cca gaa tac
tac cca aat gct ggc ttg atc atg aac tac tgc agg 4383Pro Glu Tyr Tyr
Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg 1435 1440 1445 aat cca
gat gct gtg gca gct cct tat tgt tat acg agg gat ccc 4428Asn Pro Asp
Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro 1450 1455 1460 ggt
gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca 4473Gly Val
Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala 1465 1470 1475
gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc 4518Glu
Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser 1480 1485
1490 cta gag gct cct tcc gaa caa gca ccg act gag caa agg cct ggg
4563Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly
1495 1500 1505 gtg cag gag tgc tac cat ggt aat gga cag agt tat cga
ggc aca 4608Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly
Thr 1510 1515 1520 tac tcc acc act gtc aca gga aga acc tgc caa gct
tgg tca tct 4653Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp
Ser Ser 1525 1530 1535 atg aca cca cac tcg cat agt cgg acc cca gaa
tac tac cca aat 4698Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr
Tyr Pro Asn 1540 1545 1550 gct ggc ttg atc atg aac tac tgc agg aat
cca gat gct gtg gca 4743Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro
Asp Ala Val Ala 1555 1560 1565 gct cct tat tgt tat acg agg gat ccc
ggt gtc agg tgg gag tac 4788Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly
Val Arg Trp Glu Tyr 1570 1575 1580 tgc aac ctg acg caa tgc tca gac
gca gaa ggg act gcc gtc gcg 4833Cys Asn Leu Thr Gln Cys Ser Asp Ala
Glu Gly Thr Ala Val Ala 1585 1590 1595 cct ccg act gtt acc ccg gtt
cca agc cta gag gct cct tcc gaa 4878Pro Pro Thr Val Thr Pro Val Pro
Ser Leu Glu Ala Pro Ser Glu 1600 1605 1610 caa gca ccg act gag caa
agg cct ggg gtg cag gag tgc tac cat 4923Gln Ala Pro Thr Glu Gln Arg
Pro Gly Val Gln Glu Cys Tyr His 1615 1620 1625 ggt aat gga cag agt
tat cga ggc aca tac tcc acc act gtc aca 4968Gly Asn Gly Gln Ser Tyr
Arg Gly Thr Tyr Ser Thr Thr Val Thr 1630 1635 1640 gga aga acc tgc
caa gct tgg tca tct atg aca cca cac tcg cat 5013Gly Arg Thr Cys Gln
Ala Trp Ser Ser Met Thr Pro His Ser His 1645 1650 1655 agt cgg acc
cca gaa tac tac cca aat gct ggc ttg atc atg aac 5058Ser Arg Thr Pro
Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn 1660 1665 1670 tac tgc
agg aat cca gat gct gtg gca gct cct tat tgt tat acg 5103Tyr Cys Arg
Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr 1675 1680 1685 agg
gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc 5148Arg Asp
Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys 1690 1695 1700
tca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg 5193Ser
Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro 1705 1710
1715 gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gag caa
5238Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln
1720 1725 1730 agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag
agt tat 5283Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser
Tyr 1735 1740 1745 cga ggc aca tac tcc acc act gtc aca gga aga acc
tgc caa gct 5328Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys
Gln Ala 1750 1755 1760 tgg tca tct atg aca cca cac tcg cat agt cgg
acc cca gaa tac 5373Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr
Pro Glu Tyr 1765 1770 1775 tac cca aat gct ggc ttg atc atg aac tac
tgc agg aat cca gat 5418Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys
Arg Asn Pro Asp 1780 1785 1790 gct gtg gca gct cct tat tgt tat acg
agg gat ccc ggt gtc agg 5463Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg
Asp Pro Gly Val Arg 1795 1800 1805 tgg gag tac tgc aac ctg acg caa
tgc tca gac gca gaa ggg act 5508Trp Glu Tyr Cys Asn Leu Thr Gln Cys
Ser Asp Ala Glu Gly Thr 1810 1815 1820 gcc gtc gcg cct ccg act gtt
acc ccg gtt cca agc cta gag gct 5553Ala Val Ala Pro Pro Thr Val Thr
Pro Val Pro Ser Leu Glu Ala 1825 1830 1835 cct tcc gaa caa gca ccg
act gag caa agg cct ggg gtg cag gag 5598Pro Ser Glu Gln Ala Pro Thr
Glu Gln Arg Pro Gly Val Gln Glu 1840 1845 1850 tgc tac cat ggt aat
gga cag agt tat cga ggc aca tac tcc acc 5643Cys Tyr His Gly Asn Gly
Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 1855 1860 1865 act gtc aca gga
aga acc tgc caa gct tgg tca tct atg aca cca 5688Thr Val Thr Gly Arg
Thr Cys Gln Ala Trp Ser Ser Met Thr Pro 1870 1875 1880 cac tcg cat
agt cgg acc cca gaa tac tac cca aat gct ggc ttg 5733His Ser His Ser
Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu 1885 1890 1895 atc atg
aac tac tgc agg aat cca gat gct gtg gca gct cct tat 5778Ile Met Asn
Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr 1900 1905 1910 tgt
tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg 5823Cys Tyr
Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu 1915 1920 1925
acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg act 5868Thr
Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr 1930 1935
1940 gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg
5913Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro
1945 1950 1955 act gag caa agg cct ggg gtg cag gag tgc tac cat ggt
aat gga 5958Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn
Gly 1960 1965 1970 cag agt tat cga ggc aca tac tcc acc act gtc aca
gga aga acc 6003Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly
Arg Thr 1975 1980 1985 tgc caa gct tgg tca tct atg aca cca cac tcg
cat agt cgg acc 6048Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His
Ser Arg Thr 1990 1995 2000 cca gaa tac tac cca aat gct ggc ttg atc
atg aac tac tgc agg 6093Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met
Asn Tyr Cys Arg 2005 2010 2015 aat cca gat gct gtg gca gct cct tat
tgt tat acg agg gat ccc 6138Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys
Tyr Thr Arg Asp Pro 2020 2025 2030 ggt gtc agg tgg gag tac tgc aac
ctg acg caa tgc tca gac gca 6183Gly Val Arg Trp Glu Tyr Cys Asn Leu
Thr Gln Cys Ser Asp Ala 2035 2040 2045 gaa ggg act gcc gtc gcg cct
ccg act gtt acc ccg gtt cca agc 6228Glu Gly Thr Ala Val Ala Pro Pro
Thr Val Thr Pro Val Pro Ser 2050 2055 2060 cta gag gct cct tcc gaa
caa gca ccg act gag caa agg cct ggg 6273Leu Glu Ala Pro Ser Glu Gln
Ala Pro Thr Glu Gln Arg Pro Gly 2065 2070 2075 gtg cag gag tgc tac
cat ggt aat gga cag agt tat cga ggc aca 6318Val Gln Glu Cys Tyr His
Gly Asn Gly Gln Ser Tyr Arg Gly Thr 2080 2085 2090 tac tcc acc act
gtc aca gga aga acc tgc caa gct tgg tca tct 6363Tyr Ser Thr Thr Val
Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser 2095 2100 2105 atg aca cca
cac tcg cat agt cgg acc cca gaa tac tac cca aat 6408Met Thr Pro His
Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn 2110 2115 2120 gct ggc
ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 6453Ala Gly Leu
Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 2125 2130 2135 gct
cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac 6498Ala Pro
Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr 2140 2145 2150
tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 6543Cys
Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 2155 2160
2165 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa
6588Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu
2170 2175 2180 caa gca ccg act gag caa agg cct ggg gtg cag gag tgc
tac cat 6633Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr
His 2185 2190 2195 ggt aat gga cag agt tat cga ggc aca tac tcc acc
act gtc aca 6678Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr
Val Thr 2200 2205 2210 gga aga acc tgc caa gct tgg tca tct atg aca
cca cac tcg cat 6723Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro
His Ser His 2215 2220 2225 agt cgg acc cca gaa tac tac cca aat gct
ggc ttg atc atg aac 6768Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly
Leu Ile Met Asn 2230 2235 2240 tac tgc agg aat cca gat gct gtg gca
gct cct tat tgt tat acg 6813Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala
Pro Tyr Cys Tyr Thr 2245 2250 2255 agg gat ccc ggt gtc agg tgg gag
tac tgc aac ctg acg caa tgc 6858Arg Asp Pro Gly Val Arg Trp Glu Tyr
Cys Asn Leu Thr Gln Cys 2260 2265 2270 tca gac gca gaa ggg act gcc
gtc gcg cct ccg act gtt acc ccg 6903Ser Asp Ala Glu Gly Thr Ala Val
Ala Pro Pro Thr Val Thr Pro 2275 2280 2285 gtt cca agc cta gag gct
cct tcc gaa caa gca ccg act gag caa 6948Val Pro Ser Leu Glu Ala Pro
Ser Glu Gln Ala Pro Thr Glu Gln 2290 2295 2300 agg cct ggg gtg cag
gag tgc tac cat ggt aat gga cag agt tat 6993Arg Pro Gly Val Gln Glu
Cys Tyr His Gly Asn Gly Gln Ser Tyr 2305 2310 2315 cga ggc aca tac
tcc acc act gtc aca gga aga acc tgc caa gct 7038Arg Gly Thr Tyr Ser
Thr Thr Val Thr Gly Arg Thr Cys Gln Ala 2320 2325 2330 tgg tca tct
atg aca cca cac tcg cat agt cgg acc cca gaa tac 7083Trp Ser Ser Met
Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr 2335 2340 2345 tac cca
aat gct ggc ttg atc atg aac tac tgc agg aat cca gat 7128Tyr Pro Asn
Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp 2350 2355 2360 gct
gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 7173Ala Val
Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 2365 2370 2375
tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act 7218Trp
Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr 2380 2385
2390 gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct
7263Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala
2395 2400 2405 cct tcc gaa caa gca ccg act gag caa agg cct ggg gtg
cag gag 7308Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln
Glu 2410 2415 2420 tgc tac cat ggt aat gga cag agt tat cga ggc aca
tac tcc acc 7353Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr
Ser Thr 2425 2430 2435 act gtc aca gga aga acc tgc caa gct tgg tca
tct atg aca cca 7398Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser
Met Thr Pro 2440 2445 2450 cac tcg cat agt cgg acc cca gaa tac tac
cca aat gct ggc ttg 7443His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro
Asn Ala Gly Leu 2455 2460 2465 atc atg aac tac tgc agg aat cca gat
gct gtg gca gct cct tat 7488Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala
Val Ala Ala Pro Tyr 2470 2475 2480 tgt tat acg agg gat ccc ggt gtc
agg tgg gag tac tgc aac ctg 7533Cys Tyr Thr Arg Asp Pro Gly Val Arg
Trp Glu Tyr Cys Asn Leu 2485 2490 2495 acg caa tgc tca gac gca gaa
ggg act gcc gtc gcg cct ccg act 7578Thr Gln Cys Ser Asp Ala Glu Gly
Thr Ala Val Ala Pro Pro Thr 2500 2505 2510 gtt acc ccg gtt cca agc
cta gag gct cct tcc gaa caa gca ccg 7623Val Thr Pro Val Pro Ser Leu
Glu Ala Pro Ser Glu Gln Ala Pro 2515 2520 2525 act gag caa agg cct
ggg gtg cag gag tgc tac cat ggt aat gga
7668Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly
2530 2535 2540 cag agt tat cga ggc aca tac tcc acc act gtc aca gga
aga acc 7713Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg
Thr 2545 2550 2555 tgc caa gct tgg tca tct atg aca cca cac tcg cat
agt cgg acc 7758Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser
Arg Thr 2560 2565 2570 cca gaa tac tac cca aat gct ggc ttg atc atg
aac tac tgc agg 7803Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn
Tyr Cys Arg 2575 2580 2585 aat cca gat gct gtg gca gct cct tat tgt
tat acg agg gat ccc 7848Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr
Thr Arg Asp Pro 2590 2595 2600 ggt gtc agg tgg gag tac tgc aac ctg
acg caa tgc tca gac gca 7893Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr
Gln Cys Ser Asp Ala 2605 2610 2615 gaa ggg act gcc gtc gcg cct ccg
act gtt acc ccg gtt cca agc 7938Glu Gly Thr Ala Val Ala Pro Pro Thr
Val Thr Pro Val Pro Ser 2620 2625 2630 cta gag gct cct tcc gaa caa
gca ccg act gag cag agg cct ggg 7983Leu Glu Ala Pro Ser Glu Gln Ala
Pro Thr Glu Gln Arg Pro Gly 2635 2640 2645 gtg cag gag tgc tac cac
ggt aat gga cag agt tat cga ggc aca 8028Val Gln Glu Cys Tyr His Gly
Asn Gly Gln Ser Tyr Arg Gly Thr 2650 2655 2660 tac tcc acc act gtc
act gga aga acc tgc caa gct tgg tca tct 8073Tyr Ser Thr Thr Val Thr
Gly Arg Thr Cys Gln Ala Trp Ser Ser 2665 2670 2675 atg aca cca cac
tcg cat agt cgg acc cca gaa tac tac cca aat 8118Met Thr Pro His Ser
His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn 2680 2685 2690 gct ggc ttg
atc atg aac tac tgc agg aat cca gat gct gtg gca 8163Ala Gly Leu Ile
Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 2695 2700 2705 gct cct
tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac 8208Ala Pro Tyr
Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr 2710 2715 2720 tgc
aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 8253Cys Asn
Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 2725 2730 2735
cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa 8298Pro
Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu 2740 2745
2750 caa gca ccg act gag caa agg cct ggg gtg cag gag tgc tac cat
8343Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His
2755 2760 2765 ggt aat gga cag agt tat cga ggc aca tac tcc acc act
gtc aca 8388Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val
Thr 2770 2775 2780 gga aga acc tgc caa gct tgg tca tct atg aca cca
cac tcg cat 8433Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His
Ser His 2785 2790 2795 agt cgg acc cca gaa tac tac cca aat gct ggc
ttg atc atg aac 8478Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu
Ile Met Asn 2800 2805 2810 tac tgc agg aat cca gat gct gtg gca gct
cct tat tgt tat acg 8523Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro
Tyr Cys Tyr Thr 2815 2820 2825 agg gat ccc ggt gtc agg tgg gag tac
tgc aac ctg acg caa tgc 8568Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys
Asn Leu Thr Gln Cys 2830 2835 2840 tca gac gca gaa ggg act gcc gtc
gcg cct ccg act gtt acc ccg 8613Ser Asp Ala Glu Gly Thr Ala Val Ala
Pro Pro Thr Val Thr Pro 2845 2850 2855 gtt cca agc cta gag gct cct
tcc gaa caa gca ccg act gag caa 8658Val Pro Ser Leu Glu Ala Pro Ser
Glu Gln Ala Pro Thr Glu Gln 2860 2865 2870 agg cct ggg gtg cag gag
tgc tac cat ggt aat gga cag agt tat 8703Arg Pro Gly Val Gln Glu Cys
Tyr His Gly Asn Gly Gln Ser Tyr 2875 2880 2885 cga ggc aca tac tcc
acc act gtc aca gga aga acc tgc caa gct 8748Arg Gly Thr Tyr Ser Thr
Thr Val Thr Gly Arg Thr Cys Gln Ala 2890 2895 2900 tgg tca tct atg
aca cca cac tcg cat agt cgg acc cca gaa tac 8793Trp Ser Ser Met Thr
Pro His Ser His Ser Arg Thr Pro Glu Tyr 2905 2910 2915 tac cca aat
gct ggc ttg atc atg aac tac tgc agg aat cca gat 8838Tyr Pro Asn Ala
Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp 2920 2925 2930 gct gtg
gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 8883Ala Val Ala
Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 2935 2940 2945 tgg
gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act 8928Trp Glu
Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr 2950 2955 2960
gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct 8973Ala
Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala 2965 2970
2975 cct tcc gaa caa gca ccg act gag cag agg cct ggg gtg cag gag
9018Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu
2980 2985 2990 tgc tac cac ggt aat gga cag agt tat cga ggc aca tac
tcc acc 9063Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser
Thr 2995 3000 3005 act gtc act gga aga acc tgc caa gct tgg tca tct
atg aca cca 9108Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met
Thr Pro 3010 3015 3020 cac tcg cat agt cgg acc cca gaa tac tac cca
aat gct ggc ttg 9153His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn
Ala Gly Leu 3025 3030 3035 atc atg aac tac tgc agg aat cca gat gct
gtg gca gct cct tat 9198Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val
Ala Ala Pro Tyr 3040 3045 3050 tgt tat acg agg gat ccc ggt gtc agg
tgg gag tac tgc aac ctg 9243Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp
Glu Tyr Cys Asn Leu 3055 3060 3065 acg caa tgc tca gac gca gaa ggg
act gcc gtc gcg cct ccg act 9288Thr Gln Cys Ser Asp Ala Glu Gly Thr
Ala Val Ala Pro Pro Thr 3070 3075 3080 gtt acc ccg gtt cca agc cta
gag gct cct tcc gaa caa gca ccg 9333Val Thr Pro Val Pro Ser Leu Glu
Ala Pro Ser Glu Gln Ala Pro 3085 3090 3095 act gag cag agg cct ggg
gtg cag gag tgc tac cac ggt aat gga 9378Thr Glu Gln Arg Pro Gly Val
Gln Glu Cys Tyr His Gly Asn Gly 3100 3105 3110 cag agt tat cga ggc
aca tac tcc acc act gtc act gga aga acc 9423Gln Ser Tyr Arg Gly Thr
Tyr Ser Thr Thr Val Thr Gly Arg Thr 3115 3120 3125 tgc caa gct tgg
tca tct atg aca cca cac tcg cat agt cgg acc 9468Cys Gln Ala Trp Ser
Ser Met Thr Pro His Ser His Ser Arg Thr 3130 3135 3140 cca gaa tac
tac cca aat gct ggc ttg atc atg aac tac tgc agg 9513Pro Glu Tyr Tyr
Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg 3145 3150 3155 aat cca
gat gct gtg gca gct cct tat tgt tat acg agg gat ccc 9558Asn Pro Asp
Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro 3160 3165 3170 ggt
gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca 9603Gly Val
Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala 3175 3180 3185
gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc 9648Glu
Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser 3190 3195
3200 cta gag gct cct tcc gaa caa gca ccg act gag cag agg cct ggg
9693Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly
3205 3210 3215 gtg cag gag tgc tac cac ggt aat gga cag agt tat cga
ggc aca 9738Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly
Thr 3220 3225 3230 tac tcc acc act gtc act gga aga acc tgc caa gct
tgg tca tct 9783Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp
Ser Ser 3235 3240 3245 atg aca cca cac tcg cat agt cgg acc cca gaa
tac tac cca aat 9828Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr
Tyr Pro Asn 3250 3255 3260 gct ggc ttg atc atg aac tac tgc agg aat
cca gat gct gtg gca 9873Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro
Asp Ala Val Ala 3265 3270 3275 gct cct tat tgt tat acg agg gat ccc
ggt gtc agg tgg gag tac 9918Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly
Val Arg Trp Glu Tyr 3280 3285 3290 tgc aac ctg acg caa tgc tca gac
gca gaa ggg act gcc gtc gcg 9963Cys Asn Leu Thr Gln Cys Ser Asp Ala
Glu Gly Thr Ala Val Ala 3295 3300 3305 cct ccg act gtt acc ccg gtt
cca agc cta gag gct cct tcc gaa 10008Pro Pro Thr Val Thr Pro Val
Pro Ser Leu Glu Ala Pro Ser Glu 3310 3315 3320 caa gca ccg act gag
cag agg cct ggg gtg cag gag tgc tac cac 10053Gln Ala Pro Thr Glu
Gln Arg Pro Gly Val Gln Glu Cys Tyr His 3325 3330 3335 ggt aat gga
cag agt tat cga ggc aca tac tcc acc act gtc act 10098Gly Asn Gly
Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr 3340 3345 3350 gga
aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 10143Gly
Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 3355 3360
3365 agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac
10188Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn
3370 3375 3380 tac tgc agg aat cca gat cct gtg gca gcc cct tat tgt
tat acg 10233Tyr Cys Arg Asn Pro Asp Pro Val Ala Ala Pro Tyr Cys
Tyr Thr 3385 3390 3395 agg gat ccc agt gtc agg tgg gag tac tgc aac
ctg aca caa tgc 10278Arg Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn
Leu Thr Gln Cys 3400 3405 3410 tca gac gca gaa ggg act gcc gtc gcg
cct cca act att acc ccg 10323Ser Asp Ala Glu Gly Thr Ala Val Ala
Pro Pro Thr Ile Thr Pro 3415 3420 3425 att cca agc cta gag gct cct
tct gaa caa gca cca act gag caa 10368Ile Pro Ser Leu Glu Ala Pro
Ser Glu Gln Ala Pro Thr Glu Gln 3430 3435 3440 agg cct ggg gtg cag
gag tgc tac cac gga aat gga cag agt tat 10413Arg Pro Gly Val Gln
Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 3445 3450 3455 caa ggc aca
tac ttc att act gtc aca gga aga acc tgc caa gct 10458Gln Gly Thr
Tyr Phe Ile Thr Val Thr Gly Arg Thr Cys Gln Ala 3460 3465 3470 tgg
tca tct atg aca cca cac tcg cat agt cgg acc cca gca tac 10503Trp
Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Ala Tyr 3475 3480
3485 tac cca aat gct ggc ttg atc aag aac tac tgc cga aat cca gat
10548Tyr Pro Asn Ala Gly Leu Ile Lys Asn Tyr Cys Arg Asn Pro Asp
3490 3495 3500 cct gtg gca gcc cct tgg tgt tat aca aca gat ccc agt
gtc agg 10593Pro Val Ala Ala Pro Trp Cys Tyr Thr Thr Asp Pro Ser
Val Arg 3505 3510 3515 tgg gag tac tgc aac ctg aca cga tgc tca gat
gca gaa tgg act 10638Trp Glu Tyr Cys Asn Leu Thr Arg Cys Ser Asp
Ala Glu Trp Thr 3520 3525 3530 gcc ttc gtc cct ccg aat gtt att ctg
gct cca agc cta gag gct 10683Ala Phe Val Pro Pro Asn Val Ile Leu
Ala Pro Ser Leu Glu Ala 3535 3540 3545 ttt ttt gaa caa gca ctg act
gag gaa acc ccc ggg gta cag gac 10728Phe Phe Glu Gln Ala Leu Thr
Glu Glu Thr Pro Gly Val Gln Asp 3550 3555 3560 tgc tac tac cat tat
gga cag agt tac cga ggc aca tac tcc acc 10773Cys Tyr Tyr His Tyr
Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 3565 3570 3575 act gtc aca
gga aga act tgc caa gct tgg tca tct atg aca cca 10818Thr Val Thr
Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro 3580 3585 3590 cac
cag cat agt cgg acc cca gaa aac tac cca aat gct ggc ctg 10863His
Gln His Ser Arg Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu 3595 3600
3605 acc agg aac tac tgc agg aat cca gat gct gag att cgc cct tgg
10908Thr Arg Asn Tyr Cys Arg Asn Pro Asp Ala Glu Ile Arg Pro Trp
3610 3615 3620 tgt tac acc atg gat ccc agt gtc agg tgg gag tac tgc
aac ctg 10953Cys Tyr Thr Met Asp Pro Ser Val Arg Trp Glu Tyr Cys
Asn Leu 3625 3630 3635 aca caa tgc ctg gtg aca gaa tca agt gtc ctt
gca act ctc acg 10998Thr Gln Cys Leu Val Thr Glu Ser Ser Val Leu
Ala Thr Leu Thr 3640 3645 3650 gtg gtc cca gat cca agc aca gag gct
tct tct gaa gaa gca cca 11043Val Val Pro Asp Pro Ser Thr Glu Ala
Ser Ser Glu Glu Ala Pro 3655 3660 3665 acg gag caa agc ccc ggg gtc
cag gat tgc tac cat ggt gat gga 11088Thr Glu Gln Ser Pro Gly Val
Gln Asp Cys Tyr His Gly Asp Gly 3670 3675 3680 cag agt tat cga ggc
tca ttc tct acc act gtc aca gga agg aca 11133Gln Ser Tyr Arg Gly
Ser Phe Ser Thr Thr Val Thr Gly Arg Thr 3685 3690 3695 tgt cag tct
tgg tcc tct atg aca cca cac tgg cat cag agg aca 11178Cys Gln Ser
Trp Ser Ser Met Thr Pro His Trp His Gln Arg Thr 3700 3705 3710 aca
gaa tat tat cca aat ggt ggc ctg acc agg aac tac tgc agg 11223Thr
Glu Tyr Tyr Pro Asn Gly Gly Leu Thr Arg Asn Tyr Cys Arg 3715 3720
3725 aat cca gat gct gag att agt cct tgg tgt tat acc atg gat ccc
11268Asn Pro Asp Ala Glu Ile Ser Pro Trp Cys Tyr Thr Met Asp Pro
3730 3735 3740 aat gtc aga tgg gag tac tgc aac ctg aca caa tgt cca
gtg aca 11313Asn Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Pro
Val Thr 3745 3750 3755 gaa tca agt gtc ctt gcg acg tcc acg gct gtt
tct gaa caa gca 11358Glu Ser Ser Val Leu Ala Thr Ser Thr Ala Val
Ser Glu Gln Ala 3760 3765 3770 cca acg gag caa agc ccc aca gtc cag
gac tgc tac cat ggt gat 11403Pro Thr Glu Gln Ser Pro Thr Val Gln
Asp Cys Tyr His Gly Asp 3775 3780 3785
gga cag agt tat cga ggc tca ttc tcc acc act gtt aca gga agg
11448Gly Gln Ser Tyr Arg Gly Ser Phe Ser Thr Thr Val Thr Gly Arg
3790 3795 3800 aca tgt cag tct tgg tcc tct atg aca cca cac tgg cat
cag aga 11493Thr Cys Gln Ser Trp Ser Ser Met Thr Pro His Trp His
Gln Arg 3805 3810 3815 acc aca gaa tac tac cca aat ggt ggc ctg acc
agg aac tac tgc 11538Thr Thr Glu Tyr Tyr Pro Asn Gly Gly Leu Thr
Arg Asn Tyr Cys 3820 3825 3830 agg aat cca gat gct gag att cgc cct
tgg tgt tat acc atg gat 11583Arg Asn Pro Asp Ala Glu Ile Arg Pro
Trp Cys Tyr Thr Met Asp 3835 3840 3845 ccc agt gtc aga tgg gag tac
tgc aac ctg acg caa tgt cca gtg 11628Pro Ser Val Arg Trp Glu Tyr
Cys Asn Leu Thr Gln Cys Pro Val 3850 3855 3860 atg gaa tca act ctc
ctc aca act ccc acg gtg gtc cca gtt cca 11673Met Glu Ser Thr Leu
Leu Thr Thr Pro Thr Val Val Pro Val Pro 3865 3870 3875 agc aca gag
ctt cct tct gaa gaa gca cca act gaa aac agc act 11718Ser Thr Glu
Leu Pro Ser Glu Glu Ala Pro Thr Glu Asn Ser Thr 3880 3885 3890 ggg
gtc cag gac tgc tac cga ggt gat gga cag agt tat cga ggc 11763Gly
Val Gln Asp Cys Tyr Arg Gly Asp Gly Gln Ser Tyr Arg Gly 3895 3900
3905 aca ctc tcc acc act atc aca gga aga aca tgt cag tct tgg tcg
11808Thr Leu Ser Thr Thr Ile Thr Gly Arg Thr Cys Gln Ser Trp Ser
3910 3915 3920 tct atg aca cca cat tgg cat cgg agg atc cca tta tac
tat cca 11853Ser Met Thr Pro His Trp His Arg Arg Ile Pro Leu Tyr
Tyr Pro 3925 3930 3935 aat gct ggc ctg acc agg aac tac tgc agg aat
cca gat gct gag 11898Asn Ala Gly Leu Thr Arg Asn Tyr Cys Arg Asn
Pro Asp Ala Glu 3940 3945 3950 att cgc cct tgg tgt tac acc atg gat
ccc agt gtc agg tgg gag 11943Ile Arg Pro Trp Cys Tyr Thr Met Asp
Pro Ser Val Arg Trp Glu 3955 3960 3965 tac tgc aac ctg aca cga tgt
cca gtg aca gaa tcg agt gtc ctc 11988Tyr Cys Asn Leu Thr Arg Cys
Pro Val Thr Glu Ser Ser Val Leu 3970 3975 3980 aca act ccc aca gtg
gcc ccg gtt cca agc aca gag gct cct tct 12033Thr Thr Pro Thr Val
Ala Pro Val Pro Ser Thr Glu Ala Pro Ser 3985 3990 3995 gaa caa gca
cca cct gag aaa agc cct gtg gtc cag gat tgc tac 12078Glu Gln Ala
Pro Pro Glu Lys Ser Pro Val Val Gln Asp Cys Tyr 4000 4005 4010 cat
ggt gat gga cgg agt tat cga ggc ata tcc tcc acc act gtc 12123His
Gly Asp Gly Arg Ser Tyr Arg Gly Ile Ser Ser Thr Thr Val 4015 4020
4025 aca gga agg acc tgt caa tct tgg tca tct atg ata cca cac tgg
12168Thr Gly Arg Thr Cys Gln Ser Trp Ser Ser Met Ile Pro His Trp
4030 4035 4040 cat cag agg acc cca gaa aac tac cca aat gct ggc ctg
acc gag 12213His Gln Arg Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu
Thr Glu 4045 4050 4055 aac tac tgc agg aat cca gat tct ggg aaa caa
ccc tgg tgt tac 12258Asn Tyr Cys Arg Asn Pro Asp Ser Gly Lys Gln
Pro Trp Cys Tyr 4060 4065 4070 aca acc gat ccg tgt gtg agg tgg gag
tac tgc aat ctg aca caa 12303Thr Thr Asp Pro Cys Val Arg Trp Glu
Tyr Cys Asn Leu Thr Gln 4075 4080 4085 tgc tca gaa aca gaa tca ggt
gtc cta gag act ccc act gtt gtt 12348Cys Ser Glu Thr Glu Ser Gly
Val Leu Glu Thr Pro Thr Val Val 4090 4095 4100 cca gtt cca agc atg
gag gct cat tct gaa gca gca cca act gag 12393Pro Val Pro Ser Met
Glu Ala His Ser Glu Ala Ala Pro Thr Glu 4105 4110 4115 caa acc cct
gtg gtc cgg cag tgc tac cat ggt aat ggc cag agt 12438Gln Thr Pro
Val Val Arg Gln Cys Tyr His Gly Asn Gly Gln Ser 4120 4125 4130 tat
cga ggc aca ttc tcc acc act gtc aca gga agg aca tgt caa 12483Tyr
Arg Gly Thr Phe Ser Thr Thr Val Thr Gly Arg Thr Cys Gln 4135 4140
4145 tct tgg tca tcc atg aca cca cac cgg cat cag agg acc cca gaa
12528Ser Trp Ser Ser Met Thr Pro His Arg His Gln Arg Thr Pro Glu
4150 4155 4160 aac tac cca aat gat ggc ctg aca atg aac tac tgc agg
aat cca 12573Asn Tyr Pro Asn Asp Gly Leu Thr Met Asn Tyr Cys Arg
Asn Pro 4165 4170 4175 gat gcc gat aca ggc cct tgg tgt ttt acc atg
gac ccc agc atc 12618Asp Ala Asp Thr Gly Pro Trp Cys Phe Thr Met
Asp Pro Ser Ile 4180 4185 4190 agg tgg gag tac tgc aac ctg acg cga
tgc tca gac aca gaa ggg 12663Arg Trp Glu Tyr Cys Asn Leu Thr Arg
Cys Ser Asp Thr Glu Gly 4195 4200 4205 act gtg gtc gct cct ccg act
gtc atc cag gtt cca agc cta ggg 12708Thr Val Val Ala Pro Pro Thr
Val Ile Gln Val Pro Ser Leu Gly 4210 4215 4220 cct cct tct gaa caa
gac tgt atg ttt ggg aat ggg aaa gga tac 12753Pro Pro Ser Glu Gln
Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr 4225 4230 4235 cgg ggc aag
aag gca acc act gtt act ggg acg cca tgc cag gaa 12798Arg Gly Lys
Lys Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Glu 4240 4245 4250 tgg
gct gcc cag gag ccc cat aga cac agc acg ttc att cca ggg 12843Trp
Ala Ala Gln Glu Pro His Arg His Ser Thr Phe Ile Pro Gly 4255 4260
4265 aca aat aaa tgg gca ggt ctg gaa aaa aat tac tgc cgt aac cct
12888Thr Asn Lys Trp Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro
4270 4275 4280 gat ggt gac atc aat ggt ccc tgg tgc tac aca atg aat
cca aga 12933Asp Gly Asp Ile Asn Gly Pro Trp Cys Tyr Thr Met Asn
Pro Arg 4285 4290 4295 aaa ctt ttt gac tac tgt gat atc cct ctc tgt
gca tcc tct tca 12978Lys Leu Phe Asp Tyr Cys Asp Ile Pro Leu Cys
Ala Ser Ser Ser 4300 4305 4310 ttt gat tgt ggg aag cct caa gtg gag
ccg aag aaa tgt cct gga 13023Phe Asp Cys Gly Lys Pro Gln Val Glu
Pro Lys Lys Cys Pro Gly 4315 4320 4325 agc att gta ggg ggg tgt gtg
gcc cac cca cat tcc tgg ccc tgg 13068Ser Ile Val Gly Gly Cys Val
Ala His Pro His Ser Trp Pro Trp 4330 4335 4340 caa gtc agt ctc aga
aca agg ttt gga aag cac ttc tgt gga ggc 13113Gln Val Ser Leu Arg
Thr Arg Phe Gly Lys His Phe Cys Gly Gly 4345 4350 4355 acc tta ata
tcc cca gag tgg gtg ctg act gct gct cac tgc ttg 13158Thr Leu Ile
Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu 4360 4365 4370 aag
aag tcc tca agg cct tca tcc tac aag gtc atc ctg ggt gca 13203Lys
Lys Ser Ser Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala 4375 4380
4385 cac caa gaa gtg aac ctc gaa tct cat gtt cag gaa ata gaa gtg
13248His Gln Glu Val Asn Leu Glu Ser His Val Gln Glu Ile Glu Val
4390 4395 4400 tct agg ctg ttc ttg gag ccc aca caa gca gat att gcc
ttg cta 13293Ser Arg Leu Phe Leu Glu Pro Thr Gln Ala Asp Ile Ala
Leu Leu 4405 4410 4415 aag cta agc agg cct gcc gtc atc act gac aaa
gta atg cca gct 13338Lys Leu Ser Arg Pro Ala Val Ile Thr Asp Lys
Val Met Pro Ala 4420 4425 4430 tgt ctg cca tcc cca gac tac atg gtc
acc gcc agg act gaa tgt 13383Cys Leu Pro Ser Pro Asp Tyr Met Val
Thr Ala Arg Thr Glu Cys 4435 4440 4445 tac atc act ggc tgg gga gaa
acc caa ggt acc ttt ggg act ggc 13428Tyr Ile Thr Gly Trp Gly Glu
Thr Gln Gly Thr Phe Gly Thr Gly 4450 4455 4460 ctt ctc aag gaa gcc
cag ctc ctt gtt att gag aat gaa gtg tgc 13473Leu Leu Lys Glu Ala
Gln Leu Leu Val Ile Glu Asn Glu Val Cys 4465 4470 4475 aat cac tat
aag tat att tgt gct gag cat ttg gcc aga ggc act 13518Asn His Tyr
Lys Tyr Ile Cys Ala Glu His Leu Ala Arg Gly Thr 4480 4485 4490 gac
agt tgc cag ggt gac agt gga ggg cct ctg gtt tgc ttc gag 13563Asp
Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu 4495 4500
4505 aag gac aaa tac att tta caa gga gtc act tct tgg ggt ctt ggc
13608Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly
4510 4515 4520 tgt gca cgc ccc aat aag cct ggt gtc tat gct cgt gtt
tca agg 13653Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Ala Arg Val
Ser Arg 4525 4530 4535 ttt gtt act tgg att gag gga atg atg aga aat
aat taa ttggacggga 13702Phe Val Thr Trp Ile Glu Gly Met Met Arg Asn
Asn 4540 4545 gacagagtga agcatcaacc tacttagaag ctgaaacgtg
ggtaaggatt tagcatgctg 13762gaaataatag acagcaatca aacgaagaca
ctgttcccag ctaccagcta tgccaaacct 13822tggcattttt ggtatttttg
tgtataagct tttaaggtct gactgacaaa ttctgtatta 13882aggtgtcata
gctatgacat ttgttaaaaa taaactctgc acttattttg atttga
13938525DNAArtificial SequencePCR primer 5cagctcctta ttgttatacg
aggga 25618DNAArtificial SequencePCR primer 6tgcgtctgag cattgcgt
18724DNAArtificial SequencePCR probe 7cccggtgtca ggtgggagta ctgc
24820DNAArtificial SequencePCR primer 8ggcaaattca acggcacagt
20920DNAArtificial SequencePCR primer 9gggtctcgct cctggaagat
201027DNAArtificial SequencePCR probe 10aaggccgaga atgggaagct
tgtcatc 271120DNAArtificial SequenceAntisense Oligonucleotide
11ggcaggtcct tcctgtgaca 201220DNAArtificial SequenceAntisense
Oligonucleotide 12tctgcgtctg agcattgcgt 201320DNAArtificial
SequenceAntisense Oligonucleotide 13aagcttggca ggttcttcct
201420DNAArtificial SequenceAntisense Oligonucleotide 14tcggaggcgc
gacggcagtc 201520DNAArtificial SequenceAntisense Oligonucleotide
15cggaggcgcg acggcagtcc 201620DNAArtificial SequenceAntisense
Oligonucleotide 16ggcaggttct tcctgtgaca 201720DNAArtificial
SequenceAntisense Oligonucleotide 17ataacaataa ggagctgcca
201820DNAArtificial SequenceAntisense Oligonucleotide 18gaccaagctt
ggcaggttct 201920DNAArtificial SequenceAntisense Oligonucleotide
19taacaataag gagctgccac 202020DNAArtificial SequenceAntisense
Oligonucleotide 20tgaccaagct tggcaggttc 202120DNAArtificial
SequenceAntisense Oligonucleotide 21ttctgcgtct gagcattgcg
202220DNAArtificial SequenceAntisense Oligonucleotide 22aacaataagg
agctgccaca 202320DNAArtificial SequenceAntisense Oligonucleotide
23acctgacacc gggatccctc 202420DNAArtificial SequenceAntisense
Oligonucleotide 24ctgagcattg cgtcaggttg 202520DNAArtificial
SequenceAntisense Oligonucleotide 25agtagttcat gatcaagcca
202620DNAArtificial SequenceAntisense Oligonucleotide 26gacggcagtc
ccttctgcgt 202720DNAArtificial SequenceAntisense Oligonucleotide
27ggcaggttct tccagtgaca 202820DNAArtificial SequenceAntisense
Oligonucleotide 28tgaccaagct tggcaagttc 202920DNAArtificial
SequenceAntisense Oligonucleotide 29tataacacca aggactaatc
203020DNAArtificial SequenceAntisense Oligonucleotide 30ccatctgaca
ttgggatcca 203120DNAArtificial SequenceAntisense Oligonucleotide
31tgtggtgtca tagaggacca 203220DNAArtificial SequenceAntisense
Oligonucleotide 32atgggatcct ccgatgccaa 203320DNAArtificial
SequenceAntisense Oligonucleotide 33acaccaaggg cgaatctcag
203420DNAArtificial SequenceAntisense Oligonucleotide 34ttctgtcact
ggacatcgtg 203520DNAArtificial SequenceAntisense Oligonucleotide
35cacacggatc ggttgtgtaa 203620DNAArtificial SequenceAntisense
Oligonucleotide 36acatgtcctt cctgtgacag 203720DNAArtificial
SequenceAntisense Oligonucleotide 37cagaaggagg ccctaggctt
203820DNAArtificial SequenceAntisense Oligonucleotide 38ctggcggtga
ccatgtagtc 203920DNAArtificial SequenceAntisense Oligonucleotide
39tctaagtagg ttgatgcttc 204020DNAArtificial SequenceAntisense
Oligonucleotide 40tccttaccca cgtttcagct 204120DNAArtificial
SequenceAntisense Oligonucleotide 41ggaacagtgt cttcgtttga
204220DNAArtificial SequenceAntisense Oligonucleotide 42gtttggcata
gctggtagct 204320DNAArtificial SequenceAntisense Oligonucleotide
43accttaaaag cttatacaca 204420DNAArtificial SequenceAntisense
Oligonucleotide 44atacagaatt tgtcagtcag 204520DNAArtificial
SequenceAntisense Oligonucleotide 45gtcatagcta tgacacctta
204620DNAHomo sapiens 46tgtcacagga aggacctgcc 204720DNAHomo sapiens
47acgcaatgct cagacgcaga 204820DNAHomo sapiens 48gactgccgtc
gcgcctccga 204920DNAHomo sapiens 49tgtcacagga agaacctgcc
205020DNAHomo sapiens 50tggcagctcc ttattgttat 205120DNAHomo sapiens
51agaacctgcc aagcttggtc 205220DNAHomo sapiens 52gtggcagctc
cttattgtta 205320DNAHomo sapiens 53cgcaatgctc agacgcagaa
205420DNAHomo sapiens 54gagggatccc ggtgtcaggt 205520DNAHomo sapiens
55tggcttgatc atgaactact 205620DNAHomo sapiens 56tggatcccaa
tgtcagatgg 205720DNAHomo sapiens 57tggtcctcta tgacaccaca
205820DNAHomo sapiens 58ttggcatcgg aggatcccat 205920DNAHomo sapiens
59ctgagattcg cccttggtgt 206020DNAHomo sapiens 60cacgatgtcc
agtgacagaa 206120DNAHomo sapiens 61ttacacaacc gatccgtgtg
206220DNAHomo sapiens 62ctgtcacagg aaggacatgt 206320DNAHomo sapiens
63gactacatgg tcaccgccag 206420DNAHomo sapiens 64gaagcatcaa
cctacttaga 206520DNAHomo sapiens 65agctgaaacg tgggtaagga
206620DNAHomo sapiens 66tcaaacgaag acactgttcc 206720DNAHomo sapiens
67agctaccagc tatgccaaac 206820DNAHomo sapiens 68tgtgtataag
cttttaaggt 206920DNAHomo sapiens 69taaggtgtca tagctatgac
207020DNAArtificial SequenceAntisense Oligonucleotide 70ctcttactgt
gctgtggaca 207116DNAArtificial SequencePCR primer 71ccacagtggc
cccggt 167221DNAArtificial SequencePCR primer 72acagggcttt
tctcaggtgg t 217328DNAArtificial SequencePCR probe 73ccaagcacag
aggctccttc tgaacaag 287419DNAArtificial SequencePCR primer
74gaaggtgaag gtcggagtc 197520DNAArtificial SequencePCR primer
75gaagatggtg atgggatttc 207620DNAArtificial SequencePCR probe
76caagcttccc gttctcagcc 20772732DNAH. sapiensCDS(55)..(2487)
77aacaacatcc tgggattggg acccactttc tgggcactgc tggccagtcc caaa atg
57 Met 1 gaa cat aag gaa gtg gtt ctt cta ctt ctt tta ttt ctg aaa
tca ggt 105Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys
Ser Gly 5 10 15 caa gga gag cct ctg gat gac tat gtg aat acc cag ggg
gct tca ctg 153Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly
Ala Ser Leu 20 25 30 ttc agt gtc act aag aag cag ctg gga gca gga
agt ata gaa gaa tgt 201Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly
Ser Ile Glu Glu Cys 35 40 45 gca gca aaa tgt gag gag gac gaa gaa
ttc acc tgc agg gca ttc caa 249Ala Ala Lys Cys Glu Glu Asp Glu Glu
Phe Thr Cys Arg Ala Phe Gln 50 55 60 65 tat cac agt aaa gag caa caa
tgt gtg ata atg gct gaa aac agg aag 297Tyr His Ser Lys Glu Gln Gln
Cys Val Ile Met Ala Glu Asn Arg Lys 70 75 80 tcc tcc ata atc att
agg atg aga gat gta gtt tta ttt gaa aag aaa 345Ser Ser Ile Ile Ile
Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys 85 90 95 gtg tat ctc
tca gag tgc aag act ggg aat gga aag aac tac aga ggg 393Val Tyr Leu
Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly 100 105 110 acg
atg tcc aaa aca aaa aat ggc atc acc tgt caa aaa tgg agt tcc 441Thr
Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser 115 120
125 act tct ccc cac aga cct aga ttc tca cct gct aca cac ccc tca gag
489Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu
130 135 140 145 gga ctg gag gag aac tac tgc agg aat cca gac aac gat
ccg cag ggg 537Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp
Pro Gln Gly 150 155 160 ccc tgg tgc tat act act gat cca gaa aag aga
tat gac tac tgc gac 585Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg
Tyr Asp Tyr Cys Asp 165 170 175 att ctt gag tgt gaa gag gaa tgt atg
cat tgc agt gga gaa aac tat 633Ile Leu Glu Cys Glu Glu Glu Cys Met
His Cys Ser Gly Glu Asn Tyr 180 185 190 gac ggc aaa att tcc aag acc
atg tct gga ctg gaa tgc cag gcc tgg 681Asp Gly Lys Ile Ser Lys Thr
Met Ser Gly Leu Glu Cys Gln Ala Trp 195 200 205 gac tct cag agc cca
cac gct cat gga tac att cct tcc aaa ttt cca 729Asp Ser Gln Ser Pro
His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro 210 215 220 225 aac aag
aac ctg aag aag aat tac tgt cgt aac ccc gat agg gag ctg 777Asn Lys
Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu 230 235 240
cgg cct tgg tgt ttc acc acc gac ccc aac aag cgc tgg gaa ctt tgc
825Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys
245 250 255 gac atc ccc cgc tgc aca aca cct cca cca tct tct ggt ccc
acc tac 873Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro
Thr Tyr 260 265 270 cag tgt ctg aag gga aca ggt gaa aac tat cgc ggg
aat gtg gct gtt 921Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly
Asn Val Ala Val 275 280 285 acc gtt tcc ggg cac acc tgt cag cac tgg
agt gca cag acc cct cac 969Thr Val Ser Gly His Thr Cys Gln His Trp
Ser Ala Gln Thr Pro His 290 295 300 305 aca cat aac agg aca cca gaa
aac ttc ccc tgc aaa aat ttg gat gaa 1017Thr His Asn Arg Thr Pro Glu
Asn Phe Pro Cys Lys Asn Leu Asp Glu 310 315 320 aac tac tgc cgc aat
cct gac gga aaa agg gcc cca tgg tgc cat aca 1065Asn Tyr Cys Arg Asn
Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr 325 330 335 acc aac agc
caa gtg cgg tgg gag tac tgt aag ata ccg tcc tgt gac 1113Thr Asn Ser
Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp 340 345 350 tcc
tcc cca gta tcc acg gaa caa ttg gct ccc aca gca cca cct gag 1161Ser
Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu 355 360
365 cta acc cct gtg gtc cag gac tgc tac cat ggt gat gga cag agc tac
1209Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr
370 375 380 385 cga ggc aca tcc tcc acc acc acc aca gga aag aag tgt
cag tct tgg 1257Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys
Gln Ser Trp 390 395 400 tca tct atg aca cca cac cgg cac cag aag acc
cca gaa aac tac cca 1305Ser Ser Met Thr Pro His Arg His Gln Lys Thr
Pro Glu Asn Tyr Pro 405 410 415 aat gct ggc ctg aca atg aac tac tgc
agg aat cca gat gcc gat aaa 1353Asn Ala Gly Leu Thr Met Asn Tyr Cys
Arg Asn Pro Asp Ala Asp Lys 420 425 430 ggc ccc tgg tgt ttt acc aca
gac ccc agc gtc agg tgg gag tac tgc 1401Gly Pro Trp Cys Phe Thr Thr
Asp Pro Ser Val Arg Trp Glu Tyr Cys 435 440 445 aac ctg aaa aaa tgc
tca gga aca gaa gcg agt gtt gta gca cct ccg 1449Asn Leu Lys Lys Cys
Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro 450 455 460 465 cct gtt
gtc ctg ctt cca gat gta gag act cct tcc gaa gaa gac tgt 1497Pro Val
Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys 470 475 480
atg ttt ggg aat ggg aaa gga tac cga ggc aag agg gcg acc act gtt
1545Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val
485 490 495 act ggg acg cca tgc cag gac tgg gct gcc cag gag ccc cat
aga cac 1593Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His
Arg His 500 505 510 agc att ttc act cca gag aca aat cca cgg gcg ggt
ctg gaa aaa aat 1641Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly
Leu Glu Lys Asn 515 520 525 tac tgc cgt aac cct gat ggt gat gta ggt
ggt ccc tgg tgc tac acg 1689Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly
Gly Pro Trp Cys Tyr Thr 530 535 540 545 aca aat cca aga aaa ctt tac
gac tac tgt gat gtc cct cag tgt gcg 1737Thr Asn Pro Arg Lys Leu Tyr
Asp Tyr Cys Asp Val Pro Gln Cys Ala 550 555 560 gcc cct tca ttt gat
tgt ggg aag cct caa gtg gag ccg aag aaa tgt 1785Ala Pro Ser Phe Asp
Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys 565 570 575 cct gga agg
gtt gtg ggg ggg tgt gtg gcc cac cca cat tcc tgg ccc 1833Pro Gly Arg
Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro 580 585 590 tgg
caa gtc agt ctt aga aca agg ttt gga atg cac ttc tgt gga ggc 1881Trp
Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly 595 600
605 acc ttg ata tcc cca gag tgg gtg ttg act gct gcc cac tgc ttg gag
1929Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
610 615 620 625 aag tcc cca agg cct tca tcc tac aag gtc atc ctg ggt
gca cac caa 1977Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly
Ala His Gln 630 635 640 gaa gtg aat ctc gaa ccg cat gtt cag gaa ata
gaa gtg tct agg ctg 2025Glu Val Asn Leu Glu Pro His Val Gln Glu Ile
Glu Val Ser Arg Leu 645 650 655 ttc ttg gag ccc aca cga aaa gat att
gcc ttg cta aag cta agc agt 2073Phe Leu Glu Pro Thr Arg Lys Asp Ile
Ala Leu Leu Lys Leu Ser Ser 660 665 670 cct gcc gtc atc act gac aaa
gta atc cca gct tgt ctg cca tcc cca 2121Pro Ala Val Ile Thr Asp Lys
Val Ile Pro Ala Cys Leu Pro Ser Pro 675 680 685 aat tat gtg gtc gct
gac cgg acc gaa tgt ttc atc act ggc tgg gga 2169Asn Tyr Val Val Ala
Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly 690 695 700 705 gaa acc
caa ggt act ttt gga gct ggc ctt ctc aag gaa gcc cag ctc 2217Glu Thr
Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu 710 715 720
cct gtg att gag aat aaa gtg tgc aat cgc tat gag ttt ctg aat gga
2265Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly
725 730 735 aga gtc caa tcc acc gaa ctc tgt gct ggg cat ttg gcc gga
ggc act 2313Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly
Gly Thr 740 745 750 gac agt tgc cag ggt gac agt gga ggt cct ctg gtt
tgc ttc gag aag 2361Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val
Cys Phe Glu Lys 755 760 765 gac aaa tac att tta caa gga gtc act tct
tgg ggt ctt ggc tgt gca 2409Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser
Trp Gly Leu Gly Cys Ala 770 775 780 785 cgc ccc aat aag cct ggt gtc
tat gtt cgt gtt tca agg ttt gtt act 2457Arg Pro Asn Lys Pro Gly Val
Tyr Val Arg Val Ser Arg Phe Val Thr 790 795 800 tgg att gag gga gtg
atg aga aat aat taa ttggacggga gacagagtga 2507Trp Ile Glu Gly Val
Met Arg Asn Asn 805 810 cgcactgact cacctagagg ctgggacgtg ggtagggatt
tagcatgctg gaaataactg 2567gcagtaatca aacgaagaca ctgtccccag
ctaccagcta cgccaaacct cggcattttt 2627tgtgttattt tctgactgct
ggattctgta gtaaggtgac atagctatga catttgttaa 2687aaataaactc
tgtacttaac tttgatttga gtaaattttg gtttt 27327821DNAArtificial
SequencePCR primer 78cgctgggaac tttgtgacat c 217922DNAArtificial
SequencePCR primer 79cccgctgcac aacacctcca cc 228021DNAArtificial
SequencePCR probe 80cactggtagg tgggaccaga a 218114121DNAH.
sapiensCDS(129)..(13820) 81attcccaccg ggacctgcgg ggctgagtgc
ccttctcggt tgctgccgct gaggagcccg 60cccagccagc cagggccgcg aggccgaggc
caggccgcag cccaggagcc gccccaccgc 120agctggcg atg gac ccg ccg agg
ccc gcg ctg ctg gcg ctg ctg gcg ctg 170 Met Asp Pro Pro Arg Pro Ala
Leu Leu Ala Leu Leu Ala Leu 1 5 10 cct gcg ctg ctg ctg ctg ctg ctg
gcg ggc gcc agg gcc gaa gag gaa 218Pro Ala Leu Leu Leu Leu Leu Leu
Ala Gly Ala Arg Ala Glu Glu Glu 15 20 25 30 atg ctg gaa aat gtc agc
ctg gtc tgt cca aaa gat gcg acc cga ttc 266Met Leu Glu Asn Val Ser
Leu Val Cys Pro Lys Asp Ala Thr Arg Phe 35 40 45 aag cac ctc cgg
aag tac aca tac aac tat gag gct gag agt tcc agt 314Lys His Leu Arg
Lys Tyr Thr Tyr Asn Tyr Glu Ala Glu Ser Ser Ser 50 55 60 gga gtc
cct ggg act gct gat tca aga agt gcc acc agg atc aac tgc 362Gly Val
Pro Gly Thr Ala Asp Ser Arg Ser Ala Thr Arg Ile Asn Cys 65 70 75
aag gtt gag ctg gag gtt ccc cag ctc tgc agc ttc atc ctg aag acc
410Lys Val Glu Leu Glu Val Pro Gln Leu Cys Ser Phe Ile Leu Lys Thr
80 85 90 agc cag tgc acc ctg aaa gag gtg tat ggc ttc aac cct gag
ggc aaa 458Ser Gln Cys Thr Leu Lys Glu Val Tyr Gly Phe Asn Pro Glu
Gly Lys 95 100 105 110 gcc ttg ctg aag aaa acc aag aac tct gag gag
ttt gct gca gcc atg 506Ala Leu Leu Lys Lys Thr Lys Asn Ser Glu Glu
Phe Ala Ala Ala Met 115 120 125 tcc agg tat gag ctc aag ctg gcc att
cca gaa ggg aag cag gtt ttc 554Ser Arg Tyr Glu Leu Lys Leu Ala Ile
Pro Glu Gly Lys Gln Val Phe 130 135 140 ctt tac ccg gag aaa gat gaa
cct act tac atc ctg aac atc aag agg 602Leu Tyr Pro Glu Lys Asp Glu
Pro Thr Tyr Ile Leu Asn Ile Lys Arg 145 150 155 ggc atc att tct gcc
ctc ctg gtt ccc cca gag aca gaa gaa gcc aag 650Gly Ile Ile Ser Ala
Leu Leu Val Pro Pro Glu Thr Glu Glu Ala Lys 160 165 170 caa gtg ttg
ttt ctg gat acc gtg tat gga aac tgc tcc act cac ttt 698Gln Val Leu
Phe Leu Asp Thr Val Tyr Gly Asn Cys Ser Thr His Phe 175 180 185 190
acc gtc aag acg agg aag ggc aat gtg gca aca gaa ata tcc act gaa
746Thr Val Lys Thr Arg Lys Gly Asn Val Ala Thr Glu Ile Ser Thr Glu
195 200 205 aga gac ctg ggg cag tgt gat cgc ttc aag ccc atc cgc aca
ggc atc 794Arg Asp Leu Gly Gln Cys Asp Arg Phe Lys Pro Ile Arg Thr
Gly Ile 210 215 220 agc cca ctt gct ctc atc aaa ggc atg acc cgc ccc
ttg tca act ctg 842Ser Pro Leu Ala Leu Ile Lys Gly Met Thr Arg Pro
Leu Ser Thr Leu 225 230 235 atc agc agc agc cag tcc tgt cag tac aca
ctg gac gct aag agg aag 890Ile Ser Ser Ser Gln Ser Cys Gln Tyr Thr
Leu Asp Ala Lys Arg Lys 240 245 250 cat gtg gca gaa gcc atc tgc aag
gag caa cac ctc ttc ctg cct ttc 938His Val Ala Glu Ala Ile Cys Lys
Glu Gln His Leu Phe Leu Pro Phe 255 260 265 270 tcc tac aac aat aag
tat ggg atg gta gca caa gtg aca cag act ttg 986Ser Tyr Asn Asn Lys
Tyr Gly Met Val Ala Gln Val Thr Gln Thr Leu 275 280 285 aaa ctt gaa
gac aca cca aag atc aac agc cgc ttc ttt ggt gaa ggt 1034Lys Leu Glu
Asp Thr Pro Lys Ile Asn Ser Arg Phe Phe Gly Glu Gly
290 295 300 act aag aag atg ggc ctc gca ttt gag agc acc aaa tcc aca
tca cct 1082Thr Lys Lys Met Gly Leu Ala Phe Glu Ser Thr Lys Ser Thr
Ser Pro 305 310 315 cca aag cag gcc gaa gct gtt ttg aag act ctc cag
gaa ctg aaa aaa 1130Pro Lys Gln Ala Glu Ala Val Leu Lys Thr Leu Gln
Glu Leu Lys Lys 320 325 330 cta acc atc tct gag caa aat atc cag aga
gct aat ctc ttc aat aag 1178Leu Thr Ile Ser Glu Gln Asn Ile Gln Arg
Ala Asn Leu Phe Asn Lys 335 340 345 350 ctg gtt act gag ctg aga ggc
ctc agt gat gaa gca gtc aca tct ctc 1226Leu Val Thr Glu Leu Arg Gly
Leu Ser Asp Glu Ala Val Thr Ser Leu 355 360 365 ttg cca cag ctg att
gag gtg tcc agc ccc atc act tta caa gcc ttg 1274Leu Pro Gln Leu Ile
Glu Val Ser Ser Pro Ile Thr Leu Gln Ala Leu 370 375 380 gtt cag tgt
gga cag cct cag tgc tcc act cac atc ctc cag tgg ctg 1322Val Gln Cys
Gly Gln Pro Gln Cys Ser Thr His Ile Leu Gln Trp Leu 385 390 395 aaa
cgt gtg cat gcc aac ccc ctt ctg ata gat gtg gtc acc tac ctg 1370Lys
Arg Val His Ala Asn Pro Leu Leu Ile Asp Val Val Thr Tyr Leu 400 405
410 gtg gcc ctg atc ccc gag ccc tca gca cag cag ctg cga gag atc ttc
1418Val Ala Leu Ile Pro Glu Pro Ser Ala Gln Gln Leu Arg Glu Ile Phe
415 420 425 430 aac atg gcg agg gat cag cgc agc cga gcc acc ttg tat
gcg ctg agc 1466Asn Met Ala Arg Asp Gln Arg Ser Arg Ala Thr Leu Tyr
Ala Leu Ser 435 440 445 cac gcg gtc aac aac tat cat aag aca aac cct
aca ggg acc cag gag 1514His Ala Val Asn Asn Tyr His Lys Thr Asn Pro
Thr Gly Thr Gln Glu 450 455 460 ctg ctg gac att gct aat tac ctg atg
gaa cag att caa gat gac tgc 1562Leu Leu Asp Ile Ala Asn Tyr Leu Met
Glu Gln Ile Gln Asp Asp Cys 465 470 475 act ggg gat gaa gat tac acc
tat ttg att ctg cgg gtc att gga aat 1610Thr Gly Asp Glu Asp Tyr Thr
Tyr Leu Ile Leu Arg Val Ile Gly Asn 480 485 490 atg ggc caa acc atg
gag cag tta act cca gaa ctc aag tct tca atc 1658Met Gly Gln Thr Met
Glu Gln Leu Thr Pro Glu Leu Lys Ser Ser Ile 495 500 505 510 ctc aaa
tgt gtc caa agt aca aag cca tca ctg atg atc cag aaa gct 1706Leu Lys
Cys Val Gln Ser Thr Lys Pro Ser Leu Met Ile Gln Lys Ala 515 520 525
gcc atc cag gct ctg cgg aaa atg gag cct aaa gac aag gac cag gag
1754Ala Ile Gln Ala Leu Arg Lys Met Glu Pro Lys Asp Lys Asp Gln Glu
530 535 540 gtt ctt ctt cag act ttc ctt gat gat gct tct ccg gga gat
aag cga 1802Val Leu Leu Gln Thr Phe Leu Asp Asp Ala Ser Pro Gly Asp
Lys Arg 545 550 555 ctg gct gcc tat ctt atg ttg atg agg agt cct tca
cag gca gat att 1850Leu Ala Ala Tyr Leu Met Leu Met Arg Ser Pro Ser
Gln Ala Asp Ile 560 565 570 aac aaa att gtc caa att cta cca tgg gaa
cag aat gag caa gtg aag 1898Asn Lys Ile Val Gln Ile Leu Pro Trp Glu
Gln Asn Glu Gln Val Lys 575 580 585 590 aac ttt gtg gct tcc cat att
gcc aat atc ttg aac tca gaa gaa ttg 1946Asn Phe Val Ala Ser His Ile
Ala Asn Ile Leu Asn Ser Glu Glu Leu 595 600 605 gat atc caa gat ctg
aaa aag tta gtg aaa gaa gct ctg aaa gaa tct 1994Asp Ile Gln Asp Leu
Lys Lys Leu Val Lys Glu Ala Leu Lys Glu Ser 610 615 620 caa ctt cca
act gtc atg gac ttc aga aaa ttc tct cgg aac tat caa 2042Gln Leu Pro
Thr Val Met Asp Phe Arg Lys Phe Ser Arg Asn Tyr Gln 625 630 635 ctc
tac aaa tct gtt tct ctt cca tca ctt gac cca gcc tca gcc aaa 2090Leu
Tyr Lys Ser Val Ser Leu Pro Ser Leu Asp Pro Ala Ser Ala Lys 640 645
650 ata gaa ggg aat ctt ata ttt gat cca aat aac tac ctt cct aaa gaa
2138Ile Glu Gly Asn Leu Ile Phe Asp Pro Asn Asn Tyr Leu Pro Lys Glu
655 660 665 670 agc atg ctg aaa act acc ctc act gcc ttt gga ttt gct
tca gct gac 2186Ser Met Leu Lys Thr Thr Leu Thr Ala Phe Gly Phe Ala
Ser Ala Asp 675 680 685 ctc atc gag att ggc ttg gaa gga aaa ggc ttt
gag cca aca ttg gaa 2234Leu Ile Glu Ile Gly Leu Glu Gly Lys Gly Phe
Glu Pro Thr Leu Glu 690 695 700 gct ctt ttt ggg aag caa gga ttt ttc
cca gac agt gtc aac aaa gct 2282Ala Leu Phe Gly Lys Gln Gly Phe Phe
Pro Asp Ser Val Asn Lys Ala 705 710 715 ttg tac tgg gtt aat ggt caa
gtt cct gat ggt gtc tct aag gtc tta 2330Leu Tyr Trp Val Asn Gly Gln
Val Pro Asp Gly Val Ser Lys Val Leu 720 725 730 gtg gac cac ttt ggc
tat acc aaa gat gat aaa cat gag cag gat atg 2378Val Asp His Phe Gly
Tyr Thr Lys Asp Asp Lys His Glu Gln Asp Met 735 740 745 750 gta aat
gga ata atg ctc agt gtt gag aag ctg att aaa gat ttg aaa 2426Val Asn
Gly Ile Met Leu Ser Val Glu Lys Leu Ile Lys Asp Leu Lys 755 760 765
tcc aaa gaa gtc ccg gaa gcc aga gcc tac ctc cgc atc ttg gga gag
2474Ser Lys Glu Val Pro Glu Ala Arg Ala Tyr Leu Arg Ile Leu Gly Glu
770 775 780 gag ctt ggt ttt gcc agt ctc cat gac ctc cag ctc ctg gga
aag ctg 2522Glu Leu Gly Phe Ala Ser Leu His Asp Leu Gln Leu Leu Gly
Lys Leu 785 790 795 ctt ctg atg ggt gcc cgc act ctg cag ggg atc ccc
cag atg att gga 2570Leu Leu Met Gly Ala Arg Thr Leu Gln Gly Ile Pro
Gln Met Ile Gly 800 805 810 gag gtc atc agg aag ggc tca aag aat gac
ttt ttt ctt cac tac atc 2618Glu Val Ile Arg Lys Gly Ser Lys Asn Asp
Phe Phe Leu His Tyr Ile 815 820 825 830 ttc atg gag aat gcc ttt gaa
ctc ccc act gga gct gga tta cag ttg 2666Phe Met Glu Asn Ala Phe Glu
Leu Pro Thr Gly Ala Gly Leu Gln Leu 835 840 845 caa ata tct tca tct
gga gtc att gct ccc gga gcc aag gct gga gta 2714Gln Ile Ser Ser Ser
Gly Val Ile Ala Pro Gly Ala Lys Ala Gly Val 850 855 860 aaa ctg gaa
gta gcc aac atg cag gct gaa ctg gtg gca aaa ccc tcc 2762Lys Leu Glu
Val Ala Asn Met Gln Ala Glu Leu Val Ala Lys Pro Ser 865 870 875 gtg
tct gtg gag ttt gtg aca aat atg ggc atc atc att ccg gac ttc 2810Val
Ser Val Glu Phe Val Thr Asn Met Gly Ile Ile Ile Pro Asp Phe 880 885
890 gct agg agt ggg gtc cag atg aac acc aac ttc ttc cac gag tcg ggt
2858Ala Arg Ser Gly Val Gln Met Asn Thr Asn Phe Phe His Glu Ser Gly
895 900 905 910 ctg gag gct cat gtt gcc cta aaa gct ggg aag ctg aag
ttt atc att 2906Leu Glu Ala His Val Ala Leu Lys Ala Gly Lys Leu Lys
Phe Ile Ile 915 920 925 cct tcc cca aag aga cca gtc aag ctg ctc agt
gga ggc aac aca tta 2954Pro Ser Pro Lys Arg Pro Val Lys Leu Leu Ser
Gly Gly Asn Thr Leu 930 935 940 cat ttg gtc tct acc acc aaa acg gag
gtg atc cca cct ctc att gag 3002His Leu Val Ser Thr Thr Lys Thr Glu
Val Ile Pro Pro Leu Ile Glu 945 950 955 aac agg cag tcc tgg tca gtt
tgc aag caa gtc ttt cct ggc ctg aat 3050Asn Arg Gln Ser Trp Ser Val
Cys Lys Gln Val Phe Pro Gly Leu Asn 960 965 970 tac tgc acc tca ggc
gct tac tcc aac gcc agc tcc aca gac tcc gcc 3098Tyr Cys Thr Ser Gly
Ala Tyr Ser Asn Ala Ser Ser Thr Asp Ser Ala 975 980 985 990 tcc tac
tat ccg ctg acc ggg gac acc aga tta gag ctg gaa ctg agg 3146Ser Tyr
Tyr Pro Leu Thr Gly Asp Thr Arg Leu Glu Leu Glu Leu Arg 995 1000
1005 cct aca gga gag att gag cag tat tct gtc agc gca acc tat gag
3191Pro Thr Gly Glu Ile Glu Gln Tyr Ser Val Ser Ala Thr Tyr Glu
1010 1015 1020 ctc cag aga gag gac aga gcc ttg gtg gat acc ctg aag
ttt gta 3236Leu Gln Arg Glu Asp Arg Ala Leu Val Asp Thr Leu Lys Phe
Val 1025 1030 1035 act caa gca gaa ggt gcg aag cag act gag gct acc
atg aca ttc 3281Thr Gln Ala Glu Gly Ala Lys Gln Thr Glu Ala Thr Met
Thr Phe 1040 1045 1050 aaa tat aat cgg cag agt atg acc ttg tcc agt
gaa gtc caa att 3326Lys Tyr Asn Arg Gln Ser Met Thr Leu Ser Ser Glu
Val Gln Ile 1055 1060 1065 ccg gat ttt gat gtt gac ctc gga aca atc
ctc aga gtt aat gat 3371Pro Asp Phe Asp Val Asp Leu Gly Thr Ile Leu
Arg Val Asn Asp 1070 1075 1080 gaa tct act gag ggc aaa acg tct tac
aga ctc acc ctg gac att 3416Glu Ser Thr Glu Gly Lys Thr Ser Tyr Arg
Leu Thr Leu Asp Ile 1085 1090 1095 cag aac aag aaa att act gag gtc
gcc ctc atg ggc cac cta agt 3461Gln Asn Lys Lys Ile Thr Glu Val Ala
Leu Met Gly His Leu Ser 1100 1105 1110 tgt gac aca aag gaa gaa aga
aaa atc aag ggt gtt att tcc ata 3506Cys Asp Thr Lys Glu Glu Arg Lys
Ile Lys Gly Val Ile Ser Ile 1115 1120 1125 ccc cgt ttg caa gca gaa
gcc aga agt gag atc ctc gcc cac tgg 3551Pro Arg Leu Gln Ala Glu Ala
Arg Ser Glu Ile Leu Ala His Trp 1130 1135 1140 tcg cct gcc aaa ctg
ctt ctc caa atg gac tca tct gct aca gct 3596Ser Pro Ala Lys Leu Leu
Leu Gln Met Asp Ser Ser Ala Thr Ala 1145 1150 1155 tat ggc tcc aca
gtt tcc aag agg gtg gca tgg cat tat gat gaa 3641Tyr Gly Ser Thr Val
Ser Lys Arg Val Ala Trp His Tyr Asp Glu 1160 1165 1170 gag aag att
gaa ttt gaa tgg aac aca ggc acc aat gta gat acc 3686Glu Lys Ile Glu
Phe Glu Trp Asn Thr Gly Thr Asn Val Asp Thr 1175 1180 1185 aaa aaa
atg act tcc aat ttc cct gtg gat ctc tcc gat tat cct 3731Lys Lys Met
Thr Ser Asn Phe Pro Val Asp Leu Ser Asp Tyr Pro 1190 1195 1200 aag
agc ttg cat atg tat gct aat aga ctc ctg gat cac aga gtc 3776Lys Ser
Leu His Met Tyr Ala Asn Arg Leu Leu Asp His Arg Val 1205 1210 1215
cct gaa aca gac atg act ttc cgg cac gtg ggt tcc aaa tta ata 3821Pro
Glu Thr Asp Met Thr Phe Arg His Val Gly Ser Lys Leu Ile 1220 1225
1230 gtt gca atg agc tca tgg ctt cag aag gca tct ggg agt ctt cct
3866Val Ala Met Ser Ser Trp Leu Gln Lys Ala Ser Gly Ser Leu Pro
1235 1240 1245 tat acc cag act ttg caa gac cac ctc aat agc ctg aag
gag ttc 3911Tyr Thr Gln Thr Leu Gln Asp His Leu Asn Ser Leu Lys Glu
Phe 1250 1255 1260 aac ctc cag aac atg gga ttg cca gac ttc cac atc
cca gaa aac 3956Asn Leu Gln Asn Met Gly Leu Pro Asp Phe His Ile Pro
Glu Asn 1265 1270 1275 ctc ttc tta aaa agc gat ggc cgg gtc aaa tat
acc ttg aac aag 4001Leu Phe Leu Lys Ser Asp Gly Arg Val Lys Tyr Thr
Leu Asn Lys 1280 1285 1290 aac agt ttg aaa att gag att cct ttg cct
ttt ggt ggc aaa tcc 4046Asn Ser Leu Lys Ile Glu Ile Pro Leu Pro Phe
Gly Gly Lys Ser 1295 1300 1305 tcc aga gat cta aag atg tta gag act
gtt agg aca cca gcc ctc 4091Ser Arg Asp Leu Lys Met Leu Glu Thr Val
Arg Thr Pro Ala Leu 1310 1315 1320 cac ttc aag tct gtg gga ttc cat
ctg cca tct cga gag ttc caa 4136His Phe Lys Ser Val Gly Phe His Leu
Pro Ser Arg Glu Phe Gln 1325 1330 1335 gtc cct act ttt acc att ccc
aag ttg tat caa ctg caa gtg cct 4181Val Pro Thr Phe Thr Ile Pro Lys
Leu Tyr Gln Leu Gln Val Pro 1340 1345 1350 ctc ctg ggt gtt cta gac
ctc tcc acg aat gtc tac agc aac ttg 4226Leu Leu Gly Val Leu Asp Leu
Ser Thr Asn Val Tyr Ser Asn Leu 1355 1360 1365 tac aac tgg tcc gcc
tcc tac agt ggt ggc aac acc agc aca gac 4271Tyr Asn Trp Ser Ala Ser
Tyr Ser Gly Gly Asn Thr Ser Thr Asp 1370 1375 1380 cat ttc agc ctt
cgg gct cgt tac cac atg aag gct gac tct gtg 4316His Phe Ser Leu Arg
Ala Arg Tyr His Met Lys Ala Asp Ser Val 1385 1390 1395 gtt gac ctg
ctt tcc tac aat gtg caa gga tct gga gaa aca aca 4361Val Asp Leu Leu
Ser Tyr Asn Val Gln Gly Ser Gly Glu Thr Thr 1400 1405 1410 tat gac
cac aag aat acg ttc aca cta tca tgt gat ggg tct cta 4406Tyr Asp His
Lys Asn Thr Phe Thr Leu Ser Cys Asp Gly Ser Leu 1415 1420 1425 cgc
cac aaa ttt cta gat tcg aat atc aaa ttc agt cat gta gaa 4451Arg His
Lys Phe Leu Asp Ser Asn Ile Lys Phe Ser His Val Glu 1430 1435 1440
aaa ctt gga aac aac cca gtc tca aaa ggt tta cta ata ttc gat 4496Lys
Leu Gly Asn Asn Pro Val Ser Lys Gly Leu Leu Ile Phe Asp 1445 1450
1455 gca tct agt tcc tgg gga cca cag atg tct gct tca gtt cat ttg
4541Ala Ser Ser Ser Trp Gly Pro Gln Met Ser Ala Ser Val His Leu
1460 1465 1470 gac tcc aaa aag aaa cag cat ttg ttt gtc aaa gaa gtc
aag att 4586Asp Ser Lys Lys Lys Gln His Leu Phe Val Lys Glu Val Lys
Ile 1475 1480 1485 gat ggg cag ttc aga gtc tct tcg ttc tat gct aaa
ggc aca tat 4631Asp Gly Gln Phe Arg Val Ser Ser Phe Tyr Ala Lys Gly
Thr Tyr 1490 1495 1500 ggc ctg tct tgt cag agg gat cct aac act ggc
cgg ctc aat gga 4676Gly Leu Ser Cys Gln Arg Asp Pro Asn Thr Gly Arg
Leu Asn Gly 1505 1510 1515 gag tcc aac ctg agg ttt aac tcc tcc tac
ctc caa ggc acc aac 4721Glu Ser Asn Leu Arg Phe Asn Ser Ser Tyr Leu
Gln Gly Thr Asn 1520 1525 1530 cag ata aca gga aga tat gaa gat gga
acc ctc tcc ctc acc tcc 4766Gln Ile Thr Gly Arg Tyr Glu Asp Gly Thr
Leu Ser Leu Thr Ser 1535 1540 1545 acc tct gat ctg caa agt ggc atc
att aaa aat act gct tcc cta 4811Thr Ser Asp Leu Gln Ser Gly Ile Ile
Lys Asn Thr Ala Ser Leu 1550 1555 1560 aag tat gag aac tac gag ctg
act tta aaa tct gac acc aat ggg 4856Lys Tyr Glu Asn Tyr Glu Leu Thr
Leu Lys Ser Asp Thr Asn Gly 1565 1570 1575 aag tat aag aac ttt gcc
act tct aac aag atg gat atg acc ttc 4901Lys Tyr Lys Asn Phe Ala Thr
Ser Asn Lys Met Asp Met Thr Phe 1580 1585 1590 tct aag caa aat gca
ctg ctg cgt tct gaa tat cag gct gat tac
4946Ser Lys Gln Asn Ala Leu Leu Arg Ser Glu Tyr Gln Ala Asp Tyr
1595 1600 1605 gag tca ttg agg ttc ttc agc ctg ctt tct gga tca cta
aat tcc 4991Glu Ser Leu Arg Phe Phe Ser Leu Leu Ser Gly Ser Leu Asn
Ser 1610 1615 1620 cat ggt ctt gag tta aat gct gac atc tta ggc act
gac aaa att 5036His Gly Leu Glu Leu Asn Ala Asp Ile Leu Gly Thr Asp
Lys Ile 1625 1630 1635 aat agt ggt gct cac aag gcg aca cta agg att
ggc caa gat gga 5081Asn Ser Gly Ala His Lys Ala Thr Leu Arg Ile Gly
Gln Asp Gly 1640 1645 1650 ata tct acc agt gca acg acc aac ttg aag
tgt agt ctc ctg gtg 5126Ile Ser Thr Ser Ala Thr Thr Asn Leu Lys Cys
Ser Leu Leu Val 1655 1660 1665 ctg gag aat gag ctg aat gca gag ctt
ggc ctc tct ggg gca tct 5171Leu Glu Asn Glu Leu Asn Ala Glu Leu Gly
Leu Ser Gly Ala Ser 1670 1675 1680 atg aaa tta aca aca aat ggc cgc
ttc agg gaa cac aat gca aaa 5216Met Lys Leu Thr Thr Asn Gly Arg Phe
Arg Glu His Asn Ala Lys 1685 1690 1695 ttc agt ctg gat ggg aaa gcc
gcc ctc aca gag cta tca ctg gga 5261Phe Ser Leu Asp Gly Lys Ala Ala
Leu Thr Glu Leu Ser Leu Gly 1700 1705 1710 agt gct tat cag gcc atg
att ctg ggt gtc gac agc aaa aac att 5306Ser Ala Tyr Gln Ala Met Ile
Leu Gly Val Asp Ser Lys Asn Ile 1715 1720 1725 ttc aac ttc aag gtc
agt caa gaa gga ctt aag ctc tca aat gac 5351Phe Asn Phe Lys Val Ser
Gln Glu Gly Leu Lys Leu Ser Asn Asp 1730 1735 1740 atg atg ggc tca
tat gct gaa atg aaa ttt gac cac aca aac agt 5396Met Met Gly Ser Tyr
Ala Glu Met Lys Phe Asp His Thr Asn Ser 1745 1750 1755 ctg aac att
gca ggc tta tca ctg gac ttc tct tca aaa ctt gac 5441Leu Asn Ile Ala
Gly Leu Ser Leu Asp Phe Ser Ser Lys Leu Asp 1760 1765 1770 aac att
tac agc tct gac aag ttt tat aag caa act gtt aat tta 5486Asn Ile Tyr
Ser Ser Asp Lys Phe Tyr Lys Gln Thr Val Asn Leu 1775 1780 1785 cag
cta cag ccc tat tct ctg gta act act tta aac agt gac ctg 5531Gln Leu
Gln Pro Tyr Ser Leu Val Thr Thr Leu Asn Ser Asp Leu 1790 1795 1800
aaa tac aat gct ctg gat ctc acc aac aat ggg aaa cta cgg cta 5576Lys
Tyr Asn Ala Leu Asp Leu Thr Asn Asn Gly Lys Leu Arg Leu 1805 1810
1815 gaa ccc ctg aag ctg cat gtg gct ggt aac cta aaa gga gcc tac
5621Glu Pro Leu Lys Leu His Val Ala Gly Asn Leu Lys Gly Ala Tyr
1820 1825 1830 caa aat aat gaa ata aaa cac atc tat gcc atc tct tct
gct gcc 5666Gln Asn Asn Glu Ile Lys His Ile Tyr Ala Ile Ser Ser Ala
Ala 1835 1840 1845 tta tca gca agc tat aaa gca gac act gtt gct aag
gtt cag ggt 5711Leu Ser Ala Ser Tyr Lys Ala Asp Thr Val Ala Lys Val
Gln Gly 1850 1855 1860 gtg gag ttt agc cat cgg ctc aac aca gac atc
gct ggg ctg gct 5756Val Glu Phe Ser His Arg Leu Asn Thr Asp Ile Ala
Gly Leu Ala 1865 1870 1875 tca gcc att gac atg agc aca aac tat aat
tca gac tca ctg cat 5801Ser Ala Ile Asp Met Ser Thr Asn Tyr Asn Ser
Asp Ser Leu His 1880 1885 1890 ttc agc aat gtc ttc cgt tct gta atg
gcc ccg ttt acc atg acc 5846Phe Ser Asn Val Phe Arg Ser Val Met Ala
Pro Phe Thr Met Thr 1895 1900 1905 atc gat gca cat aca aat ggc aat
ggg aaa ctc gct ctc tgg gga 5891Ile Asp Ala His Thr Asn Gly Asn Gly
Lys Leu Ala Leu Trp Gly 1910 1915 1920 gaa cat act ggg cag ctg tat
agc aaa ttc ctg ttg aaa gca gaa 5936Glu His Thr Gly Gln Leu Tyr Ser
Lys Phe Leu Leu Lys Ala Glu 1925 1930 1935 cct ctg gca ttt act ttc
tct cat gat tac aaa ggc tcc aca agt 5981Pro Leu Ala Phe Thr Phe Ser
His Asp Tyr Lys Gly Ser Thr Ser 1940 1945 1950 cat cat ctc gtg tct
agg aaa agc atc agt gca gct ctt gaa cac 6026His His Leu Val Ser Arg
Lys Ser Ile Ser Ala Ala Leu Glu His 1955 1960 1965 aaa gtc agt gcc
ctg ctt act cca gct gag cag aca ggc acc tgg 6071Lys Val Ser Ala Leu
Leu Thr Pro Ala Glu Gln Thr Gly Thr Trp 1970 1975 1980 aaa ctc aag
acc caa ttt aac aac aat gaa tac agc cag gac ttg 6116Lys Leu Lys Thr
Gln Phe Asn Asn Asn Glu Tyr Ser Gln Asp Leu 1985 1990 1995 gat gct
tac aac act aaa gat aaa att ggc gtg gag ctt act gga 6161Asp Ala Tyr
Asn Thr Lys Asp Lys Ile Gly Val Glu Leu Thr Gly 2000 2005 2010 cga
act ctg gct gac cta act cta cta gac tcc cca att aaa gtg 6206Arg Thr
Leu Ala Asp Leu Thr Leu Leu Asp Ser Pro Ile Lys Val 2015 2020 2025
cca ctt tta ctc agt gag ccc atc aat atc att gat gct tta gag 6251Pro
Leu Leu Leu Ser Glu Pro Ile Asn Ile Ile Asp Ala Leu Glu 2030 2035
2040 atg aga gat gcc gtt gag aag ccc caa gaa ttt aca att gtt gct
6296Met Arg Asp Ala Val Glu Lys Pro Gln Glu Phe Thr Ile Val Ala
2045 2050 2055 ttt gta aag tat gat aaa aac caa gat gtt cac tcc att
aac ctc 6341Phe Val Lys Tyr Asp Lys Asn Gln Asp Val His Ser Ile Asn
Leu 2060 2065 2070 cca ttt ttt gag acc ttg caa gaa tat ttt gag agg
aat cga caa 6386Pro Phe Phe Glu Thr Leu Gln Glu Tyr Phe Glu Arg Asn
Arg Gln 2075 2080 2085 acc att ata gtt gta gtg gaa aac gta cag aga
aac ctg aag cac 6431Thr Ile Ile Val Val Val Glu Asn Val Gln Arg Asn
Leu Lys His 2090 2095 2100 atc aat att gat caa ttt gta aga aaa tac
aga gca gcc ctg gga 6476Ile Asn Ile Asp Gln Phe Val Arg Lys Tyr Arg
Ala Ala Leu Gly 2105 2110 2115 aaa ctc cca cag caa gct aat gat tat
ctg aat tca ttc aat tgg 6521Lys Leu Pro Gln Gln Ala Asn Asp Tyr Leu
Asn Ser Phe Asn Trp 2120 2125 2130 gag aga caa gtt tca cat gcc aag
gag aaa ctg act gct ctc aca 6566Glu Arg Gln Val Ser His Ala Lys Glu
Lys Leu Thr Ala Leu Thr 2135 2140 2145 aaa aag tat aga att aca gaa
aat gat ata caa att gca tta gat 6611Lys Lys Tyr Arg Ile Thr Glu Asn
Asp Ile Gln Ile Ala Leu Asp 2150 2155 2160 gat gcc aaa atc aac ttt
aat gaa aaa cta tct caa ctg cag aca 6656Asp Ala Lys Ile Asn Phe Asn
Glu Lys Leu Ser Gln Leu Gln Thr 2165 2170 2175 tat atg ata caa ttt
gat cag tat att aaa gat agt tat gat tta 6701Tyr Met Ile Gln Phe Asp
Gln Tyr Ile Lys Asp Ser Tyr Asp Leu 2180 2185 2190 cat gat ttg aaa
ata gct att gct aat att att gat gaa atc att 6746His Asp Leu Lys Ile
Ala Ile Ala Asn Ile Ile Asp Glu Ile Ile 2195 2200 2205 gaa aaa tta
aaa agt ctt gat gag cac tat cat atc cgt gta aat 6791Glu Lys Leu Lys
Ser Leu Asp Glu His Tyr His Ile Arg Val Asn 2210 2215 2220 tta gta
aaa aca atc cat gat cta cat ttg ttt att gaa aat att 6836Leu Val Lys
Thr Ile His Asp Leu His Leu Phe Ile Glu Asn Ile 2225 2230 2235 gat
ttt aac aaa agt gga agt agt act gca tcc tgg att caa aat 6881Asp Phe
Asn Lys Ser Gly Ser Ser Thr Ala Ser Trp Ile Gln Asn 2240 2245 2250
gtg gat act aag tac caa atc aga atc cag ata caa gaa aaa ctg 6926Val
Asp Thr Lys Tyr Gln Ile Arg Ile Gln Ile Gln Glu Lys Leu 2255 2260
2265 cag cag ctt aag aga cac ata cag aat ata gac atc cag cac cta
6971Gln Gln Leu Lys Arg His Ile Gln Asn Ile Asp Ile Gln His Leu
2270 2275 2280 gct gga aag tta aaa caa cac att gag gct att gat gtt
aga gtg 7016Ala Gly Lys Leu Lys Gln His Ile Glu Ala Ile Asp Val Arg
Val 2285 2290 2295 ctt tta gat caa ttg gga act aca att tca ttt gaa
aga ata aat 7061Leu Leu Asp Gln Leu Gly Thr Thr Ile Ser Phe Glu Arg
Ile Asn 2300 2305 2310 gat gtt ctt gag cat gtc aaa cac ttt gtt ata
aat ctt att ggg 7106Asp Val Leu Glu His Val Lys His Phe Val Ile Asn
Leu Ile Gly 2315 2320 2325 gat ttt gaa gta gct gag aaa atc aat gcc
ttc aga gcc aaa gtc 7151Asp Phe Glu Val Ala Glu Lys Ile Asn Ala Phe
Arg Ala Lys Val 2330 2335 2340 cat gag tta atc gag agg tat gaa gta
gac caa caa atc cag gtt 7196His Glu Leu Ile Glu Arg Tyr Glu Val Asp
Gln Gln Ile Gln Val 2345 2350 2355 tta atg gat aaa tta gta gag ttg
acc cac caa tac aag ttg aag 7241Leu Met Asp Lys Leu Val Glu Leu Thr
His Gln Tyr Lys Leu Lys 2360 2365 2370 gag act att cag aag cta agc
aat gtc cta caa caa gtt aag ata 7286Glu Thr Ile Gln Lys Leu Ser Asn
Val Leu Gln Gln Val Lys Ile 2375 2380 2385 aaa gat tac ttt gag aaa
ttg gtt gga ttt att gat gat gct gtg 7331Lys Asp Tyr Phe Glu Lys Leu
Val Gly Phe Ile Asp Asp Ala Val 2390 2395 2400 aag aag ctt aat gaa
tta tct ttt aaa aca ttc att gaa gat gtt 7376Lys Lys Leu Asn Glu Leu
Ser Phe Lys Thr Phe Ile Glu Asp Val 2405 2410 2415 aac aaa ttc ctt
gac atg ttg ata aag aaa tta aag tca ttt gat 7421Asn Lys Phe Leu Asp
Met Leu Ile Lys Lys Leu Lys Ser Phe Asp 2420 2425 2430 tac cac cag
ttt gta gat gaa acc aat gac aaa atc cgt gag gtg 7466Tyr His Gln Phe
Val Asp Glu Thr Asn Asp Lys Ile Arg Glu Val 2435 2440 2445 act cag
aga ctc aat ggt gaa att cag gct ctg gaa cta cca caa 7511Thr Gln Arg
Leu Asn Gly Glu Ile Gln Ala Leu Glu Leu Pro Gln 2450 2455 2460 aaa
gct gaa gca tta aaa ctg ttt tta gag gaa acc aag gcc aca 7556Lys Ala
Glu Ala Leu Lys Leu Phe Leu Glu Glu Thr Lys Ala Thr 2465 2470 2475
gtt gca gtg tat ctg gaa agc cta cag gac acc aaa ata acc tta 7601Val
Ala Val Tyr Leu Glu Ser Leu Gln Asp Thr Lys Ile Thr Leu 2480 2485
2490 atc atc aat tgg tta cag gag gct tta agt tca gca tct ttg gct
7646Ile Ile Asn Trp Leu Gln Glu Ala Leu Ser Ser Ala Ser Leu Ala
2495 2500 2505 cac atg aag gcc aaa ttc cga gag act cta gaa gat aca
cga gac 7691His Met Lys Ala Lys Phe Arg Glu Thr Leu Glu Asp Thr Arg
Asp 2510 2515 2520 cga atg tat caa atg gac att cag cag gaa ctt caa
cga tac ctg 7736Arg Met Tyr Gln Met Asp Ile Gln Gln Glu Leu Gln Arg
Tyr Leu 2525 2530 2535 tct ctg gta ggc cag gtt tat agc aca ctt gtc
acc tac att tct 7781Ser Leu Val Gly Gln Val Tyr Ser Thr Leu Val Thr
Tyr Ile Ser 2540 2545 2550 gat tgg tgg act ctt gct gct aag aac ctt
act gac ttt gca gag 7826Asp Trp Trp Thr Leu Ala Ala Lys Asn Leu Thr
Asp Phe Ala Glu 2555 2560 2565 caa tat tct atc caa gat tgg gct aaa
cgt atg aaa gca ttg gta 7871Gln Tyr Ser Ile Gln Asp Trp Ala Lys Arg
Met Lys Ala Leu Val 2570 2575 2580 gag caa ggg ttc act gtt cct gaa
atc aag acc atc ctt ggg acc 7916Glu Gln Gly Phe Thr Val Pro Glu Ile
Lys Thr Ile Leu Gly Thr 2585 2590 2595 atg cct gcc ttt gaa gtc agt
ctt cag gct ctt cag aaa gct acc 7961Met Pro Ala Phe Glu Val Ser Leu
Gln Ala Leu Gln Lys Ala Thr 2600 2605 2610 ttc cag aca cct gat ttt
ata gtc ccc cta aca gat ttg agg att 8006Phe Gln Thr Pro Asp Phe Ile
Val Pro Leu Thr Asp Leu Arg Ile 2615 2620 2625 cca tca gtt cag ata
aac ttc aaa gac tta aaa aat ata aaa atc 8051Pro Ser Val Gln Ile Asn
Phe Lys Asp Leu Lys Asn Ile Lys Ile 2630 2635 2640 cca tcc agg ttt
tcc aca cca gaa ttt acc atc ctt aac acc ttc 8096Pro Ser Arg Phe Ser
Thr Pro Glu Phe Thr Ile Leu Asn Thr Phe 2645 2650 2655 cac att cct
tcc ttt aca att gac ttt gtc gaa atg aaa gta aag 8141His Ile Pro Ser
Phe Thr Ile Asp Phe Val Glu Met Lys Val Lys 2660 2665 2670 atc atc
aga acc att gac cag atg cag aac agt gag ctg cag tgg 8186Ile Ile Arg
Thr Ile Asp Gln Met Gln Asn Ser Glu Leu Gln Trp 2675 2680 2685 ccc
gtt cca gat ata tat ctc agg gat ctg aag gtg gag gac att 8231Pro Val
Pro Asp Ile Tyr Leu Arg Asp Leu Lys Val Glu Asp Ile 2690 2695 2700
cct cta gcg aga atc acc ctg cca gac ttc cgt tta cca gaa atc 8276Pro
Leu Ala Arg Ile Thr Leu Pro Asp Phe Arg Leu Pro Glu Ile 2705 2710
2715 gca att cca gaa ttc ata atc cca act ctc aac ctt aat gat ttt
8321Ala Ile Pro Glu Phe Ile Ile Pro Thr Leu Asn Leu Asn Asp Phe
2720 2725 2730 caa gtt cct gac ctt cac ata cca gaa ttc cag ctt ccc
cac atc 8366Gln Val Pro Asp Leu His Ile Pro Glu Phe Gln Leu Pro His
Ile 2735 2740 2745 tca cac aca att gaa gta cct act ttt ggc aag cta
tac agt att 8411Ser His Thr Ile Glu Val Pro Thr Phe Gly Lys Leu Tyr
Ser Ile 2750 2755 2760 ctg aaa atc caa tct cct ctt ttc aca tta gat
gca aat gct gac 8456Leu Lys Ile Gln Ser Pro Leu Phe Thr Leu Asp Ala
Asn Ala Asp 2765 2770 2775 ata ggg aat gga acc acc tca gca aac gaa
gca ggt atc gca gct 8501Ile Gly Asn Gly Thr Thr Ser Ala Asn Glu Ala
Gly Ile Ala Ala 2780 2785 2790 tcc atc act gcc aaa gga gag tcc aaa
tta gaa gtt ctc aat ttt 8546Ser Ile Thr Ala Lys Gly Glu Ser Lys Leu
Glu Val Leu Asn Phe 2795 2800 2805 gat ttt caa gca aat gca caa ctc
tca aac cct aag att aat ccg 8591Asp Phe Gln Ala Asn Ala Gln Leu Ser
Asn Pro Lys Ile Asn Pro 2810 2815 2820 ctg gct ctg aag gag tca gtg
aag ttc tcc agc aag tac ctg aga 8636Leu Ala Leu Lys Glu Ser Val Lys
Phe Ser Ser Lys Tyr Leu Arg 2825 2830 2835 acg gag cat ggg agt gaa
atg ctg ttt ttt gga aat gct att gag 8681Thr Glu His Gly Ser Glu Met
Leu Phe Phe Gly Asn Ala Ile Glu 2840 2845 2850
gga aaa tca aac aca gtg gca agt tta cac aca gaa aaa aat aca 8726Gly
Lys Ser Asn Thr Val Ala Ser Leu His Thr Glu Lys Asn Thr 2855 2860
2865 ctg gag ctt agt aat gga gtg att gtc aag ata aac aat cag ctt
8771Leu Glu Leu Ser Asn Gly Val Ile Val Lys Ile Asn Asn Gln Leu
2870 2875 2880 acc ctg gat agc aac act aaa tac ttc cac aaa ttg aac
atc ccc 8816Thr Leu Asp Ser Asn Thr Lys Tyr Phe His Lys Leu Asn Ile
Pro 2885 2890 2895 aaa ctg gac ttc tct agt cag gct gac ctg cgc aac
gag atc aag 8861Lys Leu Asp Phe Ser Ser Gln Ala Asp Leu Arg Asn Glu
Ile Lys 2900 2905 2910 aca ctg ttg aaa gct ggc cac ata gca tgg act
tct tct gga aaa 8906Thr Leu Leu Lys Ala Gly His Ile Ala Trp Thr Ser
Ser Gly Lys 2915 2920 2925 ggg tca tgg aaa tgg gcc tgc ccc aga ttc
tca gat gag gga aca 8951Gly Ser Trp Lys Trp Ala Cys Pro Arg Phe Ser
Asp Glu Gly Thr 2930 2935 2940 cat gaa tca caa att agt ttc acc ata
gaa gga ccc ctc act tcc 8996His Glu Ser Gln Ile Ser Phe Thr Ile Glu
Gly Pro Leu Thr Ser 2945 2950 2955 ttt gga ctg tcc aat aag atc aat
agc aaa cac cta aga gta aac 9041Phe Gly Leu Ser Asn Lys Ile Asn Ser
Lys His Leu Arg Val Asn 2960 2965 2970 caa aac ttg gtt tat gaa tct
ggc tcc ctc aac ttt tct aaa ctt 9086Gln Asn Leu Val Tyr Glu Ser Gly
Ser Leu Asn Phe Ser Lys Leu 2975 2980 2985 gaa att caa tca caa gtc
gat tcc cag cat gtg ggc cac agt gtt 9131Glu Ile Gln Ser Gln Val Asp
Ser Gln His Val Gly His Ser Val 2990 2995 3000 cta act gct aaa ggc
atg gca ctg ttt gga gaa ggg aag gca gag 9176Leu Thr Ala Lys Gly Met
Ala Leu Phe Gly Glu Gly Lys Ala Glu 3005 3010 3015 ttt act ggg agg
cat gat gct cat tta aat gga aag gtt att gga 9221Phe Thr Gly Arg His
Asp Ala His Leu Asn Gly Lys Val Ile Gly 3020 3025 3030 act ttg aaa
aat tct ctt ttc ttt tca gcc cag cca ttt gag atc 9266Thr Leu Lys Asn
Ser Leu Phe Phe Ser Ala Gln Pro Phe Glu Ile 3035 3040 3045 acg gca
tcc aca aac aat gaa ggg aat ttg aaa gtt cgt ttt cca 9311Thr Ala Ser
Thr Asn Asn Glu Gly Asn Leu Lys Val Arg Phe Pro 3050 3055 3060 tta
agg tta aca ggg aag ata gac ttc ctg aat aac tat gca ctg 9356Leu Arg
Leu Thr Gly Lys Ile Asp Phe Leu Asn Asn Tyr Ala Leu 3065 3070 3075
ttt ctg agt ccc agt gcc cag caa gca agt tgg caa gta agt gct 9401Phe
Leu Ser Pro Ser Ala Gln Gln Ala Ser Trp Gln Val Ser Ala 3080 3085
3090 agg ttc aat cag tat aag tac aac caa aat ttc tct gct gga aac
9446Arg Phe Asn Gln Tyr Lys Tyr Asn Gln Asn Phe Ser Ala Gly Asn
3095 3100 3105 aac gag aac att atg gag gcc cat gta gga ata aat gga
gaa gca 9491Asn Glu Asn Ile Met Glu Ala His Val Gly Ile Asn Gly Glu
Ala 3110 3115 3120 aat ctg gat ttc tta aac att cct tta aca att cct
gaa atg cgt 9536Asn Leu Asp Phe Leu Asn Ile Pro Leu Thr Ile Pro Glu
Met Arg 3125 3130 3135 cta cct tac aca ata atc aca act cct cca ctg
aaa gat ttc tct 9581Leu Pro Tyr Thr Ile Ile Thr Thr Pro Pro Leu Lys
Asp Phe Ser 3140 3145 3150 cta tgg gaa aaa aca ggc ttg aag gaa ttc
ttg aaa acg aca aag 9626Leu Trp Glu Lys Thr Gly Leu Lys Glu Phe Leu
Lys Thr Thr Lys 3155 3160 3165 caa tca ttt gat tta agt gta aaa gct
cag tat aag aaa aac aaa 9671Gln Ser Phe Asp Leu Ser Val Lys Ala Gln
Tyr Lys Lys Asn Lys 3170 3175 3180 cac agg cat tcc atc aca aat cct
ttg gct gtg ctt tgt gag ttt 9716His Arg His Ser Ile Thr Asn Pro Leu
Ala Val Leu Cys Glu Phe 3185 3190 3195 atc agt cag agc atc aaa tcc
ttt gac agg cat ttt gaa aaa aac 9761Ile Ser Gln Ser Ile Lys Ser Phe
Asp Arg His Phe Glu Lys Asn 3200 3205 3210 aga aac aat gca tta gat
ttt gtc acc aaa tcc tat aat gaa aca 9806Arg Asn Asn Ala Leu Asp Phe
Val Thr Lys Ser Tyr Asn Glu Thr 3215 3220 3225 aaa att aag ttt gat
aag tac aaa gct gaa aaa tct cac gac gag 9851Lys Ile Lys Phe Asp Lys
Tyr Lys Ala Glu Lys Ser His Asp Glu 3230 3235 3240 ctc ccc agg acc
ttt caa att cct gga tac act gtt cca gtt gtc 9896Leu Pro Arg Thr Phe
Gln Ile Pro Gly Tyr Thr Val Pro Val Val 3245 3250 3255 aat gtt gaa
gtg tct cca ttc acc ata gag atg tcg gca ttc ggc 9941Asn Val Glu Val
Ser Pro Phe Thr Ile Glu Met Ser Ala Phe Gly 3260 3265 3270 tat gtg
ttc cca aaa gca gtc agc atg cct agt ttc tcc atc cta 9986Tyr Val Phe
Pro Lys Ala Val Ser Met Pro Ser Phe Ser Ile Leu 3275 3280 3285 ggt
tct gac gtc cgt gtg cct tca tac aca tta atc ctg cca tca 10031Gly
Ser Asp Val Arg Val Pro Ser Tyr Thr Leu Ile Leu Pro Ser 3290 3295
3300 tta gag ctg cca gtc ctt cat gtc cct aga aat ctc aag ctt tct
10076Leu Glu Leu Pro Val Leu His Val Pro Arg Asn Leu Lys Leu Ser
3305 3310 3315 ctt cca cat ttc aag gaa ttg tgt acc ata agc cat att
ttt att 10121Leu Pro His Phe Lys Glu Leu Cys Thr Ile Ser His Ile
Phe Ile 3320 3325 3330 cct gcc atg ggc aat att acc tat gat ttc tcc
ttt aaa tca agt 10166Pro Ala Met Gly Asn Ile Thr Tyr Asp Phe Ser
Phe Lys Ser Ser 3335 3340 3345 gtc atc aca ctg aat acc aat gct gaa
ctt ttt aac cag tca gat 10211Val Ile Thr Leu Asn Thr Asn Ala Glu
Leu Phe Asn Gln Ser Asp 3350 3355 3360 att gtt gct cat ctc ctt tct
tca tct tca tct gtc att gat gca 10256Ile Val Ala His Leu Leu Ser
Ser Ser Ser Ser Val Ile Asp Ala 3365 3370 3375 ctg cag tac aaa tta
gag ggc acc aca aga ttg aca aga aaa agg 10301Leu Gln Tyr Lys Leu
Glu Gly Thr Thr Arg Leu Thr Arg Lys Arg 3380 3385 3390 gga ttg aag
tta gcc aca gct ctg tct ctg agc aac aaa ttt gtg 10346Gly Leu Lys
Leu Ala Thr Ala Leu Ser Leu Ser Asn Lys Phe Val 3395 3400 3405 gag
ggt agt cat aac agt act gtg agc tta acc acg aaa aat atg 10391Glu
Gly Ser His Asn Ser Thr Val Ser Leu Thr Thr Lys Asn Met 3410 3415
3420 gaa gtg tca gtg gca aaa acc aca aaa gcc gaa att cca att ttg
10436Glu Val Ser Val Ala Lys Thr Thr Lys Ala Glu Ile Pro Ile Leu
3425 3430 3435 aga atg aat ttc aag caa gaa ctt aat gga aat acc aag
tca aaa 10481Arg Met Asn Phe Lys Gln Glu Leu Asn Gly Asn Thr Lys
Ser Lys 3440 3445 3450 cct act gtc tct tcc tcc atg gaa ttt aag tat
gat ttc aat tct 10526Pro Thr Val Ser Ser Ser Met Glu Phe Lys Tyr
Asp Phe Asn Ser 3455 3460 3465 tca atg ctg tac tct acc gct aaa gga
gca gtt gac cac aag ctt 10571Ser Met Leu Tyr Ser Thr Ala Lys Gly
Ala Val Asp His Lys Leu 3470 3475 3480 agc ttg gaa agc ctc acc tct
tac ttt tcc att gag tca tct acc 10616Ser Leu Glu Ser Leu Thr Ser
Tyr Phe Ser Ile Glu Ser Ser Thr 3485 3490 3495 aaa gga gat gtc aag
ggt tcg gtt ctt tct cgg gaa tat tca gga 10661Lys Gly Asp Val Lys
Gly Ser Val Leu Ser Arg Glu Tyr Ser Gly 3500 3505 3510 act att gct
agt gag gcc aac act tac ttg aat tcc aag agc aca 10706Thr Ile Ala
Ser Glu Ala Asn Thr Tyr Leu Asn Ser Lys Ser Thr 3515 3520 3525 cgg
tct tca gtg aag ctg cag ggc act tcc aaa att gat gat atc 10751Arg
Ser Ser Val Lys Leu Gln Gly Thr Ser Lys Ile Asp Asp Ile 3530 3535
3540 tgg aac ctt gaa gta aaa gaa aat ttt gct gga gaa gcc aca ctc
10796Trp Asn Leu Glu Val Lys Glu Asn Phe Ala Gly Glu Ala Thr Leu
3545 3550 3555 caa cgc ata tat tcc ctc tgg gag cac agt acg aaa aac
cac tta 10841Gln Arg Ile Tyr Ser Leu Trp Glu His Ser Thr Lys Asn
His Leu 3560 3565 3570 cag cta gag ggc ctc ttt ttc acc aac gga gaa
cat aca agc aaa 10886Gln Leu Glu Gly Leu Phe Phe Thr Asn Gly Glu
His Thr Ser Lys 3575 3580 3585 gcc acc ctg gaa ctc tct cca tgg caa
atg tca gct ctt gtt cag 10931Ala Thr Leu Glu Leu Ser Pro Trp Gln
Met Ser Ala Leu Val Gln 3590 3595 3600 gtc cat gca agt cag ccc agt
tcc ttc cat gat ttc cct gac ctt 10976Val His Ala Ser Gln Pro Ser
Ser Phe His Asp Phe Pro Asp Leu 3605 3610 3615 ggc cag gaa gtg gcc
ctg aat gct aac act aag aac cag aag atc 11021Gly Gln Glu Val Ala
Leu Asn Ala Asn Thr Lys Asn Gln Lys Ile 3620 3625 3630 aga tgg aaa
aat gaa gtc cgg att cat tct ggg tct ttc cag agc 11066Arg Trp Lys
Asn Glu Val Arg Ile His Ser Gly Ser Phe Gln Ser 3635 3640 3645 cag
gtc gag ctt tcc aat gac caa gaa aag gca cac ctt gac att 11111Gln
Val Glu Leu Ser Asn Asp Gln Glu Lys Ala His Leu Asp Ile 3650 3655
3660 gca gga tcc tta gaa gga cac cta agg ttc ctc aaa aat atc atc
11156Ala Gly Ser Leu Glu Gly His Leu Arg Phe Leu Lys Asn Ile Ile
3665 3670 3675 cta cca gtc tat gac aag agc tta tgg gat ttc cta aag
ctg gat 11201Leu Pro Val Tyr Asp Lys Ser Leu Trp Asp Phe Leu Lys
Leu Asp 3680 3685 3690 gta acc acc agc att ggt agg aga cag cat ctt
cgt gtt tca act 11246Val Thr Thr Ser Ile Gly Arg Arg Gln His Leu
Arg Val Ser Thr 3695 3700 3705 gcc ttt gtg tac acc aaa aac ccc aat
ggc tat tca ttc tcc atc 11291Ala Phe Val Tyr Thr Lys Asn Pro Asn
Gly Tyr Ser Phe Ser Ile 3710 3715 3720 cct gta aaa gtt ttg gct gat
aaa ttc att act cct ggg ctg aaa 11336Pro Val Lys Val Leu Ala Asp
Lys Phe Ile Thr Pro Gly Leu Lys 3725 3730 3735 cta aat gat cta aat
tca gtt ctt gtc atg cct acg ttc cat gtc 11381Leu Asn Asp Leu Asn
Ser Val Leu Val Met Pro Thr Phe His Val 3740 3745 3750 cca ttt aca
gat ctt cag gtt cca tcg tgc aaa ctt gac ttc aga 11426Pro Phe Thr
Asp Leu Gln Val Pro Ser Cys Lys Leu Asp Phe Arg 3755 3760 3765 gaa
ata caa atc tat aag aag ctg aga act tca tca ttt gcc ctc 11471Glu
Ile Gln Ile Tyr Lys Lys Leu Arg Thr Ser Ser Phe Ala Leu 3770 3775
3780 aac cta cca aca ctc ccc gag gta aaa ttc cct gaa gtt gat gtg
11516Asn Leu Pro Thr Leu Pro Glu Val Lys Phe Pro Glu Val Asp Val
3785 3790 3795 tta aca aaa tat tct caa cca gaa gac tcc ttg att ccc
ttt ttt 11561Leu Thr Lys Tyr Ser Gln Pro Glu Asp Ser Leu Ile Pro
Phe Phe 3800 3805 3810 gag ata acc gtg cct gaa tct cag tta act gtg
tcc cag ttc acg 11606Glu Ile Thr Val Pro Glu Ser Gln Leu Thr Val
Ser Gln Phe Thr 3815 3820 3825 ctt cca aaa agt gtt tca gat ggc att
gct gct ttg gat cta aat 11651Leu Pro Lys Ser Val Ser Asp Gly Ile
Ala Ala Leu Asp Leu Asn 3830 3835 3840 gca gta gcc aac aag atc gca
gac ttt gag ttg ccc acc atc atc 11696Ala Val Ala Asn Lys Ile Ala
Asp Phe Glu Leu Pro Thr Ile Ile 3845 3850 3855 gtg cct gag cag acc
att gag att ccc tcc att aag ttc tct gta 11741Val Pro Glu Gln Thr
Ile Glu Ile Pro Ser Ile Lys Phe Ser Val 3860 3865 3870 cct gct gga
att gtc att cct tcc ttt caa gca ctg act gca cgc 11786Pro Ala Gly
Ile Val Ile Pro Ser Phe Gln Ala Leu Thr Ala Arg 3875 3880 3885 ttt
gag gta gac tct ccc gtg tat aat gcc act tgg agt gcc agt 11831Phe
Glu Val Asp Ser Pro Val Tyr Asn Ala Thr Trp Ser Ala Ser 3890 3895
3900 ttg aaa aac aaa gca gat tat gtt gaa aca gtc ctg gat tcc aca
11876Leu Lys Asn Lys Ala Asp Tyr Val Glu Thr Val Leu Asp Ser Thr
3905 3910 3915 tgc agc tca acc gta cag ttc cta gaa tat gaa cta aat
gtt ttg 11921Cys Ser Ser Thr Val Gln Phe Leu Glu Tyr Glu Leu Asn
Val Leu 3920 3925 3930 gga aca cac aaa atc gaa gat ggt acg tta gcc
tct aag act aaa 11966Gly Thr His Lys Ile Glu Asp Gly Thr Leu Ala
Ser Lys Thr Lys 3935 3940 3945 gga aca ctt gca cac cgt gac ttc agt
gca gaa tat gaa gaa gat 12011Gly Thr Leu Ala His Arg Asp Phe Ser
Ala Glu Tyr Glu Glu Asp 3950 3955 3960 ggc aaa ttt gaa gga ctt cag
gaa tgg gaa gga aaa gcg cac ctc 12056Gly Lys Phe Glu Gly Leu Gln
Glu Trp Glu Gly Lys Ala His Leu 3965 3970 3975 aat atc aaa agc cca
gcg ttc acc gat ctc cat ctg cgc tac cag 12101Asn Ile Lys Ser Pro
Ala Phe Thr Asp Leu His Leu Arg Tyr Gln 3980 3985 3990 aaa gac aag
aaa ggc atc tcc acc tca gca gcc tcc cca gcc gta 12146Lys Asp Lys
Lys Gly Ile Ser Thr Ser Ala Ala Ser Pro Ala Val 3995 4000 4005 ggc
acc gtg ggc atg gat atg gat gaa gat gac gac ttt tct aaa 12191Gly
Thr Val Gly Met Asp Met Asp Glu Asp Asp Asp Phe Ser Lys 4010 4015
4020 tgg aac ttc tac tac agc cct cag tcc tct cca gat aaa aaa ctc
12236Trp Asn Phe Tyr Tyr Ser Pro Gln Ser Ser Pro Asp Lys Lys Leu
4025 4030 4035 acc ata ttc aaa act gag ttg agg gtc cgg gaa tct gat
gag gaa 12281Thr Ile Phe Lys Thr Glu Leu Arg Val Arg Glu Ser Asp
Glu Glu 4040 4045 4050 act cag atc aaa gtt aat tgg gaa gaa gag gca
gct tct ggc ttg 12326Thr Gln Ile Lys Val Asn Trp Glu Glu Glu Ala
Ala Ser Gly Leu 4055 4060 4065 cta acc tct ctg aaa gac aac gtg ccc
aag gcc aca ggg gtc ctt 12371Leu Thr Ser Leu Lys Asp Asn Val Pro
Lys Ala Thr Gly Val Leu 4070 4075 4080 tat gat tat gtc aac aag tac
cac tgg gaa cac aca ggg ctc acc 12416Tyr Asp Tyr Val Asn Lys Tyr
His Trp Glu His Thr Gly Leu Thr 4085 4090 4095 ctg aga gaa gtg tct
tca aag ctg aga aga aat ctg cag aac aat 12461Leu Arg Glu Val Ser
Ser Lys Leu Arg Arg Asn Leu Gln Asn Asn
4100 4105 4110 gct gag tgg gtt tat caa ggg gcc att agg caa att gat
gat atc 12506Ala Glu Trp Val Tyr Gln Gly Ala Ile Arg Gln Ile Asp
Asp Ile 4115 4120 4125 gac gtg agg ttc cag aaa gca gcc agt ggc acc
act ggg acc tac 12551Asp Val Arg Phe Gln Lys Ala Ala Ser Gly Thr
Thr Gly Thr Tyr 4130 4135 4140 caa gag tgg aag gac aag gcc cag aat
ctg tac cag gaa ctg ttg 12596Gln Glu Trp Lys Asp Lys Ala Gln Asn
Leu Tyr Gln Glu Leu Leu 4145 4150 4155 act cag gaa ggc caa gcc agt
ttc cag gga ctc aag gat aac gtg 12641Thr Gln Glu Gly Gln Ala Ser
Phe Gln Gly Leu Lys Asp Asn Val 4160 4165 4170 ttt gat ggc ttg gta
cga gtt act caa aaa ttc cat atg aaa gtc 12686Phe Asp Gly Leu Val
Arg Val Thr Gln Lys Phe His Met Lys Val 4175 4180 4185 aag cat ctg
att gac tca ctc att gat ttt ctg aac ttc ccc aga 12731Lys His Leu
Ile Asp Ser Leu Ile Asp Phe Leu Asn Phe Pro Arg 4190 4195 4200 ttc
cag ttt ccg ggg aaa cct ggg ata tac act agg gag gaa ctt 12776Phe
Gln Phe Pro Gly Lys Pro Gly Ile Tyr Thr Arg Glu Glu Leu 4205 4210
4215 tgc act atg ttc ata agg gag gta ggg acg gta ctg tcc cag gta
12821Cys Thr Met Phe Ile Arg Glu Val Gly Thr Val Leu Ser Gln Val
4220 4225 4230 tat tcg aaa gtc cat aat ggt tca gaa ata ctg ttt tcc
tat ttc 12866Tyr Ser Lys Val His Asn Gly Ser Glu Ile Leu Phe Ser
Tyr Phe 4235 4240 4245 caa gac cta gtg att aca ctt cct ttc gag tta
agg aaa cat aaa 12911Gln Asp Leu Val Ile Thr Leu Pro Phe Glu Leu
Arg Lys His Lys 4250 4255 4260 cta ata gat gta atc tcg atg tat agg
gaa ctg ttg aaa gat tta 12956Leu Ile Asp Val Ile Ser Met Tyr Arg
Glu Leu Leu Lys Asp Leu 4265 4270 4275 tca aaa gaa gcc caa gag gta
ttt aaa gcc att cag tct ctc aag 13001Ser Lys Glu Ala Gln Glu Val
Phe Lys Ala Ile Gln Ser Leu Lys 4280 4285 4290 acc aca gag gtg cta
cgt aat ctt cag gac ctt tta caa ttc att 13046Thr Thr Glu Val Leu
Arg Asn Leu Gln Asp Leu Leu Gln Phe Ile 4295 4300 4305 ttc caa cta
ata gaa gat aac att aaa cag ctg aaa gag atg aaa 13091Phe Gln Leu
Ile Glu Asp Asn Ile Lys Gln Leu Lys Glu Met Lys 4310 4315 4320 ttt
act tat ctt att aat tat atc caa gat gag atc aac aca atc 13136Phe
Thr Tyr Leu Ile Asn Tyr Ile Gln Asp Glu Ile Asn Thr Ile 4325 4330
4335 ttc aat gat tat atc cca tat gtt ttt aaa ttg ttg aaa gaa aac
13181Phe Asn Asp Tyr Ile Pro Tyr Val Phe Lys Leu Leu Lys Glu Asn
4340 4345 4350 cta tgc ctt aat ctt cat aag ttc aat gaa ttt att caa
aac gag 13226Leu Cys Leu Asn Leu His Lys Phe Asn Glu Phe Ile Gln
Asn Glu 4355 4360 4365 ctt cag gaa gct tct caa gag tta cag cag atc
cat caa tac att 13271Leu Gln Glu Ala Ser Gln Glu Leu Gln Gln Ile
His Gln Tyr Ile 4370 4375 4380 atg gcc ctt cgt gaa gaa tat ttt gat
cca agt ata gtt ggc tgg 13316Met Ala Leu Arg Glu Glu Tyr Phe Asp
Pro Ser Ile Val Gly Trp 4385 4390 4395 aca gtg aaa tat tat gaa ctt
gaa gaa aag ata gtc agt ctg atc 13361Thr Val Lys Tyr Tyr Glu Leu
Glu Glu Lys Ile Val Ser Leu Ile 4400 4405 4410 aag aac ctg tta gtt
gct ctt aag gac ttc cat tct gaa tat att 13406Lys Asn Leu Leu Val
Ala Leu Lys Asp Phe His Ser Glu Tyr Ile 4415 4420 4425 gtc agt gcc
tct aac ttt act tcc caa ctc tca agt caa gtt gag 13451Val Ser Ala
Ser Asn Phe Thr Ser Gln Leu Ser Ser Gln Val Glu 4430 4435 4440 caa
ttt ctg cac aga aat att cag gaa tat ctt agc atc ctt acc 13496Gln
Phe Leu His Arg Asn Ile Gln Glu Tyr Leu Ser Ile Leu Thr 4445 4450
4455 gat cca gat gga aaa ggg aaa gag aag att gca gag ctt tct gcc
13541Asp Pro Asp Gly Lys Gly Lys Glu Lys Ile Ala Glu Leu Ser Ala
4460 4465 4470 act gct cag gaa ata att aaa agc cag gcc att gcg acg
aag aaa 13586Thr Ala Gln Glu Ile Ile Lys Ser Gln Ala Ile Ala Thr
Lys Lys 4475 4480 4485 ata att tct gat tac cac cag cag ttt aga tat
aaa ctg caa gat 13631Ile Ile Ser Asp Tyr His Gln Gln Phe Arg Tyr
Lys Leu Gln Asp 4490 4495 4500 ttt tca gac caa ctc tct gat tac tat
gaa aaa ttt att gct gaa 13676Phe Ser Asp Gln Leu Ser Asp Tyr Tyr
Glu Lys Phe Ile Ala Glu 4505 4510 4515 tcc aaa aga ttg att gac ctg
tcc att caa aac tac cac aca ttt 13721Ser Lys Arg Leu Ile Asp Leu
Ser Ile Gln Asn Tyr His Thr Phe 4520 4525 4530 ctg ata tac atc acg
gag tta ctg aaa aag ctg caa tca acc aca 13766Leu Ile Tyr Ile Thr
Glu Leu Leu Lys Lys Leu Gln Ser Thr Thr 4535 4540 4545 gtc atg aac
ccc tac atg aag ctt gct cca gga gaa ctt act atc 13811Val Met Asn
Pro Tyr Met Lys Leu Ala Pro Gly Glu Leu Thr Ile 4550 4555 4560 atc
ctc taa ttttttaaaa gaaatcttca tttattcttc ttttccaatt 13860Ile Leu
gaactttcac atagcacaga aaaaattcaa actgcctata ttgataaaac catacagtga
13920gccagccttg cagtaggcag tagactataa gcagaagcac atatgaactg
gacctgcacc 13980aaagctggca ccagggctcg gaaggtctct gaactcagaa
ggatggcatt ttttgcaagt 14040taaagaaaat caggatctga gttattttgc
taaacttggg ggaggaggaa caaataaatg 14100gagtctttat tgtgtatcat a
141218221DNAArtificial SequencePCR primer 82tgctaaaggc acatatggcc t
218323DNAArtificial SequencePCR primer 83ctcaggttgg actctccatt gag
238428DNAArtificial SequencePCR probe 84cttgtcagag ggatcctaac
actggccg 288520DNAArtificial SequenceAntisense Oligonucleotide
85cagtgtccag aaagtgtgtc 208620DNAArtificial SequenceAntisense
Oligonucleotide 86ggtttgctca gttggtgctg 208720DNAArtificial
SequenceAntisense Oligonucleotide 87ttaccatggt agcactgccg
208820DNAArtificial SequenceAntisense Oligonucleotide 88actctggcca
ttaccatggt 208920DNAArtificial SequenceAntisense Oligonucleotide
89tgtgacagtg gtggagaatg 209020DNAArtificial SequenceAntisense
Oligonucleotide 90tgacagtcgg aggagcgacc 209120DNAArtificial
SequenceAntisense Oligonucleotide 91tgcccattta tttgtccctg
209220DNAArtificial SequenceAntisense Oligonucleotide 92agttttcttg
gattcattgt 209320DNAArtificial SequenceAntisense Oligonucleotide
93gagagggata tcacagtagt 209420DNAArtificial SequenceAntisense
Oligonucleotide 94cagtcctggc ggtgaccatg 209520DNAArtificial
SequenceAntisense Oligonucleotide 95cttatagtga ttgcacactt
209620DNAArtificial SequenceAntisense Oligonucleotide 96tctggccaaa
tgctcagcac 209719DNAArtificial SequenceAntisense Oligonucleotide
97cgagaggcgg acgggaccg 199821DNAArtificial SequenceAntisense
Oligonucleotide 98cgagaggcgg acgggaccgt t 219921DNAArtificial
SequenceAntisense Oligonucleotide 99ttgctctccg cctgccctgg c
2110019DNAArtificial SequenceAntisense Oligonucleotide
100gctctccgcc tgccctggc 19
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