U.S. patent application number 09/800629 was filed with the patent office on 2002-09-12 for antisense modulation of interleukin-5 signal transduction.
Invention is credited to Dean, Nicholas M., Karras, James G., Manoharan, Muthiah, McKay, Robert.
Application Number | 20020128216 09/800629 |
Document ID | / |
Family ID | 23074712 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020128216 |
Kind Code |
A1 |
Dean, Nicholas M. ; et
al. |
September 12, 2002 |
Antisense modulation of interleukin-5 signal transduction
Abstract
Compositions and methods are provided for antisense modulation
of interleukin-5 signal transduction. Antisense compounds,
particularly antisense oligonucleotides, targeted to nucleic acids
encoding interleukin-5 and interleukin-5 receptor a are preferred.
Methods of using these compounds for modulation of interleukin-5
signal transduction and for treatment of diseases associated with
interleukin-5 signal transduction are also provided.
Inventors: |
Dean, Nicholas M.;
(Olivenhain, CA) ; Karras, James G.; (San Marcos,
CA) ; McKay, Robert; (San Diego, CA) ;
Manoharan, Muthiah; (Carlsbad, CA) |
Correspondence
Address: |
Jane Massey Licata
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
23074712 |
Appl. No.: |
09/800629 |
Filed: |
March 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09800629 |
Mar 7, 2001 |
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PCT/US00/07318 |
Mar 17, 2000 |
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PCT/US00/07318 |
Mar 17, 2000 |
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09280799 |
Mar 26, 1999 |
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6136603 |
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Current U.S.
Class: |
514/44A ;
435/6.16; 536/23.5 |
Current CPC
Class: |
C12N 15/1138 20130101;
C12N 2310/321 20130101; A61P 11/06 20180101; A61P 29/00 20180101;
A61P 43/00 20180101; C12N 15/1136 20130101; C12N 2310/341 20130101;
A61P 35/00 20180101; A61K 38/00 20130101; C12N 2310/321 20130101;
C12N 2310/3525 20130101; C12N 2310/346 20130101; C12N 2310/3341
20130101; C12N 2310/315 20130101 |
Class at
Publication: |
514/44 ; 435/6;
536/23.5 |
International
Class: |
A61K 048/00; C12Q
001/68; C07H 021/04 |
Claims
What is claimed is:
1. An antisense compound 8 to 30 nucleobases in length which
modulates interleukin-5 signal transduction.
2. The antisense compound of claim 1 which is an antisense
oligonucleotide.
3. The antisense compound of claim 1 which is targeted to a nucleic
acid molecule encoding mammalian interleukin-5, wherein said
antisense compound modulates the expression of mammalian
interleukin-5.
4. An antisense compound up to 30 nucleobases in length comprising
at least an 8-nucleobase portion of SEQ ID NO: 52, 53 or 62 which
inhibits the expression of mammalian interleukin-5.
5. The antisense compound of claim 1 which is targeted to a nucleic
acid molecule encoding a mammalian interleukin-5 receptor a,
wherein said antisense compound modulates the expression of
mammalian interleukin-5 receptor a.
6. An antisense compound up to 30 nucleobases in length comprising
at least an 8-nucleobase portion of SEQ ID NO: 162, 166, 167, 169,
170, 171 or 172 which inhibits the expression of mammalian
interleukin-5 receptor a.
7. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified internucleoside
linkage.
8. The antisense compound of claim 7 wherein the modified
internucleoside linkage of the antisense oligonucleotide is a
phosphorothioate linkage.
9. The antisense compound of claim 7 wherein the modified
internucleoside linkage of the antisense oligonucleotide is a
peptide nucleic acid.
10. The antisense compound of claim 9 which comprises at least one
basic amino acid conjugated to at least one end of the antisense
compound.
11. The antisense compound of claim 10 wherein the basic amino acid
is lysine or arginine.
12. The antisense compound of claim 10 which is less than 20
nucleobases in length.
13. The antisense compound of claim 12 comprising at least an
8-nucleobase portion of SEQ. ID NO: 209.
14. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified sugar moiety.
15. The antisense compound of claim 14 wherein the modified sugar
moiety of the antisense oligonucleotide is a 2'-O-methoxyethyl
sugar moiety.
16. The antisense compound of claim 15 wherein substantially all
sugar moieties of the antisense oligonucleotide are
2'-O-methoxyethyl sugar moieties.
17. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified nucleobase.
18. The antisense compound of claim 17 wherein the modified
nucleobase of the antisense oligonucleotide is a
5-methylcytosine.
19. The antisense compound of claim 15 wherein each
2'-O-methoxyethyl modified cytosine nucleobase of the antisense
oligonucleotide is a 5-methylcytosine.
20. The antisense compound of claim 1 which is a chimeric
oligonucleotide.
21. A pharmaceutical composition comprising the antisense compound
of claim 1 and a pharmaceutically acceptable carrier or
diluent.
22. The pharmaceutical composition of claim 21 further comprising a
colloidal dispersion system.
23. The pharmaceutical composition of claim 21 wherein the
antisense compound is an antisense oligonucleotide.
24. The antisense compound of claim 5 which is targeted to soluble
interleukin-5 receptor a and which preferentially inhibits the
expression of soluble interleukin-5 receptor a.
25. The antisense compound of claim 24 which is targeted to a
region of a nucleic acid molecule encoding soluble interleukin-5
receptor a which is not found in a nucleic acid molecule encoding
membrane interleukin-5 receptor a.
26. The antisense compound of claim 5 which is targeted to membrane
interleukin-5 receptor a and which preferentially inhibits the
expression of membrane interleukin-5 receptor a.
27. The antisense compound of claim 26 which is targeted to a
region of a nucleic acid molecule encoding membrane interleukin-5
receptor a which is not found in a nucleic acid molecule encoding
soluble interleukin-5 receptor a.
28. The antisense compound of claim 5 which inhibits the expression
of both soluble and membrane forms of interleukin-5 receptor a.
29. The antisense compound of claim 5 which alters the ratio of
interleukin-5 receptor a isoforms expressed by a cell or
tissue.
30. The antisense compound of claim 29 which increases the ratio of
the soluble form of interleukin-5 receptor a to the membrane form
of interleukin-5 receptor a expressed.
31. The antisense compound of claim 30 which is an antisense
oligonucleotide wherein substantially all sugar moieties of the
antisense oligonucleotide are 2'-O-methoxyethyl sugar moieties.
32. The antisense compound of claim 5 which promotes apoptosis.
33. An antisense compound which alters splicing of an RNA encoding
interleukin-5 receptor a, such that the ratio of interleukin-5
receptor a isoforms is altered.
34. The antisense compound of claim 33 wherein the antisense
oligonucleotide comprises at least one modified internucleoside
linkage.
35. The antisense compound of claim 34 wherein the modified
internucleoside linkage of the antisense oligonucleotide is a
phosphorothioate linkage.
36. The antisense compound of claim 34 wherein the modified
internucleoside linkage is a peptide nucleic acid.
37. The antisense compound of claim 36 which comprises at least one
basic amino acid conjugated to at least one end of the antisense
compound.
38. The antisense compound of claim 37 wherein the basic amino acid
is lysine or arginine.
39. The antisense compound of claim 38 which is less than 20
nucleobases in length.
40. The antisense compound of claim 36 comprising at least an
8-nucleobase portion of SEQ. ID NO: 209.
41. The antisense compound of claim 33 wherein the antisense
oligonucleotide comprises at least one modified sugar moiety.
42. The antisense compound of claim 41 wherein the modified sugar
moiety of the antisense oligonucleotide is a 2'-O-methoxyethyl
sugar moiety.
43. The antisense compound of claim 42 wherein substantially all
sugar moieties of the antisense oligonucleotide are
2'-O-methoxyethyl sugar moieties.
44. The antisense compound of claim 33 wherein the antisense
oligonucleotide comprises at least one modified nucleobase.
45. The antisense compound of claim 44 wherein the modified
nucleobase of the antisense oligonucleotide is a
5-methylcytosine.
46. The antisense compound of claim 42 wherein each
2'-O-methoxyethyl modified cytosine nucleobase of the antisense
oligonucleotide is a 5-methylcytosine.
47. The antisense compound of claim 33 which comprises a conjugate
group of at least one lysine or arginine linked to the antisense
compound.
48. The antisense compound of claim 33 which is a chimeric
oligonucleotide.
49. A method of modulating interleukin-5 signal transduction in
cells or tissues comprising contacting said cells or tissues with
the antisense compound of claim 1 so that interleukin-5 signal
transduction is modulated.
50. A method of modulating the expression of mammalian
interleukin-5 in mammalian cells or tissues comprising contacting
said cells or tissues with the antisense compound of claim 3 so
that expression of mammalian interleukin-5 is inhibited.
51. A method of modulating the expression of mammalian
interleukin-5 receptor a in mammalian cells or tissues comprising
contacting said cells or tissues with the antisense compound of
claim 33 so that expression of mammalian interleukin-5 receptor a
is inhibited.
52. A method of altering the ratio of the isoforms of mammalian
interleukin-5 receptor a in mammalian cells or tissues comprising
contacting said cells or tissues with the antisense compound of
claim 33 so that the ratio of the mammalian interleukin-5 receptor
a isoforms is altered.
53. A method of modulating the expression of mammalian
interleukin-5 receptor a in mammalia cells or tissues comprising
contacting said cells or tissues with the antisense compound of
claim 5 so that expression of mammalian interleukin-5 receptor a is
inhibited.
54. A method of altering the ratio of the isoforms of mammalian
interleukin-5 receptor a in mammalian cells or tissues comprising
contacting said cells or tissues with the antisense compound of
claim 31 so that the ratio of the mammalian interleukin-5 receptor
a isoforms is altered.
55. A method of treating a mammalian having a disease or condition
associated with interleukin-5 signal transduction comprising
administering to said mammal a therapeutically or prophylactically
effective amount of the antisense compound of claim 1 so that
interleukin-5 signal transduction is modulated.
56. A method of treating a mammal having a disease or condition
associated with interleukin-5 expression comprising administering
to said mammal a therapeutically or prophylactically effective
amount of the antisense compound of claim 3 so that interleukin-5
expression is modulated.
57. A method of treating a mammal having a disease or condition
associated with interleukin-5 receptor a expression comprising
administering to said mammal a therapeutically or prophylactically
effective amount of the antisense compound of claim 5 so that
interleukin-5 receptor a expression is modulated.
58. The method of claim 57 wherein the disease or condition is an
eosinophilic syndrome or asthma.
59. The method of claim 57 wherein the route of administration is
pulmonary administration.
60. A method of treating a mammal having a disease or condition
associated with interleukin-5 receptor a expression comprising
administering to said mammal a therapeutically or prophylactically
effective amount of the antisense compound of claim 33 so that the
ratio of interleukin-5 receptor a isoforms is altered.
61. The method of claim 60 wherein the disease or condition is an
eosinophilic syndrome or asthma.
62. The method of claim 60 wherein the route of administration is
pulmonary administration.
63. A method of treating a mammal having a disease or condition
characterized by a reduction in apoptosis comprising administering
to said mammal a prophylactically or therapeutically effective
amount of the antisense compound of claim 32.
64. A method of treating a mammal having a disease or condition
associated with interleukin-5 receptor a expression comprising
administering to said mammal a therapeutically or prophylactically
effective amount of the antisense compound of claim 26 so that
expression of membrane interleukin-5 receptor a is modulated.
65. The method of claim 64 wherein the disease or condition is
asthma or an eosinophilic syndrome.
66. The method of claim 64 wherein the route of administration is
pulmonary administration.
67. The pharmaceutical composition of claim 21 further comprising a
chemotherapeutic agent for the treatment of asthma.
68. A pharmaceutical composition comprising the antisense compound
of claim 28 and a pharmaceutically acceptable carrier or
diluent.
69. A pharmaceutical composition comprising the antisense compound
of claim 36 and a pharmaceutically acceptable carrier or
diluent.
70. A diagnostic kit for detecting the expression level of the
membrane versus soluble form of IL-5 Receptor a.
71. The diagnostic kit of claim 70 comprising the antisense
compound of claim 33.
72. The diagnostic kit of claim 71 wherein the antisense compound
is a peptide nucleic acid.
Description
[0001] This application is a continuation-in-part of PCT
Application No. PCT/US00/07318 filed Mar. 17, 2000 which
corresponds to U.S. application Ser. No. 09/280,799 filed Mar. 26,
1999 now issued U.S. Pat. No. 6,136,603.
FIELD OF THE INVENTION
[0002] The present invention provides compositions and methods for
modulating interleukin-5 (IL-5) signaling through antisense
modulation of IL-5 and/or IL-5 receptor a (IL-5a) expression. In
particular, this invention relates to antisense compounds,
particularly oligonucleotides, specifically hybridizable with
nucleic acids encoding IL-5 or IL-5Ra. Such oligonucleotides have
been shown to modulate the expression of IL-5 and IL-5Ra,
respectively.
BACKGROUND OF THE INVENTION
[0003] Cytokines are relatively low molecular weight,
pharmacologically active proteins that are secreted by cells for
the purpose of altering either their own functions or those of
adjacent cells. Cytokines are important regulators of
hematopoiesis. They exert their actions by binding to specific
receptors on the cell surface. Among the cytokines are a large
number of interleukins as well as growth and colony-stimulating
factors. Interleukin-5 (IL-5) is a critical cytokine for regulation
of growth, activation, maturation, and survival of eosinophils, a
type of leukocyte, and their release from the bone marrow.
Eosinophils have been implicated in the pathogenesis of certain
diseases ("eosinophilic syndromes") characterized by long-term
chronic inflammation of tissues, such as the lungs in the case of
asthma or the muscles in the case of eosinophilia myalgia. Other
eosinophilic syndromes in addition to these include allergic
rhinitis and atopic dermatitis. Eosinophils have also been noted as
a component of cellular infiltrates of malignant tumors.
Eosinophils are attracted to sites of wounding or inflammation,
where they undergo a process of activation. Because eosinophils
play a seminal role in the pathogenesis of asthma, particularly the
late-phase reaction of asthma, and other inflammatory and/or
allergic conditions, IL-5 signal transduction is of clinical
importance.
[0004] In humans, IL-5 is selective in specifically promoting
eosinophil and basophilic differentiation and maturation. Blood and
tissue eosinophilia is a characteristic abnormality in allergy and
asthma and convincing evidence implicates IL-5 as the key cytokine
regulating this selective eosinophilic inflammation. Thus,
inhibition of IL-5 production or effector function will abolish the
eosinophilic component in asthma and other eosinophilic diseases,
likely preventing further tissue damage caused by release of
eosinophil-specific inflammatory mediators and potentially
providing clinical benefit. Indeed, it has been demonstrated
neutralizing IL-5 with a monoclonal antibody can completely inhibit
bronchoalveolar eosinophilia caused by allergen challenge in guinea
pigs, mice, and monkeys. A correlation exists between pulmonary
eosinophilia and asthma in man and it is clear that selective
inhibition of IL-5 can block airway hyperresponsiveness in animal
models.
[0005] Asthma is characterized by episodic airways obstruction,
increased bronchial hyperresponsiveness, and airway inflammation.
An association has been shown between the number of activated T
cells and eosinophils in the airways and abnormalities in forced
expiratory volume in one second (FEV1), a measure of pulmonary
function, increased bronchial responsiveness, and clinical severity
in asthma. It has been documented that both interleukin-5 (IL-5)
mRNA and protein levels are increased in bronchial biopsies from
both atopic and intrinsic asthmatics. IL-5 interacts with cells via
the IL-5 receptor (IL-5R) on the cell surface. The IL-5 receptor is
a heterodimer of a- and .beta.-subunits. The IL-5 receptor
a-subunit is specific to IL-5R, whereas the .beta.-subunit is
common to IL-3, IL-5, and granulocyte/macrophage colony-stimulating
factor (GM-CSF) receptors. The human IL-5 receptor (IL-5R) is
expressed in vitro on eosinophils, basophils, and B lymphocytes,
although its role on B cells remains in question. Besides a
membrane anchored form, two forms of soluble human IL-5Ra are
produced. Only the membrane form of the a chain is complexed with
the .beta. chain, which is required for signaling.
[0006] The link between T cell derived IL-5 and lung eosinophilia
is further strengthened by the observation that increased levels of
IL-5 receptor a mRNA are also found in bronchial biopsies from
asthmatics and that the eosinophil is the predominant site of this
increased IL-5Ra expression. Further, the subset of eosinophils
that express the membrane bound form of the IL-5 receptor inversely
correlates with FEV1 while the subset expressing the soluble form
of the receptor directly correlates with FEV1. These observations
suggest that IL-5 receptor a isoform expression is of central
importance in determining clinical prognosis. The soluble form of
the receptor may be serving a beneficial role in asthmatic
patients. It is therefore presently believed that an effective
therapeutic approach to preventing eosinophilia in asthma and other
eosinophilic syndromes would entail selective inhibition of
membrane but not soluble IL-5 receptor expression. In addition,
there are several animal and lung explant models of
allergen-induced eosinophilia, late phase airway responses, and
bronchial hyperresponsiveness which collectively support a link
between IL-5 and airway eosinophilia and decreased pulmonary
function.
[0007] Several approaches to inhibition of IL-5 function have been
tried. Chimeric, humanized and other interleukin-5 (IL-5)
monoclonal antibodies (mAbs), and pharmaceutical compositions and
therapeutic methods are disclosed in WO 96/21000. Ribozymes for
cleaving IL-5 mRNA are disclosed in WO 95/23225. A 16 mer
phosphodiester oligodeoxynucleotide with two phosphorothioate
linkages, targeted to IL-5 mRNA, was used to inhibit IL-5 secretion
by human peripheral blood mononuclear cells. Weltman and Karim,
Allergy Asthma Proc., 1998, 19, 257-261; September-October 1998.
Methods of treating airway disease by administering essentially
adenosine-free antisense oligonucleotides to the airway epithelium
are disclosed in WO 96/40162. IL-5 and IL-5 receptor are among the
antisense targets disclosed.
[0008] Thus there remains a long-felt need for compositions and
methods for modulating IL-5 signal transduction, particularly in
the treatment and prevention of asthma and other reactive airway
disease.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to antisense compounds,
particularly oligonucleotides, which are targeted to a nucleic acid
encoding IL-5 or IL-5Ra, and which modulate the expression of these
gene targets. Pharmaceutical and other compositions comprising the
antisense compounds of the invention are also provided. Further
provided are methods of modulating the expression of IL-5 and/or
IL-5Ra in cells or tissues comprising contacting said cells or
tissues with one or more of the antisense compounds or compositions
of the invention. Further provided are methods of modulating IL-5
signaling in cells or tissues comprising contacting said cells or
tissues with one or more of the antisense compounds or compositions
of the invention. Further provided are methods of treating an
animal, particularly a human, suspected of having or being prone to
a disease or condition associated with IL-5 signaling or with
expression of IL-5 or IL-5Ra by administering a therapeutically or
prophylactically effective amount of one or more of the antisense
compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present invention comprehends antisense compounds
capable of modulating IL-5 signal transduction, preferably by
modulating expression of IL-5 or IL-5 receptor a. These compounds
are useful for both research and therapeutic, including
prophylactic, uses.
[0011] The human IL-5 receptor a gene contains 14 exons. A
membrane-anchored form of the receptor and two soluble forms have
been identified. The mRNA transcript encoding the membrane-anchored
form of the human IL-5 receptor a contain exons 1-10 and 12-14.
Exon 11 is spliced out by an alternative splicing event. The major
soluble isoform (soluble form 1) is generated as a result of a
normal splicing event and an in-frame stop codon in exon 11. The
other soluble form (soluble form 2) is generated by the absence of
splicing and therefore is generated by reading into intron 11.
Tuypens et al. Eur. Cytokine Netw., 1992, 3, 451-459.
[0012] The mRNA encoding the membrane form of the mouse IL-5
receptor a contains 11 exons. The transmembrane domain of the
receptor is encoded in exon 9. Two mRNAs encoding soluble
(secreted) forms of the receptor result from differential splicing
events. The mRNA encoding soluble form 1 of the receptor is missing
exon 9 (exon 8 is spliced to exon 10) and the mRNA encoding soluble
form 2 is missing exons 9 and 10 (exon 8 is spliced to exon 11).
Imamura et al., DNA and Cell Biol., 1994, 13, 283-292.
[0013] In both mouse and humans, there are both soluble forms and a
membrane-bound form of IL-5 receptor a. In mouse, the soluble form
is expressed, though experiments are usually done by addition of
exogenous recombinant soluble receptor. Recombinant murine soluble
IL-5 receptor a binds IL-5, and does not inhibit proliferation of
the IL-5-responsive Y16B cell line. In vivo, recombinant soluble
murine IL-5 receptor a suppresses antigen-induced airway
eosinophilia. In humans, recombinant human soluble IL-5 receptor a
binds human IL-5 and inhibits its biological activity in vitro,
i.e., prevents TF-1 proliferation and survival. In other words, in
the human system, the soluble IL-5 receptor a acts as a sponge to
bind the IL-5 cytokine and block its effects. Only the
membrane-bound form of IL-5 receptor a is able to transduce the
IL-5 signal. Soluble human IL-5 receptor a is not normally detected
in human biological fluids; however, a direct correlation has been
observed between the expression of soluble human IL-5 receptor a
and pulmonary function in asthmatic subjects.
[0014] The present invention employs oligomeric antisense
compounds, particularly oligonucleotides, for use in modulating
IL-5 signal transduction. In preferred embodiments this is done by
modulating the function of nucleic acid molecules encoding IL-5 or
IL-5Ra, ultimately modulating the amount of IL-5 or IL-5Ra
produced. Antisense compounds are provided which specifically
hybridize with one or more nucleic acids encoding IL-5 or IL-5Ra.
In preferred embodiments used herein, the term "nucleic acid
encoding IL-5" encompasses DNA encoding IL-5, RNA (including
pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived
from such RNA. Similarly the term "nucleic acid encoding IL-5Ra"
encompasses DNA encoding IL-5Ra, RNA (including pre-mRNA and mRNA)
transcribed from such DNA, and also cDNA derived from such RNA. In
the context of the present invention, the term "nucleic acid
target" encompasses nucleic acids encoding either IL-5 or IL-5Ra,
according to which of these the antisense compound is
complementary. The specific hybridization of an oligomeric compound
with its target nucleic acid interferes with the normal function of
the nucleic acid. This modulation of function of a target nucleic
acid by compounds which specifically hybridize to it is generally
referred to as "antisense". The functions of DNA to be interfered
with include replication and transcription. The functions of RNA to
be interfered with include all vital functions such as, for
example, translocation of the RNA to the site of protein
translation, translation of protein from the RNA, splicing of the
RNA to yield one or more mRNA species, and catalytic activity which
may be engaged in or facilitated by the RNA. The overall effect of
such interference with target nucleic acid function is modulation
of the expression of IL-5 or IL-5Ra. In the context of the present
invention, "modulation" means either an increase (stimulation) or a
decrease (inhibition) in the expression of a gene. In the context
of the present invention, inhibition is the preferred form of
modulation of gene expression and mRNA is a preferred target.
[0015] It is preferred to target specific nucleic acids for
antisense. "Targeting" an antisense compound to a particular
nucleic acid, in the context of this invention, is a multi step
process. The process usually begins with the identification of a
nucleic acid sequence whose function is to be modulated. This 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 is a nucleic acid molecule
encoding IL-5 or IL-5Ra. The targeting process also includes
determination of a site or sites within this gene for the antisense
interaction to occur such that the desired effect, e.g., detection
or modulation of expression of the protein, will result. Within the
context of the present invention, a preferred intra genic site is
the region encompassing the translation initiation or termination
codon of the open reading frame (ORF) of the gene. Since, as is
known in the art, the translation initiation codon is typically
5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding
DNA molecule), the translation initiation codon is also referred to
as the "AUG codon," the "start codon" or the "AUG start codon". A
minority of genes have a translation initiation codon having the
RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and
5'-CUG have been shown to function in vivo. Thus, the terms
"translation initiation codon" and "start codon" can encompass many
codon sequences, even though the initiator amino acid in each
instance is typically methionine (in eukaryotes) or
formylmethionine (in prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA molecule transcribed from a gene encoding
IL-5 or IL-5Ra, regardless of the sequence(s) of such codons.
[0016] It is also known in the art that a translation termination
codon (or "stop codon") of a gene may have one of three sequences,
i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences
are 5'-TAA, 5'-TAG and 5'-TGA, respectively). 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.
[0017] 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. Other target regions
include the 5' untranslated region (5'UTR), known in the art to
refer to the portion of an mRNA in the 5' direction from the
translation initiation codon, and thus including nucleotides
between the 5' cap site and the translation initiation codon of an
mRNA or corresponding nucleotides on the gene, and the 3'
untranslated region (3'UTR), known in the art to refer to the
portion of an mRNA in the 3' direction from the translation
termination codon, and thus including nucleotides between the
translation termination codon and 3' end of an mRNA or
corresponding nucleotides on the gene. The 5' cap 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. The
5' cap region may also be a preferred target region.
[0018] 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. mRNA
splice sites, i.e., intron-exon junctions, may also be preferred
target regions, and are particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular mRNA splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also preferred targets. It has also been found that
introns can also be effective, and therefore preferred, target
regions for antisense compounds targeted, for example, to DNA or
pre-mRNA.
[0019] Once one or more target sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired effect.
[0020] In the context of this invention, "hybridization" means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases. For example, adenine and thymine are
complementary nucleobases which pair through the formation of
hydrogen bonds. "Complementary," as used herein, refers to the
capacity for precise pairing between two nucleotides. For example,
if a nucleotide at a certain position of an oligonucleotide is
capable of hydrogen bonding with a nucleotide at the same position
of a DNA or RNA molecule, then the oligonucleotide and the DNA or
RNA are considered to be complementary to each other at that
position. The oligonucleotide and the DNA or RNA are complementary
to each other when a sufficient number of corresponding positions
in each molecule are occupied by nucleotides which can hydrogen
bond with each other. Thus, "specifically hybridizable" and
"complementary" are terms which are used to indicate a sufficient
degree of complementarity or precise pairing such that stable and
specific binding occurs between the oligonucleotide and the DNA or
RNA target. It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. An antisense
compound is specifically hybridizable when binding of the compound
to the target DNA or RNA molecule interferes with the normal
function of the target DNA or RNA to cause a loss of utility, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
sequences under conditions in which specific binding is desired,
i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, and, in the case of in vitro assays,
under conditions in which the assays are performed.
[0021] Antisense compounds are commonly used as research reagents
and diagnostics. For example, 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. Antisense compounds are also used, for example,
to distinguish between functions of various members of a biological
pathway. Antisense modulation has, therefore, been harnessed for
research use.
[0022] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense oligonucleotides have been employed as therapeutic
moieties in the treatment of disease states in animals and man.
Antisense oligonucleotides have been safely and effectively
administered to humans and numerous clinical trials are presently
underway. It is thus established that oligonucleotides can be
useful therapeutic modalities that can be configured to be useful
in treatment regimes of cells, tissues and animals, especially
humans. 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 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 nucleic acid target and increased
stability in the presence of nucleases.
[0023] While antisense oligonucleotides are a preferred form of
antisense compound, the present invention comprehends other
oligomeric antisense compounds, including but not limited to
oligonucleotide mimetics such as are described below. The antisense
compounds in accordance with this invention preferably comprise
from about 8 to about 30 nucleobases. Particularly preferred are
antisense oligonucleotides comprising from about 8 to about 30
nucleotides). 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 structure can be further joined to form a circular
structure. However, open linear structures are generally preferred.
Within the oligonucleotide structure, 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.
[0024] 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.
[0025] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphoro-dithioates, phosphotri-esters,
aminoalkyl-phosphotri-esters, methyl and other alkyl phosphonates
including 3-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3-5-linkages, 2-5-linked analogs of
these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3-5- to 5-3- or 2-5- to 5-2-.
Various salts, mixed salts and free acid forms are also
included.
[0026] 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; and 5,625,050, each of
which is herein incorporated by reference.
[0027] 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; 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.
[0028] 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; and
5,677,439, each of which is herein incorporated by reference.
[0029] 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 base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric 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.
[0030] Most preferred embodiments of the invention are
oligonucleotides with phosphorothioate backbones and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0031] 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.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.su- b.3)].sub.2, where n and
m are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2- position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, 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.lN.sub..beta.NH.sub.l 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 an alkoxyalkoxy group, 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504). Further preferred modifications include
2-dimethylaminooxyethoxy, i.e., a
2'-O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as
2'-DMAOE and 2'-dimethylaminoethoxyethoxy, i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2.
[0032] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (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;
5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; and
5,700,920, each of which is herein incorporated by reference.
[0033] 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 uracil and
cytosine, 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, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further
nucleobases include those disclosed in U.S. Pat. No. 3,687,808,
those disclosed in Kroschwitz, J. I., The Concise Encyclopedia Of
Polymer Science And Engineering, ed. John Wiley & Sons, 1990,
pages 858-859, those disclosed by Englisch et al., Angewandte
Chemie, International Edition, 1991, 30, 613, and those disclosed
by Sanghvi, Y. S., Crooke, S. T., and Lebleu, B. eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
289-302. Certain of these nucleobases are particularly useful for
increasing the binding affinity of the oligomeric 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.
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0034] 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; 5,681,941; and
5,750,692, each of which is herein incorporated by reference.
[0035] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937.
[0036] 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, each of which is herein incorporated by
reference.
[0037] 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.
The present invention also includes antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. 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, 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 inhibition of gene expression. Cleavage of the RNA
target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known
in the art.
[0038] Chimeric antisense compounds of the invention may be formed
as composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative United States patents
that teach the preparation of such hybrid structures include, but
are not limited to, U.S. 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, each of which is
herein incorporated by reference.
[0039] 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.
[0040] The antisense compounds of the invention are synthesized in
vitro and do not include antisense compositions of biological
origin, or genetic vector constructs designed to direct the in vivo
synthesis of antisense molecules.
[0041] 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.
[0042] 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. Accordingly,
for example, the disclosure is also drawn to prodrugs and
pharmaceutically acceptable salts of the compounds of the
invention, pharmaceutically acceptable salts of such prodrugs, and
other bioequivalents.
[0043] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of
endogenous enzymes or other chemicals and/or conditions. In
particular, prodrug versions of the oligonucleotides of the
invention are prepared as SATE [(S-acetyl-2-thioethyl)phosphate]
derivatives according to the methods disclosed in WO 93/24510 or in
WO 94/26764.
[0044] 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.
[0045] Pharmaceutically acceptable base addition salts are formed
with metals or amines, such as alkali and alkaline earth metals or
organic amines. Examples of metals used as cations are sodium,
potassium, magnesium, calcium, and the like. Examples of suitable
amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, dicyclohexylamine, ethylenediamine,
N-methylglucamine, and procaine (see, for example, Berge et al.,
"Pharmaceutical Salts," J. of Pharma Sci. , 1977, 66, 1-19). The
base addition salts of said acidic compounds are prepared by
contacting the free acid form with a sufficient amount of the
desired base to produce the salt in the conventional manner. The
free acid form may be regenerated by contacting the salt form with
an acid and isolating the free acid in the conventional manner. The
free acid forms differ from their respective salt forms somewhat in
certain physical properties such as solubility in polar solvents,
but otherwise the salts are equivalent to their respective free
acid for purposes of the present invention. As used herein, a
"pharmaceutical addition salt" includes a pharmaceutically
acceptable salt of an acid form of one of the components of the
compositions of the invention. These include organic or inorganic
acid salts of the amines. Preferred addition salts are acid salts
such as the hydrochlorides, acetates, salicylates, nitrates and
phosphates. Other suitable pharmaceutically acceptable salts are
well known to those skilled in the art and include basic salts of a
variety of inorganic and organic acids, such as, for example, with
inorganic acids, such as for example hydrochloric acid, hydrobromic
acid, sulfuric acid or phosphoric acid; with organic carboxylic,
sulfonic, sulfo or phospho acids or N-substituted sulfamic acids,
for example acetic acid, propionic acid, glycolic acid, succinic
acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric
acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic
acid, glucaric acid, glucuronic acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic
acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embolic acid,
nicotinic acid or isonicotinic acid; and with amino acids, such as
the 20 alpha-amino acids involved in the synthesis of proteins in
nature, for example glutamic acid or aspartic acid, and also with
phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,
2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,
benzenesulfonic acid, 4-methylbenzenesulfoic acid,
naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or
3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid
(with the formation of cyclamates), or with other acid organic
compounds, such as ascorbic acid. Pharmaceutically acceptable salts
of compounds may also be prepared with a pharmaceutically
acceptable cation. Suitable pharmaceutically acceptable cations are
well known to those skilled in the art and include alkaline,
alkaline earth, ammonium and quaternary ammonium cations.
Carbonates or hydrogen carbonates are also possible.
[0046] For oligonucleotides, preferred examples of pharmaceutically
acceptable salts include but are not limited to (a) salts formed
with cations such as sodium, potassium, ammonium, magnesium,
calcium, polyamines such as spermine and spermidine, etc.; (b) acid
addition salts formed with inorganic acids, for example
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; (c) salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like;
and (d) salts formed from elemental anions such as chlorine,
bromine, and iodine.
[0047] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. For therapeutics, an animal, preferably a human,
suspected of having a disease or disorder which can be treated by
modulating IL-5 signaling is treated by administering one or more
antisense compounds in accordance with this invention. The
compounds of the invention can be utilized in pharmaceutical
compositions by adding an effective amount of an antisense compound
to a suitable pharmaceutically acceptable diluent or carrier. Use
of the antisense compounds and methods of the invention may also be
useful prophylactically, e.g., to prevent or delay infection,
inflammation or tumor formation, for example.
[0048] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding IL-5 or IL-5Ra, enabling sandwich and other
assays to easily be constructed to exploit this fact. Hybridization
of the antisense oligonucleotides of the invention with a nucleic
acid encoding IL-5 or IL-5Ra can be detected by means known in the
art. Such means may include conjugation of an enzyme to the
oligonucleotide, radiolabelling of the oligonucleotide or any other
suitable detection means. Kits using such detection means for
detecting the level of IL-5 or IL-5Ra in a sample may also be
prepared.
[0049] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention. The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration.
[0050] 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.
[0051] Compositions and formulations for oral administration
include powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets or tablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable.
[0052] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0053] Pharmaceutical compositions and/or formulations comprising
the oligonucleotides of the present invention may also include
penetration enhancers in order to enhance the alimentary delivery
of the oligonucleotides. Penetration enhancers may be classified as
belonging to one of five broad categories, i.e., fatty acids, bile
salts, chelating agents, surfactants and non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8,
91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33). One or more penetration enhancers from one
or more of these broad categories may be included.
[0054] Various fatty acids and their derivatives which act as
penetration enhancers include, for example, oleic acid, lauric
acid, capric acid, myristic acid, palmitic acid, stearic acid,
linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate,
monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic
acid, arichidonic acid, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono-
and di-glycerides and 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, 8:2, 91-192; Muranishi, Critical Reviews in
Therapeutic Drug Carrier Systems, 1990, 7:1, 1-33; El-Hariri et
al., J. Pharm. Pharmacol., 1992, 44, 651-654). Examples of some
presently preferred fatty acids are sodium caprate and sodium
laurate, used singly or in combination at concentrations of 0.5 to
5%.
[0055] 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. A
presently preferred bile salt is chenodeoxycholic acid (CDCA)
(Sigma Chemical Company, St. Louis, Mo.), generally used at
concentrations of 0.5 to 2%.
[0056] Complex formulations comprising one or more penetration
enhancers may be used. For example, bile salts may be used in
combination with fatty acids to make complex formulations.
Preferred combinations include CDCA combined with sodium caprate or
sodium laurate (generally 0.5 to 5%).
[0057] Chelating agents 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, 8:2, 92-192; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1,
1-33; Buur et al., J. Control Rel., 1990, 14, 43-51). Chelating
agents have the added advantage of also serving as DNase
inhibitors.
[0058] 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, 8:2, 92-191); and perfluorochemical emulsions, such as FC-43
(Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252-257).
[0059] Non-surfactants include, for example, unsaturated cyclic
ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
8:2, 92-191); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0060] As used herein, "carrier compound" refers to a nucleic acid,
or analog thereof, which is inert (i.e., does not possess
biological activity per se) but 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 phosphorothioated oligonucleotide in
hepatic tissue is reduced when it is coadministered with
polyinosinic acid, dextran sulfate, polycytidic acid or
4-acetamido-4'-isothiocyano-stilbene-2,2'-di- sulfonic acid (Miyao
et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
[0061] In contrast to a carrier compound, a "pharmaceutically
acceptable carrier" (excipient) is a pharmaceutically acceptable
solvent, suspending agent or any other pharmacologically inert
vehicle for delivering one or more nucleic acids to an animal. The
pharmaceutically acceptable carrier 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 pharmaceutically acceptable carriers include,
but are not limited to, binding agents (e.g., pregelatinized 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.); disintegrates (e.g., starch, sodium starch glycolate, etc.);
or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained
release oral delivery systems and/or enteric coatings for orally
administered dosage forms are described in U.S. Pat. Nos.
4,704,295; 4,556,552; 4,309,406; and 4,309,404.
[0062] The compositions of the present invention 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, e.g.,
antipruritics, 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 invention.
[0063] In certain embodiments of this invention, the antisense
compounds of the invention may be administered in combination with
a conventional anti-asthma medication. Typically, two types of
medication are used in attempts to control asthma: quick-relief
medications (short-acting bronchodilators) that work fast to stop
attacks or relieve symptoms and long-term preventive medications
(especially anti-inflammatory agents) that keep symptoms and
attacks from starting. Examples of the short-acting bronchodilators
are short-acting .beta.2-agonists, for example, albuterol,
bitolterol, fenoterol isoetharine, metaproterenol, pirbuterol,
salbutamol and terbutaline; anticholinergics, for example
ipratropium bromide and oxitropium bromide; short-acting
theophyllines, for example, aminophylline; and
epinephrine/adrenaline. Examples of long-term preventive
medications are inhaled or oral corticosteroids, for example,
beclomethasone, budesonide, fluticasone triamcinolone,
prednisolone, prednisone and methylprednisolone; sodium
cromoglycate or cromolyn sodium; nedocromil; oral or inhaled
long-acting .beta.2-agonists, for example salmeterol, formoterol,
terbutaline, salbutamol; sustained-release theophyllines, for
example, aminophylline, methylxanthine and xanthine; and ketotifen.
Antisense compounds of the present inventions may be administered
in combination or conjunction with these or any of the asthma
medications known in the art.
[0064] The compounds of the invention may also be administered in
combination with another inhibitor of IL-5 signal transduction,
preferably an antibody directed to IL-5. Such antibodies are known
in the art.
[0065] Regardless of the method by which the antisense compounds of
the invention are introduced into a patient, colloidal dispersion
systems may be used as delivery vehicles to enhance the in vivo
stability of the compounds and/or to target the compounds to a
particular organ, tissue or cell type. Colloidal dispersion systems
include, but are not limited to, macromolecule complexes,
nanocapsules, microspheres, beads and lipid-based systems including
oil-in-water emulsions, micelles, mixed micelles, liposomes and
lipid:oligonucleotide complexes of uncharacterized structure. A
preferred colloidal dispersion system is a plurality of liposomes.
Liposomes are microscopic spheres having an aqueous core surrounded
by one or more outer layer(s) made up of lipids arranged in a
bilayer configuration (see, generally, Chonn et al., Current Op.
Biotech., 1995, 6, 698-708).
[0066] Certain embodiments of the invention provide for liposomes
and other compositions containing (a) one or more antisense
compounds and (b) one or more other chemotherapeutic agents which
function by a non-antisense mechanism. Examples of such
chemotherapeutic agents include, but are not limited to, anticancer
drugs such as daunorubicin, dactinomycin, doxorubicin, bleomycin,
mitomycin, nitrogen mustard, chlorambucil, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),
5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),
colchicine, vincristine, vinblastine, etoposide, teniposide,
cisplatin and diethylstilbestrol (DES). See, generally, The Merck
Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds.,
1987, Rahway, N.J., pp. 1206-1228. Anti-inflammatory drugs,
including but not limited to nonsteroidal anti-inflammatory drugs
and corticosteroids, and antiviral drugs, including but not limited
to ribovirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. See, generally, The
Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pp. 2499-2506 and 46-49, respectively.
Other non-antisense chemotherapeutic agents are also within the
scope of this invention. Two or more combined compounds may be used
together or sequentially.
[0067] 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. Two or more combined compounds may be used together or
sequentially.
[0068] The formulation of therapeutic compositions and their
subsequent administration 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, 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.
[0069] 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.
EXAMPLES
Example 1
[0070] Nucleoside Phosphoramidites for Oligonucleotide Synthesis
Deoxy and 2-alkoxy Amidites
[0071] 2-Deoxy and 2-methoxy .beta.-cyanoethyldiisopropyl
phosphoramidites were purchased from commercial sources (e.g.
Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.).
Other 2-O-alkoxy substituted nucleoside amidites are prepared as
described in U.S. Pat. No. 5,506,351, herein incorporated by
reference. For oligonucleotides synthesized using 2-alkoxy
amidites, the standard cycle for unmodified oligonucleotides was
utilized, except the wait step after pulse delivery of tetrazole
and base was increased to 360 seconds.
[0072] Oligonucleotides containing 5-methyl-2'-deoxycytidine
(5-Me-C) nucleotides were synthesized according to published
methods (Sanghvi, et. al., Nucleic Acids Research, 1993, 21,
3197-3203] using commercially available phosphoramidites (Glen
Research, Sterling Va. or ChemGenes, Needham Mass.).
[0073] 2-Fluoro Amidites
2-Fluorodeoxyadenosine Amidites
[0074] 2'-fluoro oligonucleotides are synthesized as described
previously by Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841
and U.S. Pat. No. 5,670,633, herein incorporated by reference.
Briefly, the protected nucleoside
N6-benzoyl-2'-deoxy-2'-fluoroadenosine is synthesized utilizing
commercially available 9-beta-D-arabinofuranosyladenine as starting
material and by modifying literature procedures whereby the
2-alpha-fluoro atom is introduced by a S.sub.N2-displacement of a
2-beta-trityl group. Thus
N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively
protected in moderate yield as the 3',5'-ditetrahydropyranyl (THP)
intermediate. Deprotection of the THP and N6-benzoyl groups is
accomplished using standard methodologies and standard methods are
used to obtain the 5'-dimethoxytrityl-(DMT) and
5'-DMT-3'-phosphoramidite intermediates.
2-Fluorodeoxyguanosine
[0075] The synthesis of 2'-deoxy-2'-fluoroguanosine is accomplished
using tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosylguani- ne as starting material, and
conversion to the intermediate
diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS
group is followed by protection of the hydroxyl group with THP to
give diisobutyryl di-THP protected arabinofuranosylguanine.
Selective O-deacylation and triflation is followed by treatment of
the crude product with fluoride, then deprotection of the THP
groups. Standard methodologies are used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidit- es.
2-Fluorouridine
[0076] Synthesis of 2'-deoxy-2'-fluorouridine is accomplished by
the modification of a literature procedure in which
2,2'-anhydro-1-beta-D-ara- binofuranosyluracil is treated with 70%
hydrogen fluoride-pyridine. Standard procedures were used to obtain
the 5'-DMT and 5'-DMT-3'phosphoramidites.
2-Fluorodeoxycytidine
[0077] 2'-deoxy-2'-fluorocytidine is synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures are used
to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.
2-O-(2-Methoxyethyl) Modified Amidites
[0078] 2'-O-Methoxyethyl-substituted nucleoside amidites were
prepared as follows, or alternatively, as per the methods of
Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.
2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]
[0079] 5-Methyluridine (ribosylthymine, commercially available
through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate
(90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were
added to DMF (300 mL). The mixture was heated to reflux, with
stirring, allowing the evolved carbon dioxide gas to be released in
a controlled manner. After 1 hour, the slightly darkened solution
was concentrated under reduced pressure. The resulting syrup was
poured into diethylether (2.5 L), with stirring. The product formed
a gum. The ether was decanted and the residue was dissolved in a
minimum amount of methanol (ca. 400 mL). The solution was poured
into fresh ether (2.5 L) to yield a stiff gum. The ether was
decanted and the gum was dried in a vacuum oven (60.degree. C. at 1
mm Hg for 24 hours) to give a solid that was crushed to a light tan
powder (57 g, 85% crude yield). The NMR spectrum was consistent
with the structure, contaminated with phenol as its sodium salt
(ca. 5%). The material was used as is for further reactions or
purified further by column chromatography using a gradient of
methanol in ethyl acetate (10-25%) to give a white solid, mp
222-4.degree. C.
2'-O-Methoxyethyl-5-methyluridine
[0080] 2,2'-Anhydro-5-methyluridine (195 g, 0.81 M),
tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol
(1.2 L) were added to a 2 L stainless steel pressure vessel and
placed in a pre-heated oil bath at 160.degree. C. After heating for
48 hours at 155-160.degree. C., the vessel was opened and the
solution evaporated to dryness and triturated with MeOH (200 mL).
The residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 mL) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2Cl.sub.2/Acetone/MeOH (20:5:3) containing 0.5% Et.sub.3NH.
The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL) and adsorbed
onto silica (150 g) prior to loading onto the column. The product
was eluted with the packing solvent to give 160 g (63%) of product.
Additional material was obtained by reworking impure fractions.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0081] 2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was
co-evaporated with pyridine (250 mL) and the dried residue
dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the mixture stirred at
room temperature for one hour. A second aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the reaction stirred for
an additional one hour. Methanol (170 mL) was then added to stop
the reaction. HPLC showed the presence of approximately 70%
product. The solvent was evaporated and triturated with CH.sub.3CN
(200 mL). The residue was dissolved in CHCl.sub.3 (1.5 L) and
extracted with 2.times.500 mL of saturated NaHCO.sub.3 and
2.times.500 mL of saturated NaCl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered and evaporated. 275 g of residue was
obtained. The residue was purified on a 3.5 kg silica gel column,
packed and eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5%
Et.sub.3NH. The pure fractions were evaporated to give 164 g of
product. Approximately 20 g additional was obtained from the impure
fractions to give a total yield of 183 g (57%).
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0082] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106
g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from
562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38
mL, 0.258 M) were combined and stirred at room temperature for 24
hours. The reaction was monitored by tlc by first quenching the tlc
sample with the addition of MeOH. Upon completion of the reaction,
as judged by tlc, MeOH (50 mL) was added and the mixture evaporated
at 35.degree. C. The residue was dissolved in CHCl.sub.3 (800 mL)
and extracted with 2.times.200 mL of saturated sodium bicarbonate
and 2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approx. 90% product). The residue was purified on a 3.5 kg silica
gel column and eluted using EtOAc/Hexane (4:1). Pure product
fractions were evaporated to yield 96 g (84%). An additional 1.5 g
was recovered from later fractions.
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleurid-
ine
[0083] A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methox-
yethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in
CH.sub.3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M)
was added to a solution of triazole (90 g, 1.3 M) in CH.sub.3CN (1
L), cooled to -5.degree. C. and stirred for 0.5 hours using an
overhead stirrer. POCl.sub.3 was added dropwise, over a 30 minute
period, to the stirred solution maintained at 0-10.degree. C., and
the resulting mixture stirred for an additional 2 hours. The first
solution was added dropwise, over a 45 minute period, to the latter
solution. The resulting reaction mixture was stored overnight in a
cold room. Salts were filtered from the reaction mixture and the
solution was evaporated. The residue was dissolved in EtOAc (1 L)
and the insoluble solids were removed by filtration. The filtrate
was washed with 1.times.300 mL of NaHCO.sub.3 and 2.times.300 mL of
saturated NaCl, dried over sodium sulfate and evaporated. The
residue was triturated with EtOAc to give the title compound.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0084] A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5--
methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and
NH.sub.4OH (30 mL) was stirred at room temperature for 2 hours. The
dioxane solution was evaporated and the residue azeotroped with
MeOH (2.times.200 mL). The residue was dissolved in MeOH (300 mL)
and transferred to a 2 liter stainless steel pressure vessel. MeOH
(400 mL) saturated with NH.sub.3 gas was added and the vessel
heated to 100.degree. C. for 2 hours (tlc showed complete
conversion). The vessel contents were evaporated to dryness and the
residue was dissolved in EtOAc (500 mL) and washed once with
saturated NaCl (200 mL). The organics were dried over sodium
sulfate and the solvent was evaporated to give 85 g (95%) of the
title compound.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0085] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85
g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride
(37.2 g, 0.165 M) was added with stirring. After stirring for 3
hours, tlc showed the reaction to be approximately 95% complete.
The solvent was evaporated and the residue azeotroped with MeOH
(200 mL). The residue was dissolved in CHCl.sub.3 (700 mL) and
extracted with saturated NaHCO.sub.3 (2.times.300 mL) and saturated
NaCl (2.times.300 mL), dried over MgSO.sub.4 and evaporated to give
a residue (96 g). The residue was chromatographed on a 1.5 kg
silica column using EtOAc/Hexane (1:1) containing 0.5% Et.sub.3NH
as the eluting solvent. The pure product fractions were evaporated
to give 90 g (90%) of the title compound.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amid-
ite
[0086]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
(74 g, 0.10 M) was dissolved in CH.sub.2Cl.sub.2 (1 L). Tetrazole
diisopropylamine (7.1 g) and
2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were
added with stirring, under a nitrogen atmosphere. The resulting
mixture was stirred for 20 hours at room temperature (tlc showed
the reaction to be 95% complete). The reaction mixture was
extracted with saturated NaHCO.sub.3 (1.times.300 mL) and saturated
NaCl (3.times.300 mL). The aqueous washes were back-extracted with
CH.sub.2Cl.sub.2 (300 mL), and the extracts were combined, dried
over MgSO.sub.4 and concentrated. The residue obtained was
chromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1)
as the eluting solvent. The pure fractions were combined to give
90.6 g (87%) of the title compound.
Example 2
[0087] Oligonucleotide Synthesis
[0088] Unsubstituted and substituted phosphodiester (P--O)
oligonucleotides are synthesized on an automated DNA synthesizer
(Applied Biosystems model 380B) using standard phosphoramidite
chemistry with oxidation by iodine.
[0089] Phosphorothioates (P--S) are synthesized as per the
phosphodiester oligonucleotides except the standard oxidation
bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one
1,1-dioxide in acetonitrile for the stepwise thiation of the
phosphite linkages. The thiation wait step was increased to 68
seconds and was followed by the capping step. After cleavage from
the CPG column and deblocking in concentrated ammonium hydroxide at
55.degree. C. (18 hr), the oligonucleotides were purified by
precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl
solution.
[0090] Phosphinate oligonucleotides are prepared as described in
U.S. Pat. No. 5,508,270, herein incorporated by reference.
[0091] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0092] 3-Deoxy-3-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050,
herein incorporated by reference. Phosphoramidite oligonucleotides
are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat.
No. 5,366,878, herein incorporated by reference.
[0093] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications PCT/US94/00902 and
PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively), herein incorporated by reference.
[0094] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0095] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0096] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated
by reference.
Example 3
[0097] Oligonucleoside Synthesis
[0098] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides, methylenedimethylhydrazo
linked oligonucleosides, also identified as MDH linked
oligonucleosides, 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--O or P--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.
[0099] 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.
[0100] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 4
[0101] PNA Synthesis
[0102] PNA oligomers were synthesized in a 10 .mu.mol scale on a
433A Peptide Synthesizer (ABI, Perkin-Elmer Corp.) using
commercially available Boc/Cbz-protected monomers (Perseptive
Biosystems, Perkin-Elmer Corp). The coupling reaction was performed
using 7 eqv. (70 .mu.mol) monomer (0.25 M in DMF), 6.8 eqv. (68
.mu.mol) O-(7-azabenzotriazol-1-yl)- -1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU, 0.223 M in DMF) as the condensing
reagent and a coupling time of 10 min. The coupling efficiency was
monitored qualitatively and the coupling step was repeated if the
test indicated yields below 99-100% using the following conditions:
To increase the concentration of activated monomer, HATU (68
.mu.mol, 25.9 mg) was added to the monomer solution (70 .mu.mol,
ca. 150 .mu.l) as a solid. The synthesis cycle was continued adding
DIEA (140 .mu.mol, 0.5 M in pyridine), pre-activation of the
monomer for 1 min, and a coupling time of 40 min. After cleavage
and deprotection the PNA oligomers were purified by RP-HPLC using a
306 Piston Pump System, a 811C Dynamic Mixer, a 170 Diode Array
Detector and a 215 Liquid Handler from Gilson (Middleton, Wis.).
The system was operated with Unipoint 1.8 Software. The HPLC
conditions were as follows: Column: Zorbax SB-C18 (250.times.7.8
mm, 5.mu., 300 A); column temperature: 55.degree. C.; Solvent A:
0.1% TFA in H.sub.2O; Solvent B: CH.sub.3CN/H.sub.2O (80:20);
Gradient: 0-40 min 0-40% B. After chromatographic purification the
oligomers were lyophilized and stored at -20.degree. C.
[0103] Peptide nucleic acids (PNAs), including conjugation of amino
acids to PNAs, can be prepared in accordance with any of the
various procedures referred to in Peptide Nucleic Acids (PNA):
Synthesis, Properties and Potential Applications, Bioorganic &
Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in
accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262,
herein incorporated by reference.
Example 5
[0104] Synthesis of Chimeric Oligonucleotides
[0105] 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
[0106] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 380B, 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 increasing
the wait step after the delivery of tetrazole and base to 600 s
repeated four times for RNA and twice for 2'-O-methyl. The fully
protected oligonucleotide is cleaved from the support and the
phosphate group is deprotected in 3:1 Ammonia/Ethanol at room
temperature overnight then lyophilized to dryness. Treatment in
methanolic ammonia for 24 hours at room temperature is then done to
deprotect all bases and sample was again lyophilized to dryness.
The pellet is resuspended in 1M TBAF in THF for 24 hours at room
temperature to deprotect the 2' positions. The reaction is then
quenched with 1M TEAA and the sample is then reduced to 1/2 volume
by rotovac before being desalted on a G25 size exclusion column.
The oligo recovered is then analyzed spectrophotometrically for
yield and for purity by capillary electrophoresis and by mass
spectrometry.
[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(2-Methoxyethyl)] Chimeric
Phosphorothioate Oligonucleotides
[0107] [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
[0108] [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,
oxidization with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0109] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 6
[0110] Oligonucleotide Isolation
[0111] After cleavage from the controlled pore glass column
(Applied Biosystems) and deblocking in concentrated ammonium
hydroxide at 55.degree. C. for 18 hours, the oligonucleotides or
oligonucleosides were purified by precipitation twice out of 0.5 M
NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were
analyzed by polyacrylamide gel electrophoresis on denaturing gels
and judged to be at least 85% full length material. The relative
amounts of phosphorothioate and phosphodiester linkages obtained in
synthesis were periodically checked by .sup.31P nuclear magnetic
resonance spectroscopy, and 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
[0112] Analysis of Oligonucleotide Inhibition of IL-5 or IL-5Ra
Expression
[0113] Antisense modulation of IL-5 or IL-5Ra expression can be
assayed in a variety of ways known in the art. For example, IL-5 or
IL-5Ra mRNA levels can be quantitated by Northern blot analysis,
RNAse protection assay (RPA), competitive polymerase chain reaction
(PCR), or real-time PCR (RT-PCR). RNA analysis can be performed on
total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are
taught in, for example, Ausubel, et al., Current Protocols in
Molecular Biology, Volume 1, John Wiley & Sons, Inc., 1993, pp.
4.1.1-4.2.9 and 4.5.1-4.5.3. Northern blot analysis is routine in
the art and is taught in, for example, Ausubel, et al., Current
Protocols in Molecular Biology, Volume 1, John Wiley & Sons,
Inc., 1996, pp. 4.2.1-4.2.9. Real-time quantitative (PCR) can be
conveniently accomplished using the commercially available ABI
PRISMJ 7700 Sequence Detection System, available from PE-Applied
Biosystems, Foster City, Calif. and used according to
manufacturer's instructions. Other methods of PCR are also known in
the art.
[0114] IL-5 or IL-5Ra protein levels can be quantitated in a
variety of ways well known in the art, such as immunoprecipitation,
Western blot analysis (immunoblotting), ELISA, flow cytometry or
fluorescence-activated cell sorting (FACS). Antibodies directed to
IL-5 or IL-5Ra can be identified and obtained from a variety of
sources, such as PharMingen Inc., San Diego Calif., or can be
prepared via conventional antibody generation methods. Methods for
preparation of polyclonal antisera are taught in, for example,
Ausubel, et al., Current Protocols in Molecular Biology, Volume 2,
John Wiley & Sons, Inc., 1997, pp. 11.12.1-11.12.9. Preparation
of monoclonal antibodies is taught in, for example, Ausubel, et
al., Current Protocols in Molecular Biology, Volume 2, John Wiley
& Sons, Inc., 1997, pp. 11.4.1-11.11.5.
[0115] Immunoprecipitation methods are standard in the art and can
be found at, for example, Ausubel, et al., Current Protocols in
Molecular Biology, Volume 2, John Wiley & Sons, Inc., 1998, pp.
10.16.1-10.16.11. Western blot (immunoblot) analysis is standard in
the art and can be found at, for example, Ausubel, et al., Current
Protocols in Molecular Biology, Volume 2, John Wiley & Sons,
Inc., 1997, pp. 10.8.1-10.8.21. Enzyme-linked immunosorbent assays
(ELISA) are standard in the art and can be found at, for example,
Ausubel, et al., Current Protocols in Molecular Biology, Volume 2,
John Wiley & Sons, Inc., 1991, pp. 11.2.1-11.2.22.
Example 8
[0116] Poly(A)+ mRNA Isolation
[0117] Poly(A)+ mRNA is isolated according to Miura et al., Clin.
Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA
isolation are taught in, for example, Ausubel, et al., Current
Protocols in Molecular Biology, Volume 1, John Wiley & Sons,
Inc., 1993, pp. 4.5.1-4.5.3. Briefly, for cells grown on 96-well
plates, growth medium is removed from the cells and each well is
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) is added to each well, the plate is
gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate is transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates are 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 is 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.
is added to each well, the plate is incubated on a 90.degree. C.
hot plate for 5 minutes, and the eluate is then transferred to a
fresh 96-well plate.
[0118] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 9
[0119] Total RNA Isolation
[0120] Total mRNA is isolated using an RNEASY 96J kit and buffers
purchased from Qiagen Inc. (Valencia Calif.) following the
manufacturer's recommended procedures. The kit can be used with
cells grown on a variety of sizes of plate or bottle, including
96-well plates. Briefly, for cells grown on 96-well plates, growth
medium is removed from the cells and each well is washed with 200
.mu.L cold PBS. 100 .mu.L Buffer RLT is added to each well and the
plate vigorously agitated for 20 seconds. 100 .mu.L of 70% ethanol
is then added to each well and the contents mixed by pipetting
three times up and down. The samples are then transferred to the
RNEASY 96J well plate attached to a QIAVACJ manifold fitted with a
waste collection tray and attached to a vacuum source. Vacuum is
applied for 15 seconds. 1 mL of Buffer RW1 is added to each well of
the RNEASY 96J plate and the vacuum again applied for 15 seconds. 1
mL of Buffer RPE is then added to each well of the RNEASY 96J plate
and the vacuum applied for a period of 15 seconds. The Buffer RPE
wash is then repeated and the vacuum is applied for an additional
10 minutes. The plate is then removed from the QIAVACJ manifold and
blotted dry on paper towels. The plate is then re-attached to the
QIAVACJ manifold fitted with a collection tube rack containing 1.2
mL collection tubes. RNA is then eluted by pipetting 60 .mu.L water
into each well, incubating 1 minute, and then applying the vacuum
for 30 seconds. The elution step is repeated with an additional 60
.mu.L water.
MOUSE IL-5
Example 10
[0121] Antisense Inhibition of Murine IL-5 Expression
[0122] In accordance with the present invention, a series of
antisense oligonucleotides were designed to target different
regions of murine IL-5 RNA, using published sequences (Genbank
Accession No. X06271 incorporated herein as SEQ ID NO: 1). The
oligonucleotides are shown in Table 1. Target sites are indicated
by nucleotide numbers, as given in the sequence source reference
(Genbank Accession No. X06271) to which the oligonucleotide 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
(shown in bold) are composed of 2'-O-methoxyethyl (2'-MOE)
nucleotides. The internucleoside (backbone) linkages are
phosphorothioate (P--S) throughout the oligonucleotide. Cytidine
residues in the 2'-MOE regions are 5-methylcytidines but cytidines
in the 2'-deoxy regions are unmodified unless otherwise
indicated.
1TABLE 1 Murine IL-5 Antisense Oligonucleotides SEQ ISIS NUCLEOTIDE
SEQUENCE.sup.1 ID TARGET TARGET NO. (5' -> 3') NO: SITE.sup.2
REGION 16975 CCCAAGCAATTTATTCTCTC 2 510-529 5' UTR 16976
TCAGCAAAGGAAGAGCGCAG 3 544-563 Coding 16977 CACTGTGCTCATGGGAATCT 4
654-673 Coding 16978 ACTTTACCTCATTGCTTGTC 5 718-737 Coding 16979
TCAGAGCGGTATAGCAAGGT 6 774-793 Coding 16980 CTCATCGTCTGCAAAGGAAA 7
1548- Coding 1567 16981 TATGAGTAGGGACAGGAAGC 8 1568- Coding 1587
16982 ATTTTTATGAGTAGGGACAG 9 1573- Coding 1592 16983
AGCACGGCAGTAAAGAATAA 10 1598- Coding 1617 16984
ACAAGGAAAACAAAGAGAGG 11 2380- Coding 2399 16985
CTGGTGCTGAAAGAAGATTA 12 3454- Coding 3473 16986
CCACGGACAGTTTGATCCTT 13 3513- Coding 3532 16987
AATGACAGGTTTTGGAATAG 14 3549- Coding 3568 16988
GCGGTCAATGTATTTCTTTA 15 3571- Coding 3590 16989
GGAACTTACTTTTTGGCGGT 16 3586- Coding 3605 16990
CAGACTGTCAGGTTGGCTCC 17 3644- Coding 3663 16991
TCCTCGCCACACTTCTCCTG 18 3673- Coding 3692 16992
AACTGCCTCGTCCTCCGTCT 19 3694- Coding 3713 16993
TACTCATCACACCAAGGAAC 20 3732- Coding 3751 16994
CTCAGCCTCAGCCTTCCATT 21 3762- Stop 3781 16995 TTAAATTGTGAAGTCCTGTC
22 3794- 3'-UTR 3813 16996 AAATATAAATGGAAACAGCA 23 3874- 3'-UTR
3893 16997 CTACAGGACATAAATATAAA 24 3885- 3'-UTR 3904 16998
TATACAAAAAGGTTAAACAC 25 3938- 3'-UTR 3957 16999
GGTTATCCTTGGCTACATTA 26 4001- 3'-UTR 4020 .sup.1All linkages are
phosphorothioate linkages. Residues shown in bold are
2'-methoxyethoxy, remaining residues are 2'-deoxy. All
2'-methoxyethoxy C residues are also 5-methyl C. .sup.2Nucleotide
numbers from Genbank Accession No. X06271, SEQ ID NO.1 to which the
oligonucleotide is targeted.
[0123] Oligonucleotides were tested in EL-4 T cells (ATCC TIB-39,
American Type Culture Collection, Manassas, Va.) by Northern blot
analysis as described in previous examples using a commercially
available murine IL-5 probe. These cells are PHA responsive and PMA
plus cAMP elevating agents induce a several hundredfold increase in
IL-5 synthesis by these cells. Cells were maintained and stimulated
to express IL-5 according to published methods and transfected with
oligonucleotide via electroporation.
[0124] Oligonucleotides were tested at a concentration of 10 .mu.M.
The results are shown in Table 2:
2TABLE 2 Effect of Antisense Oligonucleotides on Murine IL-5 InRNA
Levels ISIS SEQ NO. ID NO: TARGET REGION % CONTROL % INHIB 16975 2
5' UTR 89.4 10.6 16976 3 coding 93.2 6.8 16977 4 Coding 107.8 --
16978 5 Coding 95 5 16979 6 Coding 96.9 3.1 16980 7 Coding 91 9
16981 8 Coding 55.8 44.2 16982 9 Coding 60 40 16983 10 Coding 67.6
32.4 16984 11 Coding 73.2 26.8 16985 12 Coding 71.6 28.4 16986 13
Coding 74.2 25.8 16987 14 Coding 104 -- 16988 15 Coding 98.8 1.2
16989 16 Coding 107 -- 16990 17 Coding 148 -- 16991 18 Coding 107
-- 16992 19 coding 70 30 16993 20 coding 78.1 21.9 16994 21 Stop
79.4 20.6 16995 22 3'-UTR 95.7 4.3 16996 23 3'-UTR 113 -- 16997 24
3'-UTR 122 -- 16998 25 3'-UTR 110 -- 16999 26 3'-UTR 68.1 31.9 SEQ
ID NO 8, 9, 10, 19 and 26 (ISIS 16981, 16982, 16983, 16992 and
16999, respectively) showed at least 30% inhibition of IL-5
expression in this assay and are therefore preferred.
Example 11
[0125] Dose Response Comparison of ISIS 16992 and 16999 for
Reduction of Murine IL-5 mRNA Levels
[0126] ISIS 16992 and 16999 (SEQ ID NO: 19 and 26, respectively)
were screened at concentrations of 5 to 25 .mu.M in EL-4 T cells
for the ability to decrease IL-5 mRNA levels. Oligonucleotides were
introduced to cells by electroporation and mRNA levels were
measured by Northern blot analysis.
[0127] An IC50 (oligonucleotide concentration at which mRNA was
decreased by 50% compared to control) of approximately 15 .mu.M was
obtained for ISIS 16992 and approximately 18 .mu.M for ISIS
16999.
[0128] ISIS 16999 was compared to 1, 3, and 5-mismatch control
sequences (ISIS Nos 17983, 17984 and 17985; SEQ ID Nos: 30, 31 and
32, respectively) in dose-response measurements of IL-5 mRNA levels
after oligonucleotide treatment. In this experiment ISIS 16999 had
an IC50 of approximately 9 .mu.M and ISIS 17983, the 1-base
mismatch control, had an IC50 of approximately 13 .mu.M. IC50s were
not obtainable for the 3- and 5-base mismatch controls which
reduced IL-5 mRNA levels only by 8% and 17%, respectively.
Example 12
[0129] Dose Response Comparison of ISIS 16992 and 16999 for
Reduction of Murine IL-5 Protein Levels
[0130] ISIS 16992 and 16999 (SEQ ID NO: 19 and 26, respectively)
were screened at concentrations of 5 to 25 .mu.M in EL-4 T cells
for the ability to decrease IL-5 protein levels. Oligonucleotides
were introduced to cells by electroporation and protein levels were
measured by ELISA assay using a murine IL-5 ELISA kit (Endogen,
Woburn, Mass.). Starting IL-5 concentrations in the absence of
oligonucleotide were approximately 2300 pg/ml and this was
decreased to approximately 200 pg/ml at 25 .mu.M ISIS 16992 and 400
pg/ml at 25 .mu.M ISIS 16999. An IC50 of approximately 13 .mu.M was
obtained for ISIS 16992 and approximately 15 .mu.M for ISIS
16999.
Example 13
[0131] Effect of ISIS 16999 on IL-5 Secretion by EL-4 Cells
[0132] EL-4 cells were treated with ISIS 16999 at doses from 5 to
20 .mu.M as described in previous examples. Secreted IL-5 in the
medium was detected by ELISA assay as in previous examples.
[0133] Secreted IL-5 levels were reduced by 13.5-fold as
oligonucleotide concentration was increased from zero to 10 .mu.M.
ISIS 16989, which did not reduce IL-5 mRNA levels (see Table 2
above), showed much lesser reduction (approximately 2.5-fold) in
secreted IL-5 levels. IL-5 levels stayed low for at least 72 hours
after treatment with ISIS 16999.
Example 14
[0134] Optimization of Antisense Inhibition of Murine IL-5
Expression
[0135] An additional series of oligonucleotides targeted to murine
IL-5 was synthesized. The oligonucleotide sequences are those
previously tested but with modified gap placement. Sequences are
shown in Table 3. Target sites in this table refer back to the ISIS
number of the parent compound of the same sequence shown in
previous tables.
3TABLE 3 Optimization of Antisense Modulation of Murine IL-5
Expression SEQ ISIS NUCLEOTIDE SEQUENCE.sup.1 ID TARGET NO. (5'
-> 3') NO: SITE.sup.2 CHEMISTRY 17858 TATGAGTAGGGACAGGAAGC 8
ISIS P-S; 2'- 16981 MOE 17859 TATGAGTAGGGACAGGAAGC 8 ISIS P-S; 2'-
16981 MOE /2'- deoxy 17860 TATGAGTAGGGACAGGAAGC 8 ISIS P-S; 2'-
16981 MOE /2'- deoxy 17861 TATGAGTAGGGACAGGAAGC 8 ISIS P-S; 2'-
16981 MOE /2'- deoxy 17862 TATGAGTAGGGACAGGAAGC 8 ISIS P-S; 2'-
16981 MOE /2'- deoxy 17863 AACTGCCTCGTCCTCCGTCT 19 ISIS P-S; 2'-
16992 MOE 17864 AACTGCCTCGTCCTCCGTCT 19 ISIS P-S; 2'- 16992 MOE
/2'- deoxy 17865 AACTGCCTCGTCCTCCGTCT 19 ISIS P-S; 2'- 16992 MOE
/2'- deoxy 17866 AACTGCCTCGTCCTCCGTCT 19 ISIS P-S; 2'- 16992 MOE
/2'- deoxy 17867 AACTGCCTCGTCCTCCGTCT 19 ISIS P-S; 2'- 16992 MOE
/2'- deoxy 17868 GGTTATCCTTGGCTACATTA 26 ISIS P-S; 2'- 16999 MOE
17869 GGTTATCCTTGGCTACATTA 26 ISIS P-S; 2'- 16999 MOE /2'- deoxy
17870 GGTTATCCTTGGCTACATTA 26 ISIS P-S; 2'- 16999 MOE /2'- deoxy
17871 GGTTATCCTTGGCTACATTA 26 ISIS P-S; 2'- 16999 MOE /2'- deoxy
17872 GGTTATCCTTGGCTACATTA 26 ISIS P-S; 2'- 16999 MOE /2'- deoxy
17980 AACTGCCTCCTCCTCCGTCT 27 ISIS P-S; 2'- 16992 1 MOE /2'-
mismatch deoxy; 17981 AACTGCCACCTGCTCCGTCT 28 ISIS P-S; 2'- 16992 3
MOE /2'- mismatch deoxy; 17982 AACTGGCACCTGCACCGTCT 29 ISIS P-S;
2'- 16992 5 MOE /2'- mismatch deoxy; 17983 GGTTATCCTAGGCTACATTA 30
ISIS P-S; 2'- 16999 1 MOE /2'- mismatch deoxy; 17984
GGTTATCGTAGCCTACATTA 31 ISIS P-S; 2'- 16999 3 MOE /2'- mismatch
deoxy; 17985 GGTTAACGTAGCCAACATTA 32 ISIS P-S; 2'- 16999 5 MOE /2'-
mismatch deoxy; 17994 AACTGCCTCCTCCTCCGTCT 19 ISIS P-S; 2'- 16992
deoxy 17995 GGTTATCGTAGCCTACATTA 26 ISIS P-S; 2'- 16999 deoxy 18242
GGTTATCCTTGGCTACATTA 26 ISIS PS; 2'- 16999 MOE /2'- deoxy; All C-
5meC 18243 GGTTATCCTTGGCTACATTA 26 ISIS PS; 2'- 16999 MOE /2'-
deoxy; All C- 5meC 18244 AACTGCCTCGTCCTCCGTCT 19 ISIS PS; 2'- 16992
MOE /2'- deoxy; All C- 5me C 13245 AACTGCCTCGTCCTCCGTCT 19 ISIS PS;
2'- 16992 MOE 2'- deoxy; All C- Sine C 18246 TATGAGTAGGGACAGGAAGC 8
ISIS PS; 2'- 16981 MOE /2' deoxy; All C- 5me C 18247
TATGAGTAGGGACAGGAAGC 8 ISIS PS; 2'- 16981 MOE /2'- deoxy; All C-
5meC 20391 GGTTATCCTTGGCTACATTA 26 ISIS PS; 2'- 16999 MOE /2'
deoxy; All C- Sine C 20392 GGTTATCCTTGGCTACATTA 26 ISIS 2'-MOE,
16999 P-O/2'- deoxy/P- 5; All C- 5meC 20393 GCTTAACGTAGCCAACATTA 32
ISIS PS; 2'- 16999 5 MOE /2'- mismatch deoxy; All c- 5meC; 20394
GGTTAACGTAGCCAACATTA 32 ISIS 2'-MOE, 16999 5 P-O/2'- mismatch
deoxy/P- 5; All C- 5meC; 20564 GGTTATCCTTGGCTACATTA 26 ISIS P-O;
2'- 16999 MOE /2'- deoxy; All C- 5meC; 21437 GGTTATCCTTGGCTACATTA
26 ISIS P-S; 2'- 16999 MOE /2'- deoxy; 5'FITC 21882
GGTTATCCTTGGCTACATTA 26 ISIS P-O; 2'- 16999 MOE /2'- deoxy; All C-
5meC; 21966 AACTGCCTCGTCCTCCGTCT 19 ISIS 2'-MOE, 16992 P-O/2'-
deoxy/P- 5; All C- 5meC; 21967 AACTGCCTCGTCCTCCGTCT 19 ISIS PS; 2'-
16992 MOE /2'- deoxy; All C- 5meC 21968 AACTGCCTCGTCCTCCGTCT 19
ISIS P-C; 2'- 16992 MOE /2'- deoxy; All C- 5 meC 21970
GGTTAACGTAGCCAACATTA 32 ISIS P-C; 2'- 16999 5 MOE /2'- mismatch
deoxy; All C- 5meC; 22087 AACTGGCACCTGCACCGTCT 29 ISIS 2'-MOE,
16992 5 P-O/2'- mismatch deoxy/P 5; All C- 5meC; 22088
AACTGGCACCTGCACCGTCT 29 ISIS P-C; 2'- 16992 5 MOE /2'- mismatch
deoxy; All C- 5meC; 24232 AACTGGCACCTGCACCGTCT 29 ISIS PS; 2'-
16992 5 MOE /2'- mismatch deoxy; All C 5meC; .sup.1Ernboldened
residues, 2'-methoxyethoxy- residues (others are 2-deoxy-) . Unless
otherwise indicated, 2'-MOE C residues are 5'-methyl-C (5meC) and
2'-deoxy C residues are unmodified. .sup.2Target sites in this
table refer back to the ISIS number of the compound of the same
sequence shown in previous tables.
[0136] ISIS 17868, 17869, 17860, 18242 and 18243, all gap variants
of ISIS 16999 (SEQ ID NO: 26), were tested and compared to the
parent oligonucleotide, ISIS 16999 for ability to reduce IL-5 mRNA
levels in EL-4 cells. In a screen at 15 .mu.M oligonucleotide
concentration (the IC50 for ISIS 16999), ISIS 18243 gave comparable
activity to ISIS 16999. ISIS 17870 and 18242 were slightly less
active, ISIS 17869 showed modest activity and ISIS 17868 was
virtually inactive. In a subsequent dose-response assay, ISIS 17870
and 18243 showed activity comparable to or slightly better than
that of ISIS 16999.
[0137] ISIS 17858, 17859, 17860, 18246 and 18247, all gap variants
of ISIS 16981 (SEQ ID NO: 8), were tested and compared to the
parent oligonucleotide, ISIS 16981, for ability to reduce IL-5 mRNA
levels in EL-4 cells. In a screen at 15 .mu.M oligonucleotide
concentration, ISIS 17859 and 18246 showed activity comparable to
the parent, ISIS 16981, with ISIS 18247 only slightly less active.
ISIS 17858 and 17860 were more active than the parent compound. All
of the ISIS 16981 gap variants tested are therefore preferred.
[0138] ISIS 17863, 17864, 17865, 18244 and 18245, all gap variants
of ISIS 16992 (SEQ ID NO: 19), were tested and compared to the
parent oligonucleotide, ISIS 16992. In a screen at 15 .mu.M
oligonucleotide concentration, ISIS 18245 showed activity only
slightly (approx 20%) less than the parent compound. ISIS 17863 and
18244 were modestly active and ISIS 17864 and 17865 were nearly
inactive. Thus ISIS 18245 is also preferred.
[0139] ISIS 16999 was also compared to ISIS 20391, a compound of
the same sequence, backbone and gap placement but with 5-methyl
cytosines in place of every cytosine (in both the deoxy gap and the
2'-methoxyethoxy regions), and to ISIS 20392, which was identical
to ISIS 20391 except the backbone was phosphodiester (P--O) in the
2' methoxyethoxy regions and phosphorothioate (P--S) in the deoxy
gap. Oligos were compared at doses of 5, 15 and 25 .mu.M for
ability to reduce IL-5 mRNA levels in EL-4 cells. Both ISIS 20391
and 20392 showed roughly comparable activity to ISIS 16999, with
20392 slightly more active than the parent. Both of these compounds
are therefore preferred. 5-base mismatches of both ISIS 20391 and
20392 were inactive at all concentrations. ISIS 20564, a full
phosphodiester compound, was virtually inactive at these
concentrations in a separate experiment.
Example 15
[0140] Effect of IL-5 Antisense Oligonucleotide ISIS 20391 on in
vivo T Cell IL-5 mRNA Expression
[0141] IL-5 mRNA expression was measured in EL-4 T cells by
real-time quantitative PCR using the TaqMan system on a
Perkin-Elmer ABI PRISM 7700. Relative IL-5 levels were normalized
to GAPDH levels. The primer and probe sequences were as
follows:
[0142] murine IL5:
[0143] Probe: 5'-6-FAM DYE-AG TGT TCT GAC TCT CAG CTG TGT CTG
GGC-TAMRA DYE-3' (SEQ ID NO: 33)
[0144] Sense: 5'-TTC AGA GTC ATG AGA AGG ATG CTT-3' (SEQ ID
NO:34)
[0145] Antisense: 5' ACC ACT GTG CTC ATG GGA ATC T-3' (SEQ ID NO:
35) GAPDH:
[0146] Probe:5'-6-FAM DYE-AAG GCC GAG AAT GGG AAG CTT GTC ATC-TAMRA
DYE-3' (SEQ ID NO: 36)
[0147] Sense: 5'-GGC AAA TTC AAC GGC ACA GT-3' (SEQ ID NO: 37)
[0148] Antisense: 5'-GGG TCT CGC TCC TGG AAG AT-3' (SEQ ID NO:
38).
[0149] ISIS 20391 reduced IL-5 mRNA levels by 75% compared to
ovalbumin-induced IL-5 levels, whereas the mismatch oligonucleotide
ISIS 20393 reduced IL-5 mRNA by only 40%.
Example 16
[0150] Effect of ISIS 20391 (Targeted to Murine IL-5) on
Ovalbumin-induced Peritonitis in Balb/c Mice
[0151] An eosinophil peroxidase (EPO) calorimetric assay was used
to measure the effect of oligonucleotides on eosinophilia in
peritoneal lavage fluid after ovalbumin immunization and challenge.
The method used is a modification of Strath et al., J. Immunol.
Meth., 1985, 83, 209-215. Briefly, the substrate solution consists
of 0.05 M o-phenylenediamine dihydrochloride (OPD, Sigma Chem. Co.,
St. Louis, Mo.) in 0.05 M Tris buffer containing 1 mM hydrogen
peroxide and 0.1% Triton X-100. Reaction mixture is added to cells,
incubated in the dark for 30 minutes and the reaction was stopped
by addition of 1/4 volume of 4 M sulfuric acid. The EPO was
measured as the absorbance at 492 nm, blanked against substrate
solution. Using this assay, EPO levels are proportional to number
of eosinophils present. Mice were dosed chronically with
oligonucleotides. Ovalbumin challenge increased EPO levels in
peritoneal lavage fluid over sixteenfold. ISIS 20391 dosed
chronically at 5 mg/kg reduced EPO levels after ovalbumin induction
by 47%. The mismatch control reduced EPO by approximately
12.6%.
[0152] A dose-dependent reduction of EPO by ISIS 20391 was
obtained, with approximately 75% reduction at 10 mg/kg
oligonucleotide dose compared to 29% reduction by the mismatch
control. The IL-5 oligonucleotide correspondingly reduced
eosinophil infiltration into the peritoneal cavity by 86% compared
to the ovalbumin challenge control, while the mismatch only reduced
infiltration by 26%. Using chronic subcutaneous administration (5
mg/kg/day for 15 days using implanted minipumps) a slight but
reproducible inhibitory effect of the IL-5 oligonucleotide on
eosinophilia in an ovalbumin lung challenge model has also been
obtained.
Example 17
[0153] Reduction of IL-5 Protein in Peritoneal Lavage Fluid by ISIS
20391 Following 7 Day Dosing Schedule
[0154] Mice were dosed daily with ISIS 20391 at 5 or 20 mg/kg for 7
days. Following peritoneal lavage, IL-5 protein levels were
measured using an ELISA assay. IL-5 levels in ovalbumin-treated
mice were approximately 160 pg/ml. Treatment with ISIS 20391 at 5
and 20 mg/kg reduced IL-5 concentrations in peritoneal fluid to 110
and 80 pg/ml, respectively. A control oligonucleotide at 5 and 20
mg/kg reduced IL-5 levels to 160 and 130 pg/ml.
Example 18
[0155] Effect of IL-5 Antisense Oligonucleotide on
Ovalbumin-induced Murine Lung Asthma Model
[0156] Airway inflammation is observed in patients with allergic
asthma. A murine model of allergic asthma has been developed,
(Hessel et al. J. Immunol. 1998, 160, 2998-3005). Sensitization of
BALB/c mice with ovalbumin induces a high level of
ovalbumin-specific IgE in serum. Inhalation of ovalbumin in
sensitized mice causes an immediate bronchoconstrictive response.
Repeated inhalation of ovalbumin in sensitized animals induces
nonspecific airway hyperresponsiveness in vivo, and infiltration of
leukocytes in airway tissue.
[0157] Pathogen-free male BALB/c ByJ mice were obtained from
Jackson Laboratories. Active sensitization is performed by IP
injection of 20 ?g of ovalbumin (Sigma Chemical Co, St. Louis, Mo.,
grade II) in aluminum hydroxide adjuvant on days 2 and 9 of 16 days
of daily oligonucleotide treatment. This produces high titers of
total IgE in mouse serum of which 80% is ovalbumin-specific IgE
(Hessel et al., J. Immunol., 1998, 160, 2998-3005). On day 16 of
treatment, mice are exposed either 2% ovalbumin aerosol for 1
minute. The aerosol is generated with a nebulizer such as Medix
8001 (Sussex, UK). Oligonucleotides were dissolved in saline and
injected daily i.v. in the tail vein by bolus infusion at the
indicated doses from 2 days before antigen sensitization through
challenge.
[0158] Bronchoalveolar lavage (BAL) is used to measure the
leukocyte infiltration of airway tissue. 24 hours after the
ovalbumin aerosol, mice were euthanized, tracheal cannulation was
performed and saline washes collected. Percent eosinophils in BAL
were determined.
[0159] Unsensitized mice had 1.6% eosinophils in BAL fluid; after
ovalbumin sensitization this increased to 37.6%. ISIS 20391 at 5,
10 and 20 mg/kg reduced eosinophilia in BAL to 11.8%, 5.5% and
3.8%, respectively. The latter two are statistically significant
reductions. Mismatch control oligonucleotide ISIS 20393 at 10 and
20 mg/kg yielded BAL eosinophil counts of 33.6% and 28.4%,
respectively. The positive control, dexamethasone, reduced
eosinophil counts to 5.8%.
[0160] Airway responsiveness to methacholine is measured in vivo 24
hours after the last aerosol exposure. Baseline nebulized
methacholine dose response curves were constructed at day 0 before
antigen sensitization for all groups of animals. Pulmonary function
was monitored using a Buxco BioSystem Plethysmograph (Buxco, Troy
N.Y.) and expressed as enhanced pause (Penh) which correlates to
measured airway resistance (Hamelmann et al., Am. J.Respir. Crit.
Care Med., 1997, 156, 766-775). Following challenge with
aerosolized albumin, pulmonary function recordings were performed
for 30 minutes to examine the early phase allergic response. For
the late phase reaction, recordings were performed every hour from
2 hours to 9 hours after ovalbumin challenge. Airway responsiveness
was measured at 24 hours after antigen challenge by measuring the
airway response to methacholine for 3 minutes at each dose.
Post-challenge recordings were compared to baseline recordings for
each group to generate a Penh stimulation index. As a positive
control, dexamethasone was administered i.p., 25 mg/kg, 1 day
before the sensitization , 2 hours before the challenge, and 18
hours after the challenge.
[0161] Plethysmography results showed that ISIS 20391 at 10 or 20
mg/kg inhibited the methacholine-induced allergic airway
hyperresponsiveness, reducing the peak Penh index from
approximately 2.0 (no oligo) to approximately 1.25 after
oligonucleotide treatment in several experiments. Dexamethasone,
the positive control, reduced the Penh to approximately 1.0.
[0162] Data from one experiment was expressed another way, in terms
of PC100, (provocation challenge.sub.100) the concentration of
methacholine needed to give a twofold increase in airway hyper
reactivity. Unsensitized mice had a PC100 of 40.1 mg/ml
methacholine. After ovalbumin sensitization, the PC100 was 9.84,
indicating that much lower doses of methacholine caused the same
increase in airway reactivity. This effect was reversible in part
by ISIS 20391. At 5 mg/kg ISIS 20391 the PC100 was 10.6, but at 10
and 20 mg/kg the PC100 was increased to 30.7 and 41.6 mg/kg showing
a reverse in airway hyper reactivity. Dexamethasone had a PC100 of
29.8 mg/kg methacholine.
Example 19
[0163] Early and Late Phase Allergic Airway Response in Mouse Whole
Body Plethysmography Model
[0164] Ovalbumin challenge produces a two-phased response with
separate and distinct peaks in airway hyper reactivity at
approximately 2 minutes and approximately 2 hours after ovalbumin
challenge. The first peak is about a twofold increase in Penh and
the second peak is larger, a three- to four-fold increase in Penh.
The late phase response was mitigated by ISIS 20391 at doses of 10
and 20 mg/kg. In particular, the late response, in which Penh
reaches approximately 0.7 two hours after ovalbumin challenge
(compared to 0.25 for unsensitized mice) was reduced by ISIS 20391
at 10 mg/kg to a Penh of approximately 0.4, which was a
statistically significant reduction. Dexamethasone reduced the Penh
to approximately 0.3. The mismatch control, ISIS 20393 at 10 mg/kg
showed a statistically insignificant reduction of late phase Penh
to approximately 0.5. In a higher-dose experiment, ISIS 20391 at 20
mg/kg reduced the Penh 2 hours after ovalbumin challenge from 0.7
to 0.425, which was statistically significant. Mismatch control
ISIS 20393 at 20 mg/kg reduced Penh to approximately 0.6 which was
not significant, and dexamethasone (positive control) reduced the
response to approximately 0.25.
Human IL-5
Example 20
[0165] Human IL-5 Antisense Oligonucleotides
[0166] A series of antisense compounds were designed to target mRNA
encoding human IL-5. These compounds are shown in Table 4.
4TABLE 4 Nucleotide Sequences of Human IL-5 Oligonucleotides SEQ
ISIS NUCLEOTIDE SEQUENCE.sup.1 ID TARGET TARGET NO. (5' -> 3')
NO: SITE.sup.2 REGION 16071 CTTTGGCAAAGAAAGTGCAT 39 0509-0528
5'-UTR 16072 CGTTCTGCGTTTGCCTTTGG 40 0523-0542 5'-UTR 16073
TCCTCATGGCTCTGAAACGT 41 0540-0559 AUG 16074 AAGAAAATTACCTCATTGGC 42
0688-0707 Coding 16075 TTACAGCACACCAGCATTCA 43 0857-0876 Coding
16076 TCCTCAGAGTCTGGAGAGGA 44 0895-0914 Coding 16077
GGAACAGGAATCCTCAGAGT 45 0905-0924 Coding 16078 TTTAACTTACATTTTTATGT
46 0928-0947 Coding 16079 TTTACTTATTCATGCCATCA 47 0964-0983 Coding
16080 GACACGATGCTCTTTGGGAA 48 1161-1180 Coding 16081
CATTTTAATATGACCAGGCA 49 1407-1426 Coding 16082 TTCTAGGCAACAAACCACCA
50 1627-1646 Coding 16083 ACAGTTGGTGCTAAATGAGG 51 1873-1892 Coding
16084 TTCTTCAGTGCACAGTTGGT 52 1884-1903 Coding 16085
ACCCCCTTGCACAGTTTGAC 53 1932-1951 Coding 16086 TGGCCGTCAATGTATTTCTT
54 1988-2007 Coding 16087 TGTAACTTACTTTTTGGCCG 55 2002-2021 Coding
16088 TCCATAGAAATAGGCACAGC 56 2051-2070 Coding 16089
CACACTTTTTCTGTGAAAAA 57 2108-2127 Coding 16090 ATTGGTTTACTCTCCGTCTT
58 2135-2154 Coding 16091 TTATCCACTCGGTGTTCATT 59 2186-2205 Coding
16092 TCCTTCTCCTCCAAAATCTT 60 2241-2260 3'-UTR 16093
TCGCCCTCATTCTCACTGCA 61 2269-2288 3'-UTR 16094 TCTGGCAAAGTGTCAGTATG
62 2352-2371 3-'UTR 16095 TTGCCTGGAGGAAAATACTT 63 2416-2435 3'-UTR
16096 CTTTGGCAAAGAAAGTGCAT 64 0509-0528 5'-UTR 16097
CGTTCTGCGTTTGCCTTTGG 65 0523-0542 5'-UTR 16098 AAGAAAATTACCTCATTGGC
66 0688-0707 Coding 16099 TCCTCAGAGTCTGGAGAGGA 67 0895-0914 Coding
16100 TTTAACTTACATTTTTATGT 68 0928-0947 Coding 16101
ACAGTTGGTGCTAAATGAGG 69 1873-1892 Coding 16102 TGTAACTTACTTTTTGGCCG
70 2002-2021 Coding 16103 CACACTTTTTCTGTGAAAAA 71 2108-2127 Coding
17986 TCTGGCAAACTGTCAGTATG 72 mismatch 16094 17987
TCTGGCATACTCTCAGTATG 73 mismatch 16094 17988 TCTGGGATACTCTGAGTATG
74 mismatch 16094 17989 TTGCCTGGACGAAAATACTT 75 mismatch 16095
17990 TTGCCTGCACGTAAATACTT 76 mismatch 16095 17991
TTGCCAGCACGTATATACTT 77 mismatch 16095 .sup.1Emboldened residues,
2'-methoxyethoxy- residues (others are 2'-deoxy-) including "C"
residues, 5-methyl-cytosines; all linkages are phosphorothioate
linkages. .sup.2Nucleotide numbers from Genbank Accession No.
X12706, locus name AHSBCDIFFI@, SEQ ID NO.78 to which the
oligonucleotide is targeted.
[0167] These oligonucleotides were electroporated into human HSB-2
cells and tested for effect on IL-5 mRNA by Northern blot analysis
as described in previous examples. The HSB-2 T-cell line was
obtained from the American Type Culture Collection and cells are
cultured according to ATCC recommendations. They produce IL-5 upon
induction with PMA+ionomycin. Oligonucleotides were tested by
Northern blot analysis at a concentration of 10 .mu.M for their
ability to block IL-5 mRNA expression. The results are shown in
Table 5.
5TABLE 5 Activity of Antisense Oligonucleotides Targeted to Human
IL-S ISIS SEQ ID TARGET NO. NO: REGION % CONTROL % INHIB 16071 39
5'-UTR 124 -- 16072 40 5'-UTR 93.1 -- 16073 41 AUG 101 -- 16074 42
Coding 146 -- 16075 43 Coding 144 -- 16076 44 Coding 296 -- 16077
45 Coding 157 -- 16078 46 Coding 166 -- 16079 47 Coding 75 25 16080
48 Coding 224 -- 16081 49 Coding 215 -- 16082 50 Coding 94.3 5.7
16083 51 Coding 110 -- 16084 52 Coding 22.2 77.8 16085 53 coding
45.4 54.6 16086 54 Coding 158 -- 16087 55 Coding 98.7 1.3 16088 56
coding 88.4 11.6 16089 57 Coding 139 -- 16090 58 coding 72 28 16091
59 Coding 125 -- 16092 60 3'-UTR nd nd 16093 61 3'-UTR 78.5 21.5
16094 62 3'-UTR 58.1 41.9 16095 63 3'-UTR 157 -- 16096 64 5'-UTR
164 -- 16097 65 5'-UTR 286 -- 16098 66 Coding 117 -- 16099 67
Coding 157 -- 16100 68 Coding 163 -- 16101 69 Coding 94.4 5.6 16102
70 Coding 109 -- 16103 71 Coding 172 --
[0168] ISIS 16084, 16085 and 16094 inhibited IL-5 mRNA expression
by at least 40%.
[0169] A dose-response curve was generated for inhibition of human
IL-5 protein expression in HSB-2 cells by ISIS 16085. Cells
untreated with oligonucleotide were found to express approximately
47 pg/ml IL-5. After treatment with ISIS 16085 at 5, 15 and 25
.mu.M doses, IL-5 levels dropped to 21, 0 and 0 pg/ml,
respectively. Treatment with a 1-mismatch control oligonucleotide
at 5, 15 and 25 .mu.M doses gave IL-5 levels of 26, 25 and 20
pg/ml, respectively. Treatment with a 3-mismatch control
oligonucleotide at 5, 15 and 25 .mu.M doses gave IL-5 levels of 52,
48 and 46 pg/ml, respectively. A 5-mismatch oligonucleotide did not
inhibit, and at some doses stimulated, IL-5 protein expression.
Example 21
[0170] Inhibition of IL-5 Expression by ISIS 16085 in Human CEM T
Cells
[0171] Using an RNAse protection assay (RiboquantJ hCK4,
Pharmingen, La Jolla Calif.), it was determined that ISIS 16085
inhibited IL-5 expression in a second T cell line, CEM (obtained
from American Type Culture Collection) with an IC50 estimated at
approximately 25 .mu.M. IL-5 expression is induced in these cells
by treatment with PMA plus ionomycin in the presence of IL-2,
anti-CD28 crosslinking antibody, and dibutyryl cAMP. Dose response
analysis of ISIS 16085 vs. its 5-mismatch control in stimulated CEM
cells showed a dose-dependent decrease in IL-5 mRNA of about 50% at
25 .mu.M oligonucleotide, compared with about 22% reduction with
the mismatch control. No decreases were seen in other cytokine gene
products measured in this assay panel.
Example 22
[0172] Optimization of Oligonucleotides Targeted to Human IL-5
[0173] Additional 2'-methoxyethoxy gapmer oligonucleotides were
designed to optimize placement and size of 2' deoxy regions. These
are shown in Table 6.
6TABLE 6 Nucleotide Analogues of Human IL-5 Oligonucleotides SEQ
ISIS NUCLEOTIDE SEQUENCE.sup.1 ID TARGET TARGET NO. (5' -> 3')
NO: SITE.sup.2 REGION 16090 ATTGGTTTACTCTCCGTCTT 58 2135-2154
Coding 17873 ATTGGTTTACTCTCCGTCTT " " " 17874 ATTGGTTTACTCTCCGTCTT
" " " 17875 ATTGGTTTACTCTCCGTCTT " " " 17876 ATTGGTTTACTCTCCGTCTT "
" " 17877 ATTGGTTTACTCTCCGTCTT " " " 16094 TCTGGCAAAGTGTCAGTATG 62
2352-2371 3-'UTR 17878 TCTGGCAAAGTGTCAGTATG " " " 17879
TCTGGCAAAGTGTCAGTATG " " " 17880 TCTGGCAAAGTGTCAGTATG " " " 17881
TCTGGCAAAGTGTCAGTATG " " " 17882 TCTGGCAAAGTGTCAGTATG " " " 17992
TCTGGCAAAGTGTCAGTATG " " " 16095 TTGCCTGGAGGAAAATACTT 63 2416-2435
3'-UTR 17883 TTGCCTGGAGGAAAATACTT " " " 17884 TTGCCTGGAGGAAAATACTT
" " " 17885 TTGCCTGGAGGAAAATACTT " " " 17886 TTGCCTGGAGGAAAATACTT "
" " 17887 TTGCCTGGAGGAAAATACTT " " " 17993 TTGCCTGGAGGAAAATACTT " "
" 18248 TTGCCTGGAGGAAAATACTT " " " 18249 TTGCCTGGAGGAAAATACTT " " "
18250 TCTGGCAAAGTGTCAGTATG 62 2352-2371 3-'UTR 18251
TCTGGCAAAGTGTCAGTATG " " " 18252 ATTGGTTTACTCTCCGTCTT 58 2135-2154
Coding 18253 ATTGGTTTACTCTCCGTCTT " " " .sup.1Emboldened residues,
2'-methoxyethoxy- residues (others are 2'-deoxy-) including "C"
residues, 5-methyl-cytosines; all linkages are phosphorothicate
linkages. .sup.2Nucleotide numbers from Genbank Accession No.
X12706, locus name AIISBCDIFFI@, SEQ ID NO.78 to which the
oligonucleotide is targeted.
[0174] Table 7
[0175] Nucleotide Analogues of Human IL-5 Oligonucleotides
[0176] Mixed backbone [phosphorothioate (P--S) and phosphodiester
(P--O)] or all-phosphodiester (P--O) backbone analogs of ISIS 16095
and its mismatch control were also designed. These are shown in
Table 7.
7TABLE 7 SEQ ISIS NUCLEOTIDE SEQUENCE.sup.1 ID TARGET NO. (5' ->
3') NO: REGION 21883 TTGCCTGGAGGAAAATACTT 64 mixed backbone; P-O in
2' MOE regions and P-S in 2'deoxy gap 22103 TTGCCAGCACGTATATACTT 77
mixed backbone; P-O in 2' MOE regions and P-S in 2'deoxy gap; 21883
mismatch 23114 TTGCCTGGAGGAAAATACTT 63 P-O throughout 23115
TTGCCAGCACGTATATACTT 77 P-O throughout; 23114 mismatch
.sup.1Emboldened residues, 2'-methoxyethoxy- residues (others are
2'-deoxy-); all "C" and "C" residues, 5-methyl-cytosines; linkages
in 2'-deoxy gaps are phosphorothiOate linkages, linkages in 2'-MOE
regions are phosphodiester linkages.
Mouse IL-5 Receptor
Example 23
[0177] Mouse IL-5 Receptor a Oligos
[0178] The mRNA encoding the membrane form of the mouse IL-receptor
a contains 11 exons. The transmembrane domain of the receptor is
encoded in exon 9. Two mRNAs encoding soluble (secreted) forms of
the receptor result from differential splicing events. The mRNA
encoding soluble form 1 of the receptor is missing exon 9 (exon 8
is spliced to exon 10) and the mRNA encoding soluble form 2 is
missing exons 9 and 10 (exon 8 is spliced to exon 11). Imamura et
al., DNA and Cell Biology, 13, 283-292.
[0179] Murine BCL.sub.1 cells were chosen for screening antisense
oligonucleotides targeted to murine IL-5 receptor a. These are
B-cell leukemia cells derived from a spontaneously arising tumor of
BALB/c origin, and proliferate in response to murine or human IL-5.
This is a CD5+line which resembles a subset of human chronic
lymphocytic leukemia tumors and secretes IgM upon
lipopolysaccharide stimulation. Cells were obtained from the
American Type Culture Collection and cultured in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum (Sigma
Chemical Co., St. Louis, Mo.), 10 mM Hepes, pH 7.2, 50 .mu.M 2-ME,
2 mM L-glutamine, 100 U/ml penicillin and 100 .mu.g/ml streptomycin
(Gibco, Grand Island, N.Y.).
[0180] A series of antisense oligonucleotides were designed to
target the murine IL-5 receptor. All are chimeric "gapmers" with
2'-methoxyethoxy flanks and central 10-base deoxy "gaps" and a
phosphorothioate backbone throughout. Cells (1.times.10.sup.7 cells
in PBS) were transfected with oligonucleotides by electroporation
at 200V, 1000 .mu.F using a BTX Electro Cell Manipulator 600
(Genetronics, San Diego Calif.). Antisense oligonucleotide
sequences are shown in Table 8.
8TABLE 8 Nucleotide sequences of mouse IL-5 receptor a
oligonucleotides SEQ ISIS NUCLEOTIDE SEQUENCE.sup.1 ID TARGET
TARGET NO. (5' -> 3') NO: SITE REGION 16924 GACCTGTCCAGTGAGCTTCT
79 0112-0131.sup.2 5'-UTR 16925 TAGCCGAATACTGGAAAGGT 80 0281-0300
5'-UTR 16926 AACACAGGCACCATGGTAGC 81 0297-0316 AUG 16927
CTCTTGGTCAGGATTTGGGT 82 0445-0464 Coding 16928 TCCTCACGCTAGCTGCAAAG
83 0572-0591 Coding 16929 ATGGCCTTAAGTGGGTGTGG 84 0719-0738 Coding
16930 GAGCCATTAATGTGCACAGC 85 0927-0946 Coding 16931
TCCACTCGCCCCACCTTCCT 86 1250-1269 Coding 16932 AACAAGACGAAGCAGGCAGC
87 1338-1357 Coding 16933 CCGGAACCGGTGGAAACAAC 88 1400-1419 Coding
16934 CCAACCTCTTCCACACAATG 89 1500-1519 Coding 16935
TCCCATGACTTCAAATCCAA 90 1516-1535 Coding 16936 GCAAAATGCCATCAAAACGT
91 1542-1561 STOP 16937 CGAGCTCTACCACCGCCTGG 92 1651-1670 3'-UTR
16938 CAAGCTGGCCTCGAACTCAG 93 1712-1731 3'-UTR 16939
GGATGGGTTGGTGACTTGCA 94 1835-1854 3'-UTR 16940 TGAGGAAACCAAAGGCCCAT
95 1946-1965 3'-UTR 16941 TGTCTCCCACTTGCGTCAGG 96 2164-2183 3'-UTR
16942 TTGAACAGGCCTATGGAACA 97 2306-2325 3'-UTR 16943
TCTTTTTCACCCCAGGCACG 98 2359-2378 3'-UTR 16944 AATTCCCATGGATCCTCTTG
99 2515-2534 3'-UTR 16945 ATCCAGCAATCACCTCCAAA 100 2794-2813 3'-UTR
16946 TGTTCAGCCCATCAAAAAGA 101 2984-3003 3'-UTR 16947
ATTTGGCTGACAGGACCCCG 102 3140-3159 3'-UTR 16948
TCCAGAGACTGCCCCACCCA 103 3216-3235 3'-UTR 16949
CATCTGCTTCTGTATTGCCA 104 3381-3400 3'-UTR 16950
CCTTTTAGCTCCTTGGGTAC 105 3456-3475 3'-UTR 16951
CATTTCTGAGGGTTGCTGGG 106 3513-3532 3'-UTR 18278
CATCTGATTGTGTCTTGCCA 107 mismatch 16949 18279 CATCTGCTTGTGTATTGCCA
108 " " 18280 CACCTGATTGTGTCTTGTCA 109 " " 17652
TGTCCCTCCTTTTGGTGGGG 110 0741-0760.sup.3 Coding 17653
TTAGCTCTGTCTCTGCTGAT 111 0071-0090 Coding 17654
AACTGCTGGCCAGAGTTGTA 112 0611-0630 Coding 17655
CATAGTTAAAGCAATGATCT 113 1091-1110 Coding 17656
GTTTCTCATATTCAGTAACC 114 1451-1470 Coding 17657
GGAGTCCTGTATGAGTTCAT 115 1571-1590 3'-UTR 17658
TCTGTGCATCCCAGGTGCTG 116 1681-1700 3'-UTR 17659
CTGGCTGTCCTGGAACTCAC 117 1741-1760 3'-UTR 17660
TTCAAGGTAAGTCAAGCAAC 118 2001-2020 3'-UTR 17661
CTGATGGCTACCACTGGCAA 119 2081-2100 3'-UTR 17662
CACTCTCAATGAGTTCTATC 120 2121-2140 3'-UTR 17663
TGATGCTGGTTGATCAATCT 121 2411-2430 3'-UTR 17664
TCAATAGGGAATGGTGTCTT 122 2681-2700 3'-UTR 17665
TTCCAGAGTACCTAGAAGCC 123 2741-2760 3'-UTR 17666
CCAACAGGTTGCCATGAAGG 124 2851-2870 3'-UTR 17667
AGAGATTAGAATTGACTAAG 125 2881-2900 3'-UTR 17668
ACTATTGCATATACTAGCAA 126 3161-3180 3'-UTR 17669
CCATCCAATATACAACCACC 127 3191-3210 3'-UTR 17670
CTCATGGAAGGAGTTACAGA 128 3271-3290 3'-UTR 17671
TGTGGATACTTCACTGCTTC 129 3311-3330 3'-UTR 17672
ATCCAATAGATGACTGTGAG 130 3401-3420 3'-UTR 17673
GTTCATATTGTTGTTCCTGC 131 3491-3510 3'-UTR .sup.1Emboldened
residues, 2'-methoxyethoxy- residues (others are 2'-deoxy-)
including "C" residues, 5-methyl-cytosines; all linkages are
phosphorothioate linkages. .sup.2Nucleotide numbers from Genbank
Accession No. D90205, locus name AMUSIL5R@, SEQ ID NO.132 to which
the oligonucleotide is targeted. .sup.3 Nucleotide numbers from
Genbank Accession No. S69702, locus name "S69702", SEQ ID NO.133 to
which the oligonucleotide is targeted.
[0181] Total cellular RNA was isolated using the RNeasyJ kit
(Qiagen, Santa Clara Calif.). mRNA was analyzed by RNAse protection
assay (RPA) using the Riboquant Kit and a customized riboprobe
spanning exon 9 of the IL-5 receptor a (PharMingen, La Jolla
Calif.). The cDNA probes were generated from oligonucleotides
matching the exon sequences of either exons 2, 8, 9 or 10. Signals
were quantitated using a Molecular Dynamics PhosphorImager. Results
are shown in Table 9.
9TABLE 9 Antisense inhibition of mouse IL-5 receptor a mRNA
expression ISIS SEQ ID TARGET NO. NO: REGION % CONTROL % INHIB
16924 79 5'-UTR 98 2 16925 80 5'-UTR 86 14 16926 81 AUG 75 25 16927
82 Coding 74 26 16928 83 Coding 91 9 16929 84 Coding 87 13 16930 85
Coding 90 10 16931 86 Coding 108 -- 16932 87 Coding 93 7 16933 88
Coding 102 -- 16934 89 Coding 55 45 16935 90 Coding 108 -- 16936 91
STOP 76 24 16937 92 3'-UTR 91 9 16938 93 3'-UTR 80 20 16939 94
3'-UTR 83 17 16940 95 3'-UTR 81 19 16941 96 3'-UTR 98 2 16942 97
3'-UTR 91 9 16943 98 3'-UTR 81 19 16944 99 3'-UTR 88 12 16945 100
3'-UTR 65 35 16946 101 3'-UTR 82 18 16947 102 3'-UTR 75 25 16948
103 3'-UTR 89 11 16949 104 3'-UTR 52 48 16950 105 3'-UTR 87 13
16951 106 3'-UTR 99 1
[0182] In this assay, ISIS 16926, 16927, 16934, 16936, 16945, 16947
and 16949 gave at least approximately 25% inhibition of IL-5Ra mRNA
expression and are preferred. Of these, ISIS 16934, 16945 and 16949
gave at least 35% inhibition and are more preferred.
[0183] ISIS 16934, 16945 and 16949 were chosen for further study.
These demonstrated IC50s for inhibition of murine IL-5 receptor a
mRNA in BCL.sub.1 cells of approximately 2.5 .mu.M, 1.5 .mu.M and 1
.mu.M, respectively. ISIS 16949 was tested for effects on IL-5
receptor a protein expression and showed nearly complete
inhibition.
Example 24
[0184] Antisense Oligonucleotides Targeted to Exon 9 of Mouse IL-5
Receptor
[0185] A series of antisense oligonucleotides were designed to
"walk" the entire exon 9 of the coding region of murine IL-5
receptor a mRNA. Oligonucleotides were targeted to regions starting
approximately every 10 nucleobases along the exon 9 sequence, which
extends from nucleotides 1288 to 1381 on the sequence given as
Genbank accession no. D90205. Oligonucleotides are shown in Table
10.
10TABLE 10 Nucleotide Sequences of Mouse IL-5R Oligonucleotides- 2'
MOE gapmers SEQ ISIS NUCLEOTIDE SEQUENCE.sup.1 ID TARGET TARGET NO.
(5' -> 3') NO: SITE.sup.2 REGION 18001 CAAGGACTTCCTTTCCTTTC 134
1288-1307 Coding /exon 9 18002 GCCATTCTACCAAGGACTTC 135 1298-1317
Coding /exon 9 18003 ACAATGAGATGCCATTCTAC 136 1308-1327 Coding
/exon 9 18004 TGTTGGGAGCACAATGAGAT 137 1318-1337 Coding /exon 9
18005 AGCAGGCAGCTGTTGGGAGC 138 1328-1347 Coding /exon 9 18006
TGAGAAGATTAACAAGACGA 139 1348-1367 Coding /exon 9 18007
TGCAGATGAGTGAGAAGATT 140 1358-1377 Coding /exon 9 18008
ACTCTGCAGATGAGTGAGAA 141 1362-1381 Coding /exon 9 .sup.1Emboldened
residues, 2'-methoxyethoxy- residues (others are 2'-deoxy-)
including "C" residues, 5-methyl-cytosines; all linkages are
phosphorothioate linkages. .sup.2Nucleotide numbers from Genbank
Accession No.D90205, locus name "MTJSIL5R," to which the
oligonucleotide is targeted.
[0186] Effect of these compounds on both membrane and soluble forms
of murine IL-5 receptor a were measured and are shown in Table 11.
Oligonucleotides were screened in BCL.sub.1 cells at a dose of 10
.mu.M and IL-5 receptor a mRNA was measured by RPA. Percent
inhibition is compared to untreated (no oligonucleotide)
control.
11TABLE 11 Effect of 2'-MOE gapmers targeted to murine IL-5
receptor a mRNA exon 9 on membrane and soluble IL-5 receptor a mRNA
expression ISIS % inhibition of % inhibition of SEQ NO. membrane
IL-5 Ra soluble.sup.1 IL-5 Ra ID NO: 18001 35 39 134 18002 5 8 135
18003 15 20 136 18004 10 20 137 18005 55 59 138 18006 59 65 139
18007 65 65 140 18008 75 75 141 .sup.1Only one soluble form is
detectable by RPA; the RPA probe does not distinguish between the
two soluble forms. These gapmers were able to reduce both membrane
and soluble orms and each oligonucleotide reduced the two forms
approximately equally.
Example 25
[0187] Effect of Fully 2'-MOE Oligonucleotides Targeted to Murine
IL-5 Receptor a mRNA Exon 9 on Membrane and Soluble IL-5 Receptor a
mRNA Expression
[0188] Additional oligonucleotides were designed to target exon 9
and intron/exon boundaries; these were uniformly 2'-methoxyethoxy
modified with phosphorothioate backbones throughout. These are
shown in Table 12 below.
12TABLE 12 Nucleotide Sequences of Mouse IL-5R Oligonucleotides
uniform 2' MOE SEQ ISIS NUCLEOTIDE SEQUENCE.sup.1 ID TARGET TARGET
NO. (5' -> 3') NO: SITE REGION 21750 GACTTCCTTTCCTTTCCTGG 142
1284-1303.sup.2 I8/E9 21751 CAAGGACTTCCTTTCCTTTC 134 1288-1307
18001 21752 GCCATTCTACCAAGGACTTC 135 1298-1317 18002 21753
ACAATGAGATGCCATTCTAC 136 1308-1327 18003 21754 TGTTGGGAGCACAATGAGAT
137 1318-1337 18004 21755 AGCAGGCAGCTGTTGGGAGC 138 1328-1347 18005
21756 AACAAGACGAAGCAGGCAGC 143 1338-1357 Exon 9 21757
TGAGAAGATTAACAAGACGA 139 1348-1367 18006 21758 TGCAGATGAGTGAGAAGATT
140 1358-1377 18007 21759 ACTCTGCAGATGAGTGAGAA 141 1362-1381 18008
21760 CTACACTCTGCAGATGAGTG 144 1366-1383 E9/E10 21761
CGATCAGTTTTTCCTTCTAA 145 1145-1164.sup.3 E7/E8 21762
TCACCCACATAAATAGGTTG 146 1272-1288 E8/E9 21763 GGTCCATAAATGACACCTGA
147 1382-1397 E9/E10 21764 TTACCTCATATTCAGTAACC 148 1451-1466
E10/E11 23235 GCCATTCTATCAAGGACTTC 149 mismatch 21752 23236
GCCATGCTATCAAGCACTTC 150 " " 23237 GCTATCCTATCAAGCACGTC 151 " "
23238 GACTTCCTTACCTTTCCTGG 152 " 21750 23239 GACTTCCTCTTCTTCCCTGG
153 " " 23240 GACCTCTTTCCCTCTTCTGG 154 " " .sup.1Emboldened
residues, 2'-methoxyethoxy- residues (others are 2'-deoxy-)
including "C" residues, 5-methyl-cytosines; all linkages are
phosphorothicate linkages. .sup.2Co-ordinates from Genbank
Accession No. D90205, locus name ANUSILSR@, SEQ ID NO.132.
.sup.3ISIS 21761-21764 were designed to hybridize to intron-exon
border sequences provided in Table 1 of Itnarnura, F., et al., DNA
Cell Biol., 1994, 13, 283-292.
[0189] BCL.sub.1 cells were treated with 10 .mu.M of the
full-2'-methoxyethoxy, full phosphorothioate oligonucleotides for
24 hours and total RNA was extracted and analyzed by RPA. Results
are shown in Table 13.
13TABLE 13 Effect of 2' MOE uniformly modified oligonucleotides
targeted to murine IL-5 receptor a mRNA exon on IL-5 mRNA % % %
control % inhib'n control inhib'n SEQ ISIS membrane membrane
soluble soluble ID NO. IL-5 Ra IL-5 Ra IL-5 Ra IL-5 Ra NO: 21750 8
92 197 -- 142 21751 9 91 191 -- 134 21752 6 94 194 -- 135 21753 6
94 175 -- 136 21754 8 92 184 -- 137 21755 16 84 181 -- 138 21756 6
94 166 -- 143 21757 19 81 144 -- 139 21758 31 69 116 -- 140 21759
34 66 134 -- 141 21760 55 45 116 -- 144
[0190] All of the fully modified 2'-methoxyethoxy oligonucleotides
targeted to murine IL-5 receptor a mRNA exon reduced expression of
the membrane form of IL-5 receptor a and increased expression of
the soluble form of the receptor. The potencies of these concurrent
effects were coordinately diminished as the antisense target site
moved toward the 3' end of the exon. The overall amount of IL-5
receptor a transcription is unaffected. This demonstrates that
fully 2'-methoxyethoxy-modified oligonucleotides targeted to exon 9
just distal to the intronic 3' splice acceptor site blocked
inclusion of exon 9 in the splice product and redirect the splicing
machinery to the next downstream splice acceptor site (in intron
9). Reduction of the membrane form of IL-5 receptor a, particularly
with no decrease or more particularly with an increase in the
soluble form, is believed to have therapeutic utility in diseases
associated with IL-5 signal transduction, especially asthma. These
results show that splicing has been redirected by use of uniformly
2'-methoxyethoxy oligonucleotides targeted to exon 9 to cause
exclusion (skipping) of exon 9 from the spliced mRNA products,
resulting in controlled alteration of the ratio of soluble/membrane
IL-5 receptor produced.
[0191] It was also shown that conversion of an RNAse H-dependent
compound (the 2' MOE gapmer ISIS 18002) to an RNAse H-independent
compound (the fully-2' MOE compound 21752) converted this
oligonucleotide sequence from an inhibitor of both forms of IL-5
receptor a to one which selectively inhibits of the membrane form
via splice redirection.
[0192] ISIS 21752 was chosen for further study. In dose response
experiments, an IC50 of approximately 4 .mu.M was obtained for
inhibition of the membrane form of IL-5 receptor a mRNA. A 1-base
mismatch (ISIS 23235) gave an IC50 of approximately 10.5 .mu.M and
3- and 5-base mismatches did not inhibit membrane IL-5 receptor
mRNA at any concentration.
Example 26
[0193] Effect of Fully 2'-MOE Peptide Nucleic Acid Oligonucleotides
Targeted to Murine IL-5 Receptor a mRNA Exon 9 on Membrane and
Soluble IL-5 Receptor a mRNA Expression
Example 27
[0194] Oligonucleotides Targeted to Exon-exon Boundaries of Various
Forms of Mouse IL-5 Receptor a mRNA
[0195] Oligonucleotides, either 2' MOE gapmers or uniform 2' MOE,
were designed to target exon-exon boundaries of the mature IL-5
receptor a mRNA. The mRNA encoding the membrane form of the mouse
IL-5 receptor a contains 11 exons. The transmembrane domain of the
receptor is encoded in exon 9. Two mRNAs encoding soluble
(secreted) forms of the receptor result from differential splicing
events. The mRNA encoding soluble form 1 of the receptor is missing
exon 9 (exon 8 is spliced to exon 10)and the mRNA encoding soluble
form 2 is missing exons 9 and 10 (exon 8 is spliced to exon 11). In
Table 14, the target region designated "E7-E8" indicates that the
oligonucleotide is targeted to the exon 7-8 boundary, and so
forth.
14TABLE 14 Nucleotide Sequences of Mouse IL-5R Oligonucleotides SEQ
ISIS NUCLEOTIDE SEQUENCE.sup.1 ID TARGET TARGET NO. (5' -> 3')
NO: SITE.sup.2 REGION 21847 GTTTTTCCTTCTGAATGTGA 155 1139-1158
E7-E8 21848 GTTTTTCCTTCTGAATGTGA " 21847 21849 CTTTCCTTTCCCACATAAAT
156 1278-1297 E8-E9 21850 CTTTCCTTTCCCACATAAAT " 21849 21851
TAAATGACACACTCTGCAGA 157 1372-1391 E9-E10 21852
TAAATGACACACTCTGCAGA " 21851 21853 TAAATGACACCCACATAAAT 158 E8-E10
(soluble form 1) 21854 TAAATGACACCCACATAAAT " 21853 21855
TCGAAGGTTTCCACATAAAT 159 E8-E11 (soluble form 2) 21856
TCGAAGGTTTCCACATAAAT " 21855 21969 CACCTGATTGTGTCTTGTCA 109
mismatch 16949 21972 CATCTGCTTCTGTATTGCCA 104 16949 22089
TTACCTCATATTCAGTAACC 148 21764 22090 GGTCCATAAATGACACCTGA 147 21763
22091 TCACCCACATAAATAGGTTG 146 21762 22092 CGATCAGTTTTTCCTTCTAA 145
21761 22093 CTACACTCTGCAGATGAGTG 144 21760 22094
GACTTCCTTTCCTTTCCTGG 142 21750 23232 GCCATTCTATCAAGGACTTC 149
mismatch 21752 23233 GCCATGCTATCAAGCACTTC 150 " " 23234
GCTATCCTATCAAGCACGTC 151 " " .sup.1EnTh oldened residues,
2'-methoxyethoxy- residues (others are 2'-deoxy-), all "C" and "C"
residues, 5-methyl-cytosines; all linkages are phosphorothicate
linkages. .sup.2Nucleotide numbers from Genbank Accession No.
D90205, locus name AMUSTL5R@, SEQ ID NO.132.
[0196] These compounds were tested at 10 .mu.M dose for ability to
reduce membrane or soluble IL-5 receptor a mRNA by RPA. Results for
compounds tested are shown in Table 15.
15TABLE 15 Activity of Mouse IL-5R Oligonucleotides against Soluble
and Membrane IL-5 receptor a mRNA % INHIB'N % INHIB'N SEQ MEMBRANE
SOLUBLE ISIS ID CHEM- IL-5 IL-5 TARGET NO. NO: ISTRY RECEPTOR
RECEPTOR REGION 21847 155 uniform 23 20 E7-E8 2'-MOE (common) 21848
155 2' MOE 89 86 21847 /deoxy gapmer 21849 156 uniform 70 5 E8-E9
2'-MOE (membrane) 21850 156 2' MOE 39 25 21849 /deoxy gapmer 21851
157 uniform 61 0 E9-E10 2'-MOE (membrane) 21852 157 2' MOE 20 14
21851 /deoxy gapmer 21853 158 uniform 14 45 E8-E10 2'-MOE (soluble
form 1) 21854 158 2' MOE 11 14 21853 /deoxy gapmer 21855 159
uniform 14 25 E8-E11 2'-MOE (soluble form 2)
[0197] As shown in Table 15, selective reduction of expression of
the soluble form of IL-5 receptor a could be achieved with
antisense oligonucleotides targeted to the exon 8-exon 10 boundary,
or, to a lesser extent to the exon 8-exon 11 boundary, both of
which junctions are only found in the soluble receptor mRNA.
Selective reduction of expression of the membrane form of IL-5
receptor a could be achieved with antisense oligonucleotides
targeted to the exon 8-exon 9 boundary or exon 9-exon 10 boundary,
both of which are only present in the mRNA targeting the membrane
form of IL-5 receptor a. Placement of the fully-2' MOE
oligonucleotides across the intron/exon boundaries of exon 9
resulted in similar effects as were obtained with fully-modified
oligonucleotides positioned inside exon 9.
Example 28
[0198] Effect of Antisense Oligonucleotides on Expression of
Membrane Form of IL-5 Receptor a Protein in Murine BCL.sub.1
Cells
[0199] BCL.sub.1 cells were treated with antisense oligonucleotide
for 48 hours. Oligonucleotides used were ISIS 16949 ("common"
oligonucleotide targeted to both soluble and membrane forms of IL-5
receptor), ISIS 21752, targeted only to the membrane form and ISIS
21853 and 21855, targeted only to the soluble forms of IL-5
receptor a. Oligonucleotides were introduced by electroporation as
described in previous examples. Effect on levels of the membrane
form of the receptor was examined by Western blot analysis.
Membrane-enriched fractions were prepared as Triton X-100 insoluble
material and separated by SDS-PAGE using 8% gels. Antibody to mouse
IL-5 receptor a was purchased from Santa Cruz Biotechnology (Santa
Cruz, Calif.) and used at 1:1000 dilution.
[0200] Compared to control (no oligonucleotide), ISIS 21752 nearly
completely ablated the membrane IL-5 receptor. ISIS 21853 and 21855
together had little to no effect; both target the soluble receptor
isoforms specifically. The common sequence oligonucleotide, ISIS
16949, reduced the soluble receptor by 75%.
[0201] Transfection with a fully 2'-MOE oligonucleotide targeted to
the 5' intron splice site for either exon 8, 9 or 10 resulted in
specific exclusion of that particular downstream exon but not
others adjacent or upstream. Thus targeting the 5' intron splice
sites with high-affinity antisense compounds such as fully 2'-MOE
oligonucleotides allows selective deletion of individual exons of
the mRNA transcript.
Example 29
[0202] Reduction of Eosinophils in Blood and Peritoneal Lavage
Fluid of Mice Treated with IL-5 Receptor a Antisense
Oligonucleotide
[0203] Mice received daily injections of recombinant mouse IL-5 for
5 days, with or without ISIS 21972 or its mismatch control, ISIS
21969. Percent eosinophils in blood and peritoneal lavage fluid
were measured. In control mice (no IL-5, no oligonucleotide)
eosinophil levels were 4% in peritoneal lavage fluid and 2% in
blood. After IL-5 treatment, eosinophils increased to 13.5% in
lavage fluid and 9.5% in blood. Treatment with mismatch
oligonucleotide did not change this significantly (13.5% in lavage
fluid, 10.5% in blood) but treatment with IL-5 receptor a antisense
oligonucleotide reduced eosinophil levels to 8.5% in peritoneal
lavage fluid and 7% in blood.
Human IL-5 Receptor
Example 30
[0204] Antisense Oligonucleotides Targeted to Human IL-5 Receptor
a
[0205] The human IL-5 receptor a gene contains 14 exons. A
membrane-anchored form of the receptor and two soluble forms have
been identified. The membrane form is active in signal transduction
and the soluble forms can act antagonistically. The mRNA transcript
encoding the membrane-anchored form of the human IL-5 receptor a
contain exons 1-10 and 12-14. Exon 11 is spliced out by an
alternative splicing event. The major soluble isoform (soluble form
1) is generated as a result of a normal splicing event and an
in-frame stop codon in exon 11. The other soluble form (soluble
form 2) is generated by the absence of splicing and therefore is
generated by reading into intron 11.
[0206] mRNA transcripts encoding the membrane form of the human
IL-5 receptor a contain exons 1-10 and 12-14. Exon 11 is spliced
out. It is, therefore, possible to target sequences in exons 1-10
which are common to both soluble and membrane forms of the
receptor, or to selectively target sequences only present in the
membrane form (exons 12-14). A series of antisense oligonucleotides
were designed to be specific to only the membrane form of human
IL-5 receptor a (IL-5Ra). These oligonucleotides target regions
downstream of exon 11 (i.e., exons 12-14 and intervening introns,
stop codon and 3' untranslated region). Tavernier et al., Proc.
Natl. Acad. Sci., 1992, 89, 7041-7045. These are shown in Table
16.
16TABLE 16 Nucleotide Sequences of Human IL-5 receptor a
membrane-specific antisense oligonucleotides SEQ ISIS NUCLEOTIDE
SEQUENCE.sup.1 ID TARGET TARGET NO. (5' ->3') NO: SITE.sup.2
REGION 16767 AACCACTCTCTCAAGGGCTT 160 1070-1089 Coding 16768
TGCTGGAATTGGTGGAAACA 161 1173-1192 Coding 17769
GTCTCAACTCCAGGCTTCTC 162 1283-1302 Coding 16770
TCAAAACACAGAATCCTCCA 163 1305-1324 STOP 16771 AGGATGCCAAAGTGACAGTC
164 1323-1342 STOP 16772 ATCCCTGTTCTTTTCACTGA 165 1371-1390 3'-UTR
16773 CGCAGGTAAATTGAGTGTTG 166 1426-1445 3'-UTR 16774
TGAGGCGATTTGGATGAAGC 167 1495-1514 3'-UTR 16775
TGGACGTTAGCCTTAAAAGC 168 1651-1670 3'-UTR 16776
AGCTTAAACAGCCAAACGGG 169 1693-1712 3'-UTR 16777
CTCCAGGCTGATGCAAAATG 170 1751-1770 3'-UTR 16778
GGGTGAGGAATTTGTGGCTC 171 1817-1836 3'-UTR 16779
CTGGATCAGGCCTCTGGAGC 172 1936-1955 3'-UTR 18012
GGGTGAGGATTTTGTGGCTC 173 mismatch 16778 18013 GGGTGATGATTTGGTGGCTC
174 " " 18014 GGCTGATGATTTGGTGGGTC 175 " " .sup.1Emboldened
residues, 2'-methoxyethoxy- residues (others are 2'-deoxy-)
including "C" residues, 5-methyl-cytosines; all linkages are
phosphorothioate linkages. .sup.2Nucleotide numbers from Genbank
Accession No. X61176, locus name AHSIL5RG@, SEQ ID NO.176, to which
oligonucleotides are targeted.
[0207] These cells were tested in an IL-5 receptor-expressing
subclone of TF-1 cells (provided by Dr. Christoph Walker, Novartis
Research Centre, Horsham, UK. Cells were cultured in RPMI 1640
medium supplemented with 10% heat-inactivated fetal bovine serum
(Sigma Chemical Company, St.Louis, Mo.), 10 mM Hepes, pH 7.2, 50
.mu.M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin (Gibco, Grand Island, N.Y.) and 10 ng/ml recombinant
human IL-5 (R & D Systems, Minneapolis, Minn.) added every
48-72 hours. TF-1 cells (1.times.10.sup.7 cells in PBS) were
transfected with oligonucleotides by electroration at 250V, 1000
.mu.F using a BTX ElectroCell tor 600 (Genetronics, San Diego
Calif.).
[0208] Total cellular RNA was isolated using the RNeasyJ kit
(Qiagen, Santa Clarita Calif.). Northern blotting was performed
using standard methods using a full-length cDNA probe or a cDNA
probe corresponding to the membrane isoform-specific exon sequences
prepared from HL-60 cell RNA by standard RT-PCR followed by a
nested primer reaction. Signals were quantitated using a Molecular
Dynamics PhosphorImager. Results are shown in Table 17.
17TABLE 17 Activity of Human IL-5 receptor a membrane-specific
antisense oligonucleotides on IL-5 receptor mRNA expression % % %
control % inhib. control inhib. SEQ ISIS membrane membrane soluble
soluble ID NO. IL-5 Ra IL-5 Ra IL-5 Ra IL-5 Ra NO: 16767 86 14 95 5
160 16768 72 28 97 3 161 16769 48 52 100 0 162 16770 69 31 84 16
163 16771 66 34 78 22 164 16772 66 34 92 8 165 16773 48 52 84 16
166 16774 55 45 103 -- 167 16775 100 0 95 5 168 16776 59 41 81 19
169 16777 31 69 84 16 170 16778 41 59 92 8 171 16779 55 45 95 5
172
[0209] ISIS 16769, 16773, 16774, 16776, 16777, 16778 and 16779
inhibited the membrane form of IL-5 receptor a by at least 40% and
are preferred. Of these, ISIS 16769, 16774, 16778 and 16779 are
more preferred because of their minimal effect on the soluble form
of IL-5Ra.
[0210] The effect of ISIS 16778 on expression of human IL-5
receptor a protein on the surface of TF-1 cells was measured by
flow cytometry. Following electroporation with oligonucleotide,
TF-1 cells were incubated for 24 hours or as indicated, collected
by centrifugation and washed with cold PBS. Cells were transferred
to 12.times.75 mm polystyrene tubes and washed in 2% bovine serum
albumin, 0.2% sodium azide in PBS at 41 C. Cells were centrifuged
at 200.times.g and the supernatant was decanted. Specific antibody
was then added at 1:100 for human IL-5 receptor a phycoerythrin and
the isotype control antibody in 0.1 mL of the above buffer.
Antibodies were incubated with the cells for 30 minutes at 41 C. in
the dark with gentle agitation. Cells were then washed as above and
resuspended in 0.3 mL of FacsFlow buffer (Becton Dickinson,
Franklin Lakes, N.J.) with 0.5% formaldehyde. Cells were analyzed
on a Becton-Dickinson FACScan. Results are expressed as the
percentage of control expression based on mean fluorescence
intensity, subtracting basal expression.
[0211] In dose-response experiments to determine the effect of this
oligonucleotide on human IL-5 receptor a cell surface protein
expression in TF-1 cells, ISIS 16778 demonstrated an IC50 of
approximately 5 .mu.M. A 1-mismatch control had an IC50 of 7.5
.mu.M and 3- and 5-mismatch controls did not inhibit IL-5 receptor
a below 75% of control.
[0212] An additional set of oligonucleotides was designed to target
both membrane and soluble forms of human IL-5 receptor. These
oligonucleotides, targeted to exons 1-10 and intervening introns,
are sometimes referred to as "common" IL-5 receptor
oligonucleotides. Sequences are shown in Table 18.
18TABLE 18 Human IL-5R "Common" Antisense Oligonucleotides SEQ ISIS
NUCLEOTIDE SEQUENCE.sup.1 ID TARGET TARGET NO. (5' -> 3') NO:
SITE.sup.2 REGION 16780 CCTGAGAAATGCGGTGGCCA 177 0019-0038 5'-UTR
16781 GTGTCTATGCTCGTGGCTGC 178 0093-0112 5'-UTR 16782
CGATCCTCTTGTTCCGACCA 179 0148-0167 5'-UTR 16783
ATGCGCCACGATGATCATAT 180 0248-0267 AUG 16784 GCAGTATCTCAGTGGCCCCC
181 0285-0304 Coding 16785 TGCTCTTGATCAGGATTTGG 182 0403-0422
Coding 16786 CAGGATGGTCCGCACACTTG 183 0536-0555 Coding 16787
GGGCATGAAGTTCAGCAGAA 184 0591-0610 Coding 16788
GCCAGGTGCAGTGAAGGGAA 185 0702-0721 Coding 16789
CTCCCCAGTGTGTCTTTGCT 186 0805-0824 Coding 16790
AAGCCAGTCACGCCCTTTGC 187 0863-0882 Coding 16791
AAACAGCTGATCAAAGGGCC 188 0923-0942 Coding 16792
ATGGATTGGAAAAGCAGACA 189 1034-1053 Coding 16793
TCTGCACATGGAGCTCACTG 190 1181-1200 Coding 16794
AGGTTGGCTCCACTCACTCC 191 1214-1233 Coding 18015
TCTGCACATGTAGCTCACTG 192 mismatch 16793 18016 TCTGCACGTGTAACTCACTG
193 " " 18017 TATGCACGTGTAACTCCCTG 194 " " .sup.1Emboldened
residues, 2'-methoxyethoxy- residues (others are 2'-deoxy-)
including "C" residues, 5-methyl-cytosines; all linkages are
phosphorothioate linkages. .sup.2Nucleotide numbers from Genbank
Accession No. M96652, locus name AHUMIL5RB@, SEQ ID NO.195, to
which oligonucleotides are targeted. Note: these sequences are also
common to GenBank accession nos. M96651 and X61176.
[0213]
19TABLE 19 Activity of Human IL-5 receptor a "Common" antisense
oligonucleotides on IL-5 receptor mRNA expression % % % control %
inhib'n control inhib'n SEQ ISIS membrane membrane soluble soluble
ID NO. IL-5 Ra IL-5 Ra IL-5 Ra IL-5 Ra NO: 16780 86 14 84 16 177
16781 42 58 39 61 178 16782 41 59 39 61 179 16783 49 51 47 53 180
16784 92 8 89 11 181 16785 19 81 32 68 182 16786 14 86 13 87 183
16787 49 51 47 53 184 16788 22 78 21 79 185 16789 14 86 12 88 186
16790 22 78 21 79 187 16791 46 54 45 55 188 16792 35 65 34 66 189
16793 14 86 13 87 190 16794 38 62 37 63 191
[0214] In this assay, ISIS 16781, 16782, 16783, 16785, 16786,
16,787, 16788, 16789, 16790, 16791, 16792, 16793 and 16794
inhibited both membrane and soluble IL-5 receptor a isoforms by
greater than 50% and are preferred. Of these, ISIS 16786, 16188,
16789, 16790 and 16793 inhibited both isoforms by greater than
75%.
[0215] ISIS 16793 was chosen for further study. It totally
inhibited expression of both soluble and membrane forms of human
IL-5 receptor a mRNA. This compound was found to have am IC50 of
approximately 2 .mu.M for reduction of IL-5 receptor a cell surface
protein in TF-1 cells. A 1-mismatch control had an IC50 of
approximately 3 .mu.M and 3- and 5-mismatch controls did not
inhibit IL-S receptor a expression below 75% of control.
Example 30
[0216] Antisense Oligonucleotides Targeted to Splice Sites in the
Human IL-5 Receptor a mRNA
[0217] The human IL-5 receptor a gene contains 14 exons. A
membrane-anchored form of the receptor and two soluble forms have
been identified. As with the mouse receptor, the membrane form is
active in signal transduction and the soluble forms are not, and
can act antagonistically. The mRNA transcript encoding the
membrane-anchored form of the human IL-5 receptor a contain exons
1-10 and 12-14. Exon 11 is spliced out by an alternative splicing
event. The major soluble isoform (soluble form 1) is generated as a
result of a normal splicing event and an in-frame stop codon in
exon 11. The other soluble form (soluble form 2) is generated by
the absence of splicing and therefore is generated by reading into
intron 11.
[0218] Transcripts encoding soluble forms of human IL-5 receptor a
do not contain exons 12, 13 or 14. It is, therefore, possible to
target sequences in exons 1-10 which are common to both soluble and
membrane forms of the receptor, or to selectively target sequences
only present in the membrane form (exons 12-14). Oligonucleotides
were also designed to target various intron/exon boundaries
downstream of exon 11, with the intention of preventing successful
splicing downstream of exon 11 and thus redirecting splice products
away from the membrane form and in favor of the soluble form of
IL-5 receptor a. A series of oligonucleotides were designed to
target various splice sites or (intron-exon boundaries) in the IL-5
receptor mRNA. These are shown in Table 20 and their effect on IL-5
receptor mRNA and cell surface protein levels is shown in Tables 21
and 22.
20TABLE 20 Nucleotide Sequences of Human IL-5R Oligonucleotides
ISIS NUCLEOTIDE SEQUENCE.sup.1 SEQ TARGET NO. (5' -> 3') ID NO:
REGION.sup.2 16746 ACCCAGCTTTCTGCAAAACA 196 I13/E14 16747
ACCCAGCTTTCTGCAAAACA " " 16748 ACCCAGCTTTCTGCAAAACA " " 16749
TCAACATTACCTCATAGTTA 197 E13/I13 16750 TCAACATTACCTCATAGTTA " "
16751 TCAACATTACCTCATAGTTA " " 16752 TAAATGACATCTGAAAACAG 198
I12/E13 16753 TAAATGACATCTGAAAACAG " " 16754 TAAATGACATCTGATAACAG "
" 16755 GAACACTTACATTTTACAGA 199 E12/I12 16756 GAACACTTACATTTTACAGA
" " 16757 GAACACTTACATTTTACAGA " " 16758 TCATCATTTCCTGGTGGAAA 200
I11/E12 16759 TCATCATTTCCTGGTGGAAA " " 16760 TCATCATTTCCTGGTGGAAA "
" 18009 TCATCATTTACTGGTGGAAA 201 mismatch 18010
TCAGCATTTACTGGTGTAAA 202 mismatch 18011 TCAGCAGTTACTTGTGTAAA 203
mismatch .sup.1Emboldened residues, 2'-methoxyethoxy- residues
(others are 2'-deoxy-) including "C" residues, 5-methyl-cytosines;
all linkages are phosphorothioate linkages. .sup.2Target regions
refer to intron/exon. junctions (splice sites) to which
oligonucleotides are targeted. I13/E14 indicates the junction
between the 3' end of intron 13 and the 5' end of exon 14. E13/I13
indicates the junction between the 3' end of exon 13 and the 5' end
of intron 13. I12/E13 indicates the junction between the 3' end of
intron 12 and the 5' end of exon 13. E12/I12 indicates the junction
between the 3' end of
[0219]
21TABLE 21 Modulation of Human IL-5 receptor a membrane form mRNA
expression by Splice Site Oligonucleotides (18 hr) ISIS SEQ TARGET
NO. ID NO: REGION % of CONTROL % INHIB 16746 196 I13/E14 36% 64%
16747 " 66 34 16748 " 25 75 16749 197 E13/I13 101 -- 16750 " 96 4
16751 " 96 4 16752 198 I12/E13 101 -- 16753 " 98 2 16754 " 101 --
16755 199 E12/I12 15.5 84 16756 " 96 4 16757 " 91 9 16758 200
I11/E12 176 -- 16759 " 81 19 16760 " 76 24
[0220] ISIS 16746, 16748 and 16755 inhibited IL-5 membrane receptor
mRNA expression by over 50% and are therefore preferred in this
assay. Northern blot analysis indicated that ISIS 16755 inhibited
the membrane receptor transcript without significantly inhibiting
the soluble form. Thus it is believed that ISIS 16755 redirects
splicing in favor of the membrane form, as is consistent with data
obtained with other non-RNAse H (e.g., uniform 2'-methoxyethoxy)
oligonucleotides targeted to splice sites.
22TABLE 22 Modulation of Human IL-5 receptor a protein expression
on the Cell Surface by Splice Site Oligonucleotides (36 hr) ISIS
NUCLEOTIDE SEQUENCE.sup.1 SEQ ID TARGET % of % NO. (5' -> 3')
NO: REGION.sup.2 CONTROL INHIB 16746 ACCCAGCTTTCTGCAAAACA 196
I13/E14 35 65% 16747 ACCCAGCTTTCTGCAAAACA " 80.5 19.5 16748
ACCCAGCTTTCTGCAAAACA " 40.5 59.5 16749 TCAACATTACCTCATAGTTA 197
E13/I13 75 25 16750 TCAACATTACCTCATAGTTA " 91 9 16751
TCAACATTACCTCATAGTTA " 101 -- 16752 TAAATGACATCTGAAAACAG 198
I12/E13 100.5 -- 16753 TAAATGACATCTGAAAACAG " 96 4 16754
TAAATGACATCTGAAAACAG " 100.5 -- 16755 GAACACTTACATTTTACAGA 199
E12/I12 10.5 89.5 16756 GAACACTTACATTTTACAGA " 101 -- 16757
GAACACTTACATTTTACAGA " 81 19 16758 TCATCATTTCCTGGTGGAAA 200 I11/E12
5.5 94.5 16759 TCATCATTTCCTGGTGGAAA " 75.5 24.5 16760
TCATCATTTCCTGGTGGAAA " 71 29 .sup.1Emboldened residues,
2'-methoxyethoxy- residues (others are 2'-deoxy-) including "C"
residues, 5-methyl-cytosines; all linkages are phosphorothioate
linkages. .sup.2Target regions refer to intron/exon junctions
(splice sites) to which oligonucleotides are targeted. 113/E14
indicates the junction between the 3' end of intron 13 and the 5'
end of exon 14. E13/113 indicates the junction between the 3' end
of exon 13 and the 5' end of intron 13. 112/E13 indicates the
junction between the 3' end of intron 12 and the 5' end of exon 13.
E12/112 indicates the junction between the 3' end of exon 12 and
the 5' end of intron 12. I11/E12 indicates the junction between the
3' end of intron 11 and the 5' end of exon 12.
[0221] ISIS 16746, 16748, 16755 and 16758 inhibited human IL-5
receptor a protein by over 50% in this assay and are therefore
preferred. ISIS 16758 and 16755 were chosen for further study. ISIS
16758 was found to have an IC50 of approximately 5 .mu.M for
reduction of IL-5 receptor a cell surface protein in TF-1 cells. A
1-mismatch control had an IC50 of 10 .mu.M and 3- and 5-mismatch
controls did not inhibit IL-5 receptor a expression. ISIS 16758
inhibited IL-5 receptor a protein expression without reducing mRNA
levels, consistent with an RNAse H-independent mechanism as
predicted for a uniformly 2'-methoxyethoxy modified
oligonucleotide.
Example 31
[0222] Induction of Apoptosis in TF-1 Cells Treated with IL-5
Receptor a Oligonucleotide
[0223] 1.times.10.sup.6 TF-1 cells cultured in IL-5 (0.5 ng/ml)
were collected 48 hours following oligonucleotide treatment
(transfection was by electroporation as described in previous
examples) and phosphatidylserine expression was detected as a
measure of apoptosis using the Annexin-V flow cytometry kit
(Clontech, Palo Alto, Calif.) according to the manufacturer's
instructions. Briefly, cells were resuspended in 0.2 ml of staining
buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 5 mM CaCl.sub.2) and 10
.mu.M of propidium iodide (50 .mu.g/ml) and 5 .mu.l of Annexin V
reagent were added at 41 C. for 10 minutes. The samples were
diluted with FacsFlow (Becton Dickinson, Franklin Lakes N.J.)
buffer and analyzed on a Becton Dickinson FACScan. Results are
shown in Table 23.
23TABLE 23 Apoptosis induction mediated by antisense to human IL-5
receptor a ISIS Oligo dose % Apoptotic SEQ ID No. Chemistry (.mu.M)
cells NO: No 14 oligo 16793 2'-MOE gapmer 5 19.8 190 "common"
sequence " 10 49.2 " " 15 62.3 " 18017 5-mismatch 5 20.5 194 for
16793 " 10 17.5 " " 15 20.3 " 16758 Uniform 2'- 10 33.1 200 MOE "
15 40.1 " " 20 50.4 " 18011 5-mismatch 10 19 203 for 16758 " 15
23.6 " " 20 21.8 " 16778 2'-MOE gapmer/ 7.5 29.9 171 Membrane-
specific " 12.5 49.2 " 18014 5-mismatch 7.5 38 175 for 16778 " 12.5
32.2 "
[0224] Apoptosis was shown to be induced in TF-1 cells cultured in
the presence of IL-5 by antisense oligonucleotide inhibitors of
IL-5 receptor a.
Example 32
[0225] Effect of IL-5 Receptor Oligonucleotides on Cell
Proliferation
[0226] 2.5.times.10.sup.4 TF-1 cells were incubated in 96-well
plates in 200 .mu.l complete RPMI in the absence of IL-5 for 16
hours following electroporation. IL-5 (0.5 ng/ml) was added and the
cultures were pulsed with 1 .mu.Ci of [.sup.3H]-thymidine for the
last 8 hours of a 48-hour culture period. The cells were harvested
on glass fiber filters and analyzed for thymidine incorporation
(proportional to cell proliferation) by liquid scintillation
counting. Results are shown in Table 24. Results are compared to
thymidine incorporation in untreated controls.
24TABLE 24 Inhibition of IL-5-induced TF-l cell proliferation by
human IL-5 receptor a antisense oligonucleotides % of control Oligo
thymidine dose incorpora- SEQ ID ISIS No. Chemistry (.mu.M) tion
NO: 16793 2'-MOE 5 44.5 190 gapmer "common" sequence " 10 11.1 "
18017 5- 5 89.1 194 mismatch for 16793 " 10 92.8 " 16758 Uniform 10
42.8 200 2'-MOE " 15 39.2 " " 20 19.9 " 18011 5- 10 95.6 203
mismatch for 16758 " 15 97.9 " " 20 84.6 "
[0227] These data demonstrate that antisense inhibitors of IL-5
receptor a greatly reduce cellular response to IL-5, i.e., cell
proliferation in response to IL-5. Control oligonucleotides were
ineffective.
Example 33
[0228] Oligonucleotides Targeted to Human IL-5 Receptor a
[0229] Oligonucleotides were designed to target the 5' untranslated
region of the IL-5 receptor a. These are shown in Table 25. Both
2'-methoxyethoxy gapmers and uniform 2'-methoxyethoxy compounds
were designed.
25TABLE 25 Nucleotide Sequences of Human IL-SR Oligonucleotides
ISIS NUCLEOTIDE SEQUENCE.sup.1 SEQ TARGET TARGET NO. (5' -> 3')
ID NO: SITE.sup.2 REGION 16963 AGCGGCAGAGCATTGAGAAC 204 0562-0581
5'-UTR 16964 AGCGGCAGAGCATTGAGAAC 205 " " 16965
GAAGCAGCGGCAGAGCATTG 206 0567-0586 5'-UTR 16966
GAAGCAGCGGCAGAGCATTG 207 " " .sup.1Emlboldened residues,
2'-methoxyethoxy- residues (others are 2'-deoxy-) including "C"
residues, 5-methyl-cytosines; all linkages are phosphorothioate
linkages. .sup.2Nucleotide numbers are from Genbank Accession No.
U18373, locus name AHSUl8373@, SEQ ID NO.208 to which
oligonucleotides are targeted.
Example 34
[0230] Mixed Backbone Oligonucleotides Were Designed to Target
Human IL-5 Receptor. These are Shown in Table 26.
26TABLE 26 Mixed Backbone Nucleotide Analogues of Human IL-SR
Oligonucleotides ISIS NUCLEOTIDE SEQUENCE.sup.1 BACKBONE SEQ TARGET
NO. (5' -> 3') CHEMISTRY ID NO: REGION 18018
TCATCATTTCCTGGTGGAAA P-S 200 16758 18019 TCATCATTTCCTGGTGGAAA P-O "
" 18020 GGGTGAGGAATTTGTGGCTC P-S 171 16778 18021
GGGTGAGGAATTTGTGGCTC P-O/P-S " " 18022 TCTGCACATGGAGCTCACTG P-S 190
16793 18023 TCTGCACATGGAGCTCACTG P-O/P-S " " .sup.1Emboldened
residues, 2'-methoxyethoxy- residues (others are 2'-deoxy-)
including "C" residues, 5-methyl-cytosines; P-O/P-S indicates
phosphodiester linkages in the 2'-MOE regions and phosphorothioate
linkages in the 2'-deoxy gap.
Example 35
[0231] Optimization of Human IL-5 Receptor a Oligonucleotides
[0232] A series of antisense oligonucleotides were designed based
on active sequences, with various placements of 2' methoxyethoxy
regions. These are shown in Table 27.
27TABLE 27 Nucleotide Analogues of Human IL-SR Oligonucleotides
ISIS NUCLEOTIDE SEQUENCE.sup.1 SEQ ID TARGET NO. (5' -> 3 ) NO:
REGION 18024 AGCTTAAACAGCCAAACGGG 169 16776 18025
AGCTTAAACAGCCAAACGGG " " 18026 AGCTTAAACAGCCAAACGGG " " 18027
AGCTTAAACAGCCAAACGGG " " 18028 AGCTTAAACAGCCAAACGGG " " 18029
AGCTTAAACAGCCAAACGGG " " 18030 CGCAGGTAAATTGAGTGTTG 166 16773 18031
CGCAGGTAAATTGAGTGTTG " " 18032 CGCAGGTAAATTGAGTGTTG " " 18033
CGCAGGTAAATTGAGTGTTG " " 18034 CGCAGGTAAATTGAGTGTTG " " 18035
CGCAGGTAAATTGAGTGTTG " " 18036 GGGTGAGGAATTTGTGGCTC 172 16778 18037
GGGTGAGGAATTTGTGGCTC " " 18038 GGGTGAGGAATTTGTGGCTC " " 18039
GGGTGAGGAATTTGTGGCTC " " 18040 GGGTGAGGAATTTGTGGCTC " " 18041
GGGTGAGGAATTTGTGGCTC " " 18042 AAGCCAGTCACGCCCTTTGC 187 16790 18043
AAGCCAGTCACGCCCTTTGC " " 18044 AAGCCAGTCACGCCCTTTGC " " 18045
AAGCCAGTCACGCCCTTTGC " " 18046 AAGCCAGTCACGCCCTTTGC " " 18047
AAGCCAGTCACGCCCTTTGC " " 18048 CAGGATGGTCCGCACACTTG 183 16786 18049
CAGGATGGTCCGCACACTTG " " 18050 CAGGATGGTCCGCACACTTG " " 18051
CAGGATGGTCCGCACACTTG " " 18052 CAGGATGGTCCGCACACTTG " " 18053
CAGGATGGTCCGCACACTTG " " 18054 TCTGCACATGGAGCTCACTG 190 16793 18055
TCTGCACATGGAGCTCACTG " " 18056 TCTGCACATGGAGCTCACTG " " 18057
TCTGCACATGGAGCTCACTG " " 18058 TCTGCACATGGAGCTCACTG " " 18059
TCTGCACATGGAGCTCACTG " " 18060 GAACACTTACATTTTACAGA 199 16755 18061
GAACACTTACATTTTACAGA " " 18062 GAACACTTACATTTTACAGA " " 18063
GAACACTTACATTTTACAGA " " 18064 TCATCATTTCCTGGTGGAAA 200 16758 18065
TCATCATTTCCTGGTGGAAA " " 18066 TCATCATTTCCTGGTGGAAA " " 18067
TCATCATTTCCTGGTGGAAA " " .sup.1Ernboldened residues,
2'-methoxyethoxy- residues (others are 2'-deoxy-) including "C"
residues, 5-methyl-cytosines; all linkages are phosphorothioate
linkages.
Example 36
[0233] Modulation of mRNA Splicing of IL-5 Receptor a by Antisense
Peptide Nucleic Acids (PNAs)
[0234] In order to determine the effectiveness of peptide nucleic
acids as selective modulators of alternative mRNA splicing, a
series of PNA oligonucleotide mimetics having the same nucleobase
sequence (SEQ ID NO: 135) as an antisense sequence shown to produce
exclusion of exon 9 from the IL-5 Receptor a processed mRNA were
synthesized and evaluated.
[0235] Murine BCL.sub.1 cells were chosen for screening PNA
oligonucleotides targeted to murine IL-5 receptor a and were
maintained in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal bovine serum (Sigma Chemical Company, St.
Louis, Mo.), 10 mM Hepes, pH 7.2, 50 uM 2-ME, 2 mM L-glutamine, 100
U/mL penicillin and 100 ug/mL streptomycin.
[0236] BCL.sub.1 cells were transfected by electroporation as
described previously with 0.25, 0.5, 1, 5 and 10 .mu.M of each of
the compounds in Table 28. ISIS 110790 (SEQ ID NO: 209) is a
shortmer (15 bp) of ISIS 21752 (SEQ ID NO: 135, described
previously) lacking the first five nucleobases and having the same
internucleoside linkages and modifications as ISIS 21752. ISIS
32297 (SEQ ID NO: 209) is a peptide nucleic acid with the
nucleobase sequence of ISIS 110790 while ISIS 28496, a peptide
nucleic acid with the same nucleobase sequence of ISIS 32297,
contains the amino acid lysine conjugated to the COOH terminal end.
The control peptide nucleic acid, ISIS 32304 (SEQ ID NO: 210) is a
3 base pair mismatch of ISIS 28496. At 24 hours, total RNA was
extracted and analyzed by RPA. The results are shown in Table 29.
Expression data for both isoforms are expressed as a percent of
control. "N.D." indicates no data.
28TABLE 28 PNA oligonucleotide mimetics SEQ ISIS ID Number
Nucleotide Sequence NO: Backbone 21752 GCCATTCTACCAAGGACTTC 135
2'-O-MOE/P-S 110790 TCTACCAAGGACTTC 209 2'-O-MOE/P-S 32297
H-TCTACCAAGGACTTC-NH.sub.2 209 PNA 28496
H-TCTACCAAGGACTTC-Lys-NH.sub.2 209 PNA 32304
H-TCAACCTAGAACTTC-Lys-NH.sub.2 210 PNA
[0237]
29TABLE 29 Alteration of splicing IL5Ra splicing pattern by PNAs
ISIS Membrane Isoform Soluble Isoform Number 0.25 0.5 1 5 10 0.25
0.5 1 5 10 21752 N.D. 58 35 5 3 N.D. 119 150 170 160 110790 N.D. 75
59 7 7 N.D. 119 140 158 160 32297 78 55 41 15 N.D. 110 122 135 140
N.D. 28496 85 59 42 6 N.D. 119 135 150 138 N.D. 32304 110 102 95 95
N.D. 110 105 95 100 N.D.
[0238] These data show that peptide nucleic acids (PNAs) of shorter
length and/or with the additional lysine modification are more
potent in reducing expression and redirecting splicing of IL-5
Receptor a than their 2'-O-MOE-modified counterparts of the same
sequence. Treatment of cells with antisense PNA resulted in
dose-dependent, specific down regulation of the membrane isoform
and enhanced expression of the soluble isoform with an effective
concentration (EC50) lower than that observed with the
corresponding 2'-O-MOE antisense oligonucleotides. These properties
makes PNAs and modified PNAs a promising new class of lower
molecular weight splicing modulators.
Sequence CWU 1
1
210 1 6727 DNA Mus musculus 1 tgtacctccc acatctgctg gtgtgtacca
ccacacctag taagatattc tcaacattta 60 tgtattttag cctaaccctg
ttggaggtat acatttgaat acattttttc tcactttatc 120 aggaattgag
tttaacacat attaaagcag gtgtggggca gggagggggg gataaaaaag 180
aaggtgctca agaaaagccg atcacgctcc caagagtgtg agcatgggcg tctctagaga
240 gatccgccat atatgcacaa cttttaaaga gaaattcaat aaccagaatg
gagtgtaaat 300 gtggatcaaa gttgtagaaa cattctttta tgttatagaa
aatgcttttt aagcaggggt 360 gggggtcaag atgttaacta ttattaaaga
gcaaaaaaaa aaaaatgcat tttgtttgaa 420 gacccagggc actggaaacc
ctgagtttca ggactcgcct ttattaggtg tcctctatct 480 gattgttagc
aattattcat ttcctcagag agagaataaa ttgcttgggg attcggccct 540
gctctgcgct cttcctttgc tgaaggccag cgctgaagac ttcagagtca tgagaaggat
600 gcttctgcac ttgagtgttc tgactctcag ctgtgtctgg gccactgcca
tggagattcc 660 catgagcaca gtggtgaaag agaccttgac acagctgtcc
gctcaccgag ctctgttgac 720 aagcaatgag gtaaagtata acttattcct
tcagctttgt ttttaagatc aggaccttgc 780 tataccgctc tgactggcct
caaacttgct atgtagggta ggctgtccta acccctacca 840 gatctcctta
cctatgtctc ccaaatacta ggattacaga cacattacct tgcctgacgc 900
tatggttctt cagaatgcat aaatagctgc atttggcctt taatcccaga acttgggagg
960 cagggtcagg tggatctctg tgagttcaag gccagacttg tctacgtggc
cagttacagg 1020 acagccagag ctaaagcaag accctgattc aaaataattt
tttttcaaaa caaaaaaaaa 1080 aaacccaaac catttgtggc aattcatttc
taaacataaa gatctgcttt aaatagtgca 1140 attatggctt gttcccttgc
cttcttgctc ccgttctgtc ctcttgtccc actctctccc 1200 cattccaccc
ccaccatgtg ctcatggccc gcatctctac ttctctactc tctttctctc 1260
cctctcccct ccttcttcct ttccctctct ctctccctct tcttctcctc ctctctttct
1320 ctctctctcc ctctctctct ctctttctct ctctctctgc ttttttctat
ctctactacc 1380 ctctcaactc ccctctccat gccctgaata agctctattc
tatactaaaa aaaaaaaagt 1440 gcaattatga atgtgttagt gttaatgcac
aggtgataac cctatcacca gcaagcattg 1500 cattaaaaaa ggcaacggac
tctctttagg atgaccctat gatgttcttt cctttgcaga 1560 cgatgaggct
tcctgtccct actcataaaa atgtaagtta ttctttactg ccgtgcttgc 1620
atgagtaagt cagcttcgca tactaagcta taagtcatct gcatctagct ttctggtgtt
1680 gtgtgtgtct gggatgggga cctctctagg tctcaagctc ctgggttcaa
gtgattctct 1740 tgccttgata gagcagctgg gacacaggcc tgtgccacca
cacccagcag agcttttgat 1800 ttcagttaaa ctgtttgact ttcttggaaa
agaaaattta tgtaggtaga tatgaaagtt 1860 tgtgcttata aataaaaaga
atatgagagt ggcaaattat gtaatcccag tacttgggag 1920 ccaaaggcag
gggtagtctg agtctagggc cagcttagat acattgccct gtatgtatca 1980
aaagtaaatc ctataaataa ataaacaaaa acattagagg gctggagata taagctctgt
2040 tgatagatgg cctaatatgc tgggttgact cttagcaccc cataaactaa
acatggaagt 2100 acctggctgt aatctcatga tggtgaaatg gaggcgggaa
gatcataggt tcaaggtcat 2160 cctcagctac atttttgagc tagaggccag
cctgggctat gagacacgca aaaaccacca 2220 gccaattaat attaggaatg
gctttgagct agatctgtta tgtaagtggc cagctggagc 2280 tgtcagtcat
acatctcaca gcctcacaag attctttgca tggcgagagg tcctgctggg 2340
ctccctttgg ctctgtccat ggctctcttc atcctagtgc ctctctttgt tttccttgtc
2400 ttatttctta ctgctgagga tcaagcccag ggccttcagt gtgtgaagtg
agcactctac 2460 cactgaattc cagagcccgc ccactctaat gcctttctga
aagtattaag agtttagggt 2520 tatatattcc ttttgtttat tttatgtgta
tgagcatttt gcctgcatat atatatatat 2580 atatatatat atatatatat
gtgtgtgtgt gtgtgtgtgt gtgtgtatat atatatgtat 2640 gtatgtatgt
atgtatgtat gtatgtatat gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 2700
gttccacgta tgtgtctatg tgtctggtgt tcctgaaggc taaaagaagg gcatcagatc
2760 acctggggct ggatatgcag atggttgtga gccaaccatc tggatgctgg
gaactgcatc 2820 aagtgttctt aaccactgag ccatctctcc cgctcagagg
gttatattct taggtaatga 2880 tagaaagaca taaaaatatc atgaatgcct
ttattaataa tttctaaaca gtttaatgaa 2940 tatgactatg tagtgatatt
gtatacattt caatattatc ttattctagc gtaaagtaca 3000 ttatttaact
ttttctaaat agaagaaaat tcatcagcct aaatttcaaa agaaaatatt 3060
aatatgggtg tggtaccact cacctttaat ccagatggtt gtgagccacc acaagggtgc
3120 tggtaactga acccaggtcc tctggaagag gacccagtga tcttaaccac
tgagccatct 3180 ccccagcccc aatcctaact ttgggttcat ttttttgaaa
tgatctcatg tagcactagc 3240 tggcctcaaa ctctatgtat cagaggctgg
ccttcaactc ctgatcctct tacctcaact 3300 tcctgaatgc tggcattaca
gataagcacc atcacatctt gtattgtctg gggtttttta 3360 ttgatgcatt
taaattgcat gtatttattg catatggcat gatatttcaa aatatgtgta 3420
cgttgtgggc agtctgatct atttgcttct tgataatctt ctttcagcac cagctatgca
3480 ttggagaaat ctttcagggg ctagacatac tgaagaatca aactgtccgt
gggggtactg 3540 tggaaatgct attccaaaac ctgtcattaa taaagaaata
cattgaccgc caaaaagtaa 3600 gttccccagg gaccctgtga atccggctgc
agctggttct ccaggagcca acctgacagt 3660 ctgttctttt cacaggagaa
gtgtggcgag gagagacgga ggacgaggca gttcctggat 3720 tacctgcaag
agttccttgg tgtgatgagt acagagtggg caatggaagg ctgaggctga 3780
gctgctccat ggtgacagga cttcacaatt taagttaaat tgtcaacaga tgcaaaaacc
3840 ccacaaaact gtgcaaatgc aagggatacc atatgctgtt tccatttata
tttatgtcct 3900 gtagtcagtt aaacctatct atgtccatat atgcaaagtg
tttaaccttt ttgtatacgc 3960 ataaaagaaa ttcctgtagc gcaggctggc
ctcaaactgg taatgtagcc aaggataacc 4020 ttgaatttct gatcctcctg
cctcctcttc ctgaaggctg aggttacaga catgcaccat 4080 tgccactagt
tcatgaagtg ctggagatgg aacccaaggc tttgtgcatg ttaccaactg 4140
agttatactc cctccccctc atcctcttcg ttgcatcagg gtctcaagta ttccaggctg
4200 actttgaact cagtgtgtag ccaagggtga ccctgaactc ttggtccaga
tggacgcagg 4260 aggatcacat acccaacctt agcatccttt ctcctagccc
ctttagatag atgatactta 4320 atgactctct tgctgaggga tgccacaccg
gggcttcctg ctcctatcta acttcaattt 4380 aatacccact agtcaatctc
tcctcaactc cctgctactc tccccaaact ctagtaagcc 4440 cacttctatt
tcttggggag agagaaggtt gacttttctt atgtcctatg tatgaatcag 4500
actgtgccat gactgtgcct ctgtgcctgg agcagctgga ttttggaaaa gaaaagggac
4560 atctccttgc agtgtgaatg agagccagcc acatgctggg ccttacttct
ccgtgtaact 4620 gaacttaaga agcaaagtaa ataccacaac cttactaccc
catgccaaca gaaagcataa 4680 aatggttggg atgttattca ggtatcaggg
tcactggaga agcctccccc agtttactcc 4740 aggaaaaaca gatgtatgct
tttatttaat tctgtaagat gttcatatta tttatgatgg 4800 attcagtaag
ttaatattta ttacaacgta tataatattc taataaagca gaagggacaa 4860
ctcaaattca gtttgctatt ggtcttttct aaccctgggt gtgtgcaggg acccagagga
4920 gagactgagt atgtcctgac taagcacttt cagctcctta gagcttcagg
gagcaccaag 4980 ggtggacttg gtagtggtat cgggagcaag aacaagggct
gggactgagc ctggatctcc 5040 ctatgtagga gtatgtccag atggctcagg
gtgaacagga gaggaatgaa tgagaggatg 5100 aatgaatgaa tgaataaatg
aatgaatggg agatcgctcc attaataaag tgcttgctgt 5160 acaaggatga
agagctgagt tcgagctcca aaacccattt cagaaagctg ggcatggtgg 5220
gggcacactt gtagtcctga cactgggaga cagaaatagc cagatccctg gggctctctg
5280 ttcagccaac ctaaatgaat tggtgagttc tggaccagtg agagatcttc
tctcaaaaag 5340 caaggtggaa gccgagcgtg gtgacacacg cctttaattc
cagcacttgg gaggcagagg 5400 caggcggatt tctgagttcg aggccagcct
ggtctacaaa gtgagttcca ggacagccag 5460 ggctacacag agaaaccctg
tctcaaaaaa caaacaaaca aacaaacaaa caaaccacca 5520 tgaactacct
gtgtatgcat gttgtgtgtg cttgcattgt gcaggtcaaa tgaacacact 5580
gggactcttc cactaacact ctctacctcg ttccctaaga gggtctcctg ctgaacatgg
5640 agtttcccat ttcttttggt taggctggca gccagccagc aagtcccagc
gatcctcctg 5700 tctcctcttc ctcctgctca gccccagggg tggagtctta
ggtatgcgtg gccatgccag 5760 gctttttcca tgggtgctgg agatccagac
gcagcttctc atgttcgcgc agtggcactc 5820 ttgcccactg aagcatcttc
catcttgccc actgaagcat ctcccatctt acccactcaa 5880 gcatcttcca
tcttacccac tcaagcatct tccatcttac ccactcaagc atcttccatc 5940
ttacccactc aagcatcttc cagctcctta gtatgttttt tttttaaaca tgtacttggc
6000 tttttaaaat tgtaataaac taaaggtata caatatgtat tgattgatat
gcttacttat 6060 gtatttatct ttattttctt atttttttaa aaaatttatt
ttatttatat gaatacactg 6120 tagctgactt cagacacacc agaacagggc
attggatccc attacggatg gttgtgagcc 6180 accatgtggt tgctgggaat
tgaactcagg acctttggaa gaacagtctc tctggctctg 6240 tagttatctt
tcagtatact tttccttgaa aattttatat gtctgtgcga tctattctgg 6300
tcctaccatt cactctcact cttcctggac ttcccagtat ggccccctcc cgatttcaaa
6360 tcttctcact cttatttttt agcccactga gttcagttag tgttgtccct
atgagcacgt 6420 gtggaccatc tacttgagct taggcaacct accagtggcc
acatccctac aggaaaggta 6480 ctcttcctct cttggtggcc ataaaccccc
aacgggtcct cacatagggc aggagcctta 6540 ggagtttccc tccccattca
tactaaactt tggttggctt gatggtgtga agataaccac 6600 agctgctgtg
aggtcctgag tacaagggcc aagtcacgtc caggaggcag catctcacag 6660
tacttacccc cagtctctgg ctcgaacatc cttcccacca tcccccttca tcatgttcct
6720 taagctt 6727 2 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 2 cccaagcaat ttattctctc 20 3 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 3
tcagcaaagg aagagcgcag 20 4 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 4 cactgtgctc atgggaatct 20 5 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 5
actttacctc attgcttgtc 20 6 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 6 tcagagcggt atagcaaggt 20 7 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 7
ctcatcgtct gcaaaggaaa 20 8 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 8 tatgagtagg gacaggaagc 20 9 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 9
atttttatga gtagggacag 20 10 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 10 acaaggaaaa taaagaataa 20 11 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
11 acaaggaaaa caaagagagg 20 12 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 12 ctggtgctga
aagaagatta 20 13 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 13 ccacggacag tttgatcctt 20 14 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 14
aatgacaggt tttggaatag 20 15 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 15 gcggtcaatg tatttcttta 20 16 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
16 ggaacttact ttttggcggt 20 17 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 17 cagactgtca
ggttggctcc 20 18 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 18 tcctcgccac acttctcctg 20 19 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 19
aactgcctcg tcctccgtct 20 20 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 20 tactcatcac accaaggaac 20 21 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
21 ctcagcctca gccttccatt 20 22 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 22 ttaaattgtg
aagtcctgtc 20 23 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 23 aaatataaat ggaaacagca 20 24 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 24
ctacaggaca taaatataaa 20 25 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 25 tatacaaaaa ggttaaacac 20 26 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
26 ggttatcctt ggctacatta 20 27 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 27 aactgcctcc
tcctccgtct 20 28 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 28 aactgccacc tgctccgtct 20 29 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 29
aactggcacc tgcaccgtct 20 30 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 30 ggttatccta ggctacatta 20 31 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
31 ggttatcgta gcctacatta 20 32 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 32 ggttaacgta
gccaacatta 20 33 29 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 33 agtgttctga ctctcagctg tgtctgggc 29
34 24 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 34 ttcagagtca tgagaaggat gctt 24 35 22 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 35
accactgtgc tcatgggaat ct 22 36 27 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 36 aaggccgaga
atgggaagct tgtcatc 27 37 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 37 ggcaaattca acggcacagt 20 38 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 38
gggtctcgct cctggaagat 20 39 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 39 ctttggcaaa gaaagtgcat 20 40 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
40 cgttctgcgt ttgcctttgg 20 41 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 41 tcctcatggc
tctgaaacgt 20 42 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 42 aagaaaatta cctcattggc 20 43 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 43
ttacagcaca ccagcattca 20 44 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 44 tcctcagagt ctggagagga 20 45 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
45 ggaacaggaa tcctcagagt 20 46 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 46 tttaacttac
atttttatgt 20 47 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 47 tttacttatt catgccatca 20 48 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 48
gacacgatgc tctttgggaa 20 49 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 49 cattttaata tgaccaggca 20 50 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
50 ttctaggcaa caaaccacca 20 51 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 51 acagttggtg
ctaaatgagg 20 52 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 52 ttcttcagtg cacagttggt 20 53 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 53
acccccttgc acagtttgac 20 54 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 54 tggccgtcaa tgtatttctt 20 55 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
55 tgtaacttac tttttggccg 20 56 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 56 tccatagaaa
taggcacagc 20 57 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 57 cacacttttt ctgtgaaaaa 20 58 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 58
attggtttac tctccgtctt 20 59 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 59 ttatccactc ggtgttcatt 20 60 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
60 tccttctcct ccaaaatctt 20 61 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 61 tggccctcat
tctcactgca 20 62 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 62 tctggcaaag tgtcagtatg 20 63 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 63
ttgcctggag gaaaatactt 20 64 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 64 ctttggcaaa gaaagtgcat 20 65 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
65 cgttctgcgt ttgcctttgg 20 66 20 DNA Artificial Sequence
Description
of Artificial SequenceSynthetic 66 aagaaaatta cctcattggc 20 67 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
67 tcctcagagt ctggagagga 20 68 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 68 tttaacttac
atttttatgt 20 69 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 69 acagttggtg ctaaatgagg 20 70 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 70
tgtaacttac tttttggccg 20 71 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 71 cacacttttt ctgtgaaaaa 20 72 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
72 tctggcaaac tgtcagtatg 20 73 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 73 tctggcatac
tctcagtatg 20 74 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 74 tctgggatac tctgagtatg 20 75 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 75
ttgcctggac gaaaatactt 20 76 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 76 ttgcctgcac gtaaatactt 20 77 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
77 ttgccagcac gtatatactt 20 78 3230 DNA Homo sapiens 78 atcctaatca
agaccccagt gaacagaact cgaccctgcc aaggcttggc atttccattt 60
caatcactgt cttcccacca gtattttcaa tttcttttaa gacagattaa tctagccaca
120 gtcatagtag aacatagccg atcttgaaaa aaaacattcc caatatttat
gtattttagc 180 ataaaattct gtttagtggt ctaccttata ctttgttttg
cacacatctt ttaagaggaa 240 gttaattttc tgattttaag aaatgcaaat
gtggggcaat gatgtattaa cccaaagatt 300 ccttccgtaa tagaaaatgt
ttttaaaggg gggaaacagg gatttttatt attaaaagat 360 aaaagtaaat
ttatttttta agatataagg cattggaaac atttagtttc acgatatgcc 420
attattaggc attctctatc tgattgttag aaattattca tttcctcaaa gacagacaat
480 aaattgactg gggacgcagt cttgtactat gcactttctt tgccaaaggc
aaacgcagaa 540 cgtttcagag ccatgaggat gcttctgcat ttgagtttgc
tagctcttgg agctgcctac 600 gtgtatgcca tccccacaga aattcccaca
agtgcattgg tgaaagagac cttggcactg 660 ctttctactc atcgaactct
gctgatagcc aatgaggtaa ttttctttat gattcctaca 720 gtctgtaaag
tgcataggta atcatttgtg atggttcctt tactatatat agagatctgt 780
tataaataat aagattctga gcacattagt acatgggtga taactacatc accagcaaac
840 attctgttaa aagttatgaa tgctggtgtg ctgtaaaaat gattgtattt
cctttcctct 900 ccagactctg aggattcctg ttcctgtaca taaaaatgta
agttaaatta tgattcagta 960 aaatgatggc atgaataagt aaatttcctg
ttttaagctg taaatcatta gttatcattg 1020 gaactattta attttctata
ttttgttttc atatgggtgg ctgtgaatgt ctgtacttat 1080 aaatatgagg
aatgactttt tatcaagtag aatcctttaa acaagtggat taggctcttt 1140
ggtgatgttg ttagtttgcc ttcccaaaga gcatcgtgtc aggattcttt ccagaaggat
1200 tccacactga gtgagaggtg cgtgctagtc tccgtgcagt tctgactctt
tctcactcta 1260 acgtgtttct gaaagtatta gcaactcaga attatatttt
tagaaccatg atcagtagac 1320 attaaaatat ataacaaatg ccctatatta
ataattctgc atacttaaat aattatgact 1380 atatgatggt gtgtatgcat
tgaatatgcc tggtcatatt aaaatgtaaa atatatagtt 1440 tattagtcta
aatagaataa aactaccagc tagaactgta gaaacacatt gatatgagtt 1500
taatgtataa tgcattacac ttccaaaaca tttttttcca gttacataat taagttatat
1560 cctttataaa actcctcagt aatcatataa gcttcatcta ctttttgaaa
attttatctt 1620 aatatgtggt ggtttgttgc ctagaaaaca aacaaaaaac
tctttggaga agggaactca 1680 tgtaaatacc acaaaacaaa gcctaacttt
gtggaccaaa attgttttaa taattatttt 1740 ttaattgatg aattaaaaag
tatatatatt tattgtgtac aatatgatgt tttgaagtat 1800 gtatacattg
cagaatggac aatggaccaa atttttatac cttgtcttga ttatttgcat 1860
tttaaaaatt ttcctcattt agcaccaact gtgcactgaa gaaatctttc agggaatagg
1920 cacactggag agtcaaactg tgcaaggggg tactgtggaa agactattca
aaaacttgtc 1980 cttaataaag aaatacattg acggccaaaa agtaagttac
acacattcaa tggaagctat 2040 atttgtcctg gctgtgccta tttctatgga
attgacagtt tcctgtaata cctattgtca 2100 tttttctttt ttcacagaaa
aagtgtggag aagaaagacg gagagtaaac caattcctag 2160 actacctgca
agagtttctt ggtgtaatga acaccgagtg gataatagaa agttgagact 2220
aaactggttt gttgcagcca aagattttgg aggagaagga cattttactg cagtgagaat
2280 gagggccaag aaagagtcag gccttaattt tcaatataat ttaacttcag
agggaaagta 2340 aatatttcag gcatactgac actttgccag aaagcataaa
attcttaaaa tatatttcag 2400 atatcagaat cattgaagta ttttcctcca
ggcaaaattg atatactttt ttcttattta 2460 acttaacatt ctgtaaaatg
tctgttaact taatagtatt tatgaaatgg ttaagaattt 2520 ggtaaattag
tatttattta atgttatgtt gtgttctaat aaaacaaaaa tagacaactg 2580
ttcaatttgc tgctggcctc tgtccttagc aatttgaagt tagcacagtc cattgagtac
2640 atgcccagtt tggaggaagg gtctgagcac atgtggctga gcatccccat
ttctctggag 2700 aagtctcaag gttgcaaggc acaccagagg tggaagtgat
ctagcaggac ttagtgggga 2760 tgtggggagc agggacacag gcaggaggtg
aacctggttt tctctctaca gtatatccag 2820 aacctgggat ggtcgaaggg
taaatggtag ggaataaatg aatgaatgtc gtttccaaga 2880 tgattgtaga
actaaaatga gttgtaagct cccctggaag aagggatgtg gaacctgtaa 2940
ctaggttcct gcccagcctg tgagaagaat ttggcagatc atctcattgc cagtatagag
3000 aggaagccag aaaccctctc tgccaaggcc tgcaggggtt cttaccacct
gaccctgcac 3060 cataacaaaa ggacagagag acatggtagg gcagtcccat
tagaaagact gagttccgta 3120 ttcccggggc agggcagcac caggccgcac
aacatccatt ctgcctgctt atggctatca 3180 gtagcatcac tagagattct
tctgtttgag aaaacttctc tcaaggatcc 3230 79 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 79 gacctgtcca
gtgagcttct 20 80 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 80 tagccgaata ctggaaaggt 20 81 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 81
aacacaggca ccatggtagc 20 82 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 82 ctcttggtca ggatttgggt 20 83 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
83 tcctcacgct agctgcaaag 20 84 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 84 atggccttaa
gtgggtgtgg 20 85 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 85 gagccattaa tgtgcacagc 20 86 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 86
tccactcgcc ccaccttcct 20 87 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 87 aacaagacga agcaggcagc 20 88 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
88 ccggaaccgg tggaaacaac 20 89 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 89 ccaacctctt
ccacacaatg 20 90 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 90 tcccatgact tcaaatccaa 20 91 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 91
gcaaaatgcc atcaaaacgt 20 92 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 92 cgagctctac caccgcctgg 20 93 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
93 caagctggcc tcgaactcag 20 94 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 94 ggatgggttg
gtgacttgca 20 95 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 95 tgaggaaacc aaaggcccat 20 96 20 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 96
tgtctcccac ttgcgtcagg 20 97 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 97 ttgaacaggc ctatggaaca 20 98 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
98 tctttttcac cccaggcacg 20 99 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 99 aattcccatg
gatcctcttg 20 100 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 100 atccagcaat cacctccaaa 20 101 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
101 tgttcagccc atcaaaaaga 20 102 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 102 atttggctga
caggaccccg 20 103 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 103 tccagagact gccccaccca 20 104 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
104 catctgcttc tgtattgcca 20 105 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 105 ccttttagct
ccttgggtac 20 106 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 106 catttctgag ggttgctggg 20 107 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
107 catctgattg tgtcttgcca 20 108 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 108 catctgcttg
tgtattgcca 20 109 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 109 cacctgattg tgtcttgtca 20 110 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
110 tgtccctcct tttggtgggg 20 111 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 111 ttagctctgt
ctctgctgat 20 112 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 112 aactgctggc cagagttgta 20 113 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
113 catagttaaa gcaatgatct 20 114 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 114 gtttctcata
ttcagtaacc 20 115 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 115 ggagtcctgt atgagttcat 20 116 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
116 tctgtgcatc ccaggtgctg 20 117 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 117 ctggctgtcc
tggaactcac 20 118 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 118 ttcaaggtaa gtcaagcaac 20 119 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
119 ctgatggcta ccactggcaa 20 120 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 120 cactctcaat
gagttctatc 20 121 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 121 tgatgctggt tgatcaatct 20 122 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
122 tcaataggga atggtgtctt 20 123 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 123 ttccagagta
cctagaagcc 20 124 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 124 ccaacaggtt gccatgaagg 20 125 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
125 agagattaga attgactaag 20 126 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 126 actattgcat
atactagcaa 20 127 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 127 ccatccaata tacaaccacc 20 128 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
128 ctcatggaag gagttacaga 20 129 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 129 tgtggatact
tcactgcttc 20 130 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 130 atccaataga tgactgtgag 20 131 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
131 gttcatattg ttgttcctgc 20 132 3571 DNA Mus musculus 132
gaaataattg gtaaacacag aaaatgtttc aatagaaaaa agaggaaaca gaacactgtg
60 tagccctgtt atcagcagag acagagctaa cgctggggat accaaactag
aagaagctca 120 ctggacaggt cccggtatgc agttctattt ttgttgatgg
ctctgtatct aatgtgttca 180 tttgtaccaa ggatctaacc agggtcttcc
agagtctgag caagcttctc ccactgagct 240 acatcacagc cccctgttta
ttggaagaag aaatacttac acctttccag tattcggcta 300 ccatggtgcc
tgtgttacta attcttgtgg gagctttggc aacactgcaa gctgacttac 360
ttaatcacaa aaagttttta cttctaccac ctgtcaattt taccattaaa gccactggat
420 tagctcaagt tcttttacac tgggacccaa atcctgacca agagcaaagg
catgttgatc 480 tagagtatca cgtgaaaata aatgccccac aagaagacga
atatgatacc agaaagactg 540 aaagcaaatg tgtgaccccc cttcatgaag
gctttgcagc tagcgtgagg accattctga 600 agagcagcca tacaactctg
gccagcagtt gggtttctgc tgaactcaaa gctccaccag 660 gatctcctgg
aacctcggtt acgaatttaa cttgtaccac acacactgtt gtaagtagcc 720
acacccactt aaggccatac caagtgtccc ttcgttgcac ctggcttgtt gggaaggatg
780 cccctgagga cacacagtat ttcctatact acaggtttgg tgttttgact
gaaaaatgcc 840 aagaatacag cagagatgca ctgaacagaa atactgcatg
ctggtttccc aggacattta 900 tcaacagcaa agggtttgaa cagcttgctg
tgcacattaa tggctcaagc aagcgtgctg 960 caatcaagcc ctttgatcag
ctgttcagtc cacttgccat tgaccaagtg aatcctccaa 1020 ggaatgtcac
agtggaaatt gaaagcaatt ctctctatat acagtgggag aaaccacttt 1080
ctgcctttcc agatcattgc tttaactatg agctgaaaat ttacaacaca aaaaatggtc
1140 acattcagaa ggaaaaactg atcgccaata agttcatctc aaaaattgat
gatgtttcta 1200 catattccat tcaagtgaga gcagctgtga gctcaccttg
cagaatgcca ggaaggtggg 1260 gcgagtggag tcaacctatt tatgtgggaa
aggaaaggaa gtccttggta gaatggcatc 1320 tcattgtgct cccaacagct
gcctgcttcg tcttgttaat cttctcactc atctgcagag 1380 tgtgtcattt
atggaccagg ttgtttccac cggttccggc cccaaagagt aacatcaaag 1440
atctccctgt ggttactgaa tatgagaaac cttcgaatga aaccaaaatt gaagttgtac
1500 attgtgtgga agaggttgga tttgaagtca tgggaaattc cacgttttga
tggcattttg 1560 ccattctgaa atgaactcat acaggactcc gtgataagag
caaggactgc tatttcttgg 1620 caaggaggta tttcaaatga acactcagag
ccaggcggtg gtagagctcg cctttaatac 1680 cagcacctgg gatgcacaga
cgggaggatt tctgagttcg aggccagctt ggtctataaa 1740 gtgagttcca
ggacagccag agctacacag agaaaccctg tctcgaaaaa acaaacaaac 1800
aaacaaacaa acaaaaatga acactcaatt tgaatgcaag tcaccaaccc atccagacat
1860 gagtcaccaa tgtcccattt cataaagtgt gcatgcctca ctcaaacctc
cttgctcaca 1920 gcatagcacc agactcaccc agagcatggg cctttggttt
cctacccaga gtaccatgtt 1980 ataccagtgt gtctttgaaa gttgcttgac
ttaccttgaa ctttttgcac aggagacagt 2040 ttttttaagc taatgtcaca
catgtttact ttgggttaag ttgccagtgg tagcactcag 2100 ctacagtgac
aggaggaaag gatagaactc attgagagtg aacccaaatt caagactgtc 2160
tttcctgacg caagtgggag acacaatttc atggtgcttt tcccctttca gttctagaat
2220 agtttccttt ctagaactgt gcctgtgtct taaagcataa ggtaacattg
aggcaaaaac 2280 aaagactatg tcccacatgt ccctgtgttc cataggcctg
ttcaaggaaa tgtctaagcc 2340 aaagtaagtt taagtcaccg tgcctggggt
gaaaaagatg gttcagatga cgaagaagca 2400 tgagggcctg agattgatca
accagcatca agaaacaaca acaacaacag cagcagcaac 2460 aacaaaacag
tgcaagaagc acattcctat aaccccagag ttgggagata aagacaagag 2520
gatccatggg aattgtagtt caaccagttt agccaattat gttatctcta ggttcactga
2580 gagaaatggt cttaaaaatt taaggtggag agtgactagg cagatcctct
gatactgact 2640 tctgccctaa atatgcatac acatgtacac acacaacaca
aagacaccat tccctattga 2700 gagagaagac agaagcttgt tcaaggatta
aattcttcaa ggcttctagg tactctggaa 2760 atgacctgag aaagacattg
aaaataattc tgctttggag gtgattgctg gatctagaat 2820 gtacttccca
aagagatgtt gatgaaagag ccttcatggc aacctgttgg tcaactcatg 2880
cttagtcaat tctaatctct taaattaggg tttcctatac atattacaat tgtataaaaa
2940 tgtattctct aaatatcttc attaatgaag ctgtatctat aggtcttttt
gatgggctga 3000 acatagaagc aaacacactt atgtgttggg aagaggaata
agtagtgata gagggaccta 3060 gtggtagtta ttttacatag tcctgaagag
ctaaagacaa tgaaagaaga aatggtactc 3120 acaagagaga gagctatgtc
ggggtcctgt cagccaaatc ttgctagtat atgcaatagt 3180 gtctgggttt
ggtggttgta tattggatgg ttccctgggt ggggcagtct ctggatggtc 3240
tttccttcca tcacagctct gaaatttgtc tctgtaactc cttccatgag tattttgttc
3300 cccattctaa gaagcagtga agtatccaca ctttggtctt
ccttcttctt gagtttcatg 3360 tgttttgcaa attgtgtgcc tggcaataca
gaagcagatg ctcacagtca tctattggat 3420 gaaacacagg gcccctaatg
aaggagccag agaaagtacc caaggagcta aaagggtctg 3480 caaccctata
gcaggaacaa caatatgaac tacccagcaa ccctcagaaa tgtaaatgaa 3540
gaaaatatct aataaaaaaa aaaaaaaaaa a 3571 133 965 DNA Mus musculus
133 gccttggaga ctgtcactgt cagggctgat gacggatgag ctgggtcagg
ctagatagac 60 cctagcaatt tattagagcc agactcctag gcaattctct
ctctacatgt tcacttaagg 120 gttcagagct tcataacaaa gcagaagtca
ggagtctcag aaatgcactt caaaatcagg 180 gtggaggaac ctgcccatgt
gtcaggccct gtgacctatc aactcacaag ccttctgttg 240 ggatattgac
caaacacagt atctttgctt atatgcaagc acacacttgc gtgcaacaca 300
cacacacaca cacacacaca cacacacaca cacacacaca cacacaccag gctaaagctc
360 gcagagttct cagattgtgg tatatgaagg agcaagcctt tgtcagtgaa
cagtatgatc 420 actaagactc tagtgtgggc cctctctaat gggttgctct
cttgggaatc ttcttccaaa 480 gagcagttgt gtggtctttc cattgtaaga
gaaactgcag gtgtcttctt aaccatgaca 540 gttctgatga tgaaagtgta
aagaacccgc cttaaagtca aacaccagtg cacccagaaa 600 gtagatgcac
agctgcaggc tcagagctcg gcagccactg tacttcttag taaccaggaa 660
tcaaacgttt gactcactgt ggggttggta gggcagataa ataccttttt ctatgactag
720 gctggagaca cgcccaggac ccccaccaaa aggagggaca ggaaaagaga
aataattggt 780 aaacacagaa aatgtttcaa tagaaaaaag aggaaacaga
acactgtgta gccctgttat 840 cagcagagac agagctaacg ctggggatac
caaactagaa gaagctcact ggacaggtcc 900 cggtatgcag ttctattttt
gttgatggct ctgtatctaa tgtgttcatt tgtaccaagg 960 tgagt 965 134 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
134 caaggacttc ctttcctttc 20 135 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 135 gccattctac
caaggacttc 20 136 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 136 acaatgagat gccattctac 20 137 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
137 tgttgggagc acaatgagat 20 138 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 138 agcaggcagc
tgttgggagc 20 139 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 139 tgagaagatt aacaagacga 20 140 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
140 tgcagatgag tgagaagatt 20 141 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 141 actctgcaga
tgagtgagaa 20 142 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 142 gacttccttt cctttcctgg 20 143 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
143 aacaagacga agcaggcagc 20 144 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 144 ctacactctg
cagatgagtg 20 145 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 145 cgatcagttt ttccttctaa 20 146 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
146 tcacccacat aaataggttg 20 147 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 147 ggtccataaa
tgacacctga 20 148 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 148 ttacctcata ttcagtaacc 20 149 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
149 gccattctat caaggacttc 20 150 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 150 gccatgctat
caagcacttc 20 151 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 151 gctatcctat caagcacgtc 20 152 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
152 gacttcctta cctttcctgg 20 153 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 153 gacttcctct
tcttccctgg 20 154 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 154 gacctctttc cctcttctgg 20 155 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
155 gtttttcctt ctgaatgtga 20 156 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 156 ctttcctttc
ccacataaat 20 157 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 157 taaatgacac actctgcaga 20 158 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
158 taaatgacac ccacataaat 20 159 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 159 tcgaaggttt
ccacataaat 20 160 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 160 aaccactctc tcaagggctt 20 161 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
161 tgctggaatt ggtggaaaca 20 162 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 162 gtctcaactc
caggcttctc 20 163 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 163 tcaaaacaca gaatcctcca 20 164 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
164 aggatgccaa agtgacagtc 20 165 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 165 atccctgttc
ttttcactga 20 166 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 166 cgcaggtaaa ttgagtgttg 20 167 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
167 tgaggcgatt tggatgaagc 20 168 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 168 tggacgttag
ccttaaaagc 20 169 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 169 agcttaaaca gccaaacggg 20 170 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
170 ctccaggctg atgcaaaatg 20 171 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 171 gggtgaggaa
tttgtggctc 20 172 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 172 ctggatcagg cctctggagc 20 173 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
173 gggtgaggat tttgtggctc 20 174 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 174 gggtgatgat
ttggtggctc 20 175 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 175 ggctgatgat ttggtgggtc 20 176 2006
DNA Homo sapiens 176 cggtcctcgc catcttctgt tgagtactgg tcggaacaag
aggatcgtct gtagacagga 60 tatgatcatc gtggcgcatg tattactcat
ccttttgggg gccactgaga tactgcaagc 120 tgacttactt cctgatgaaa
agatttcact tctcccacct gtcaatttca ccattaaagt 180 tactggtttg
gctcaagttc ttttacaatg gaaaccaaat cctgatcaag agcaaaggaa 240
tgttaatcta gaatatcaag tgaaaataaa cgctccaaaa gaagatgact atgaaaccag
300 aatcactgaa agcaaatgtg taaccatcct ccacaaaggc ttttcagcaa
gtgtgcggac 360 catcctgcag aacgaccact cactactggc cagcagctgg
gcttctgctg aacttcatgc 420 cccaccaggg tctcctggaa cctcagttgt
gaatttaact tgcaccacaa acactacaga 480 agacaattat tcacgtttaa
ggtcatacca agtttccctt cactgcacct ggcttgttgg 540 cacagatgcc
cctgaggaca cgcagtattt tctctactat aggtatggct cttggactga 600
agaatgccaa gaatacagca aagacacact ggggagaaat atcgcatgct ggtttcccag
660 gacttttatc ctcagcaaag ggcgtgactg gcttgcggtg cttgttaacg
gctccagcaa 720 gcactctgct atcaggccct ttgatcagct gtttgccctt
cacgccattg atcaaataaa 780 tcctccactg aatgtcacag cagagattga
aggaactcgt ctctctatcc aatgggagaa 840 accagtgtct gcttttccaa
tccattgctt tgattatgaa gtaaaaatac acaatacaag 900 gaatggatat
ttgcagatag aaaaattgat gaccaatgca ttcatctcaa taattgatga 960
tctttctaag tacgatgttc aagtgagagc agcagtgagc tccatgtgca gagaggcagg
1020 gctctggagt gagtggagcc aacctattta tgtgggaaat gatgaacaca
agcccttgag 1080 agagtggttt gtcattgtga ttatggcaac catctgcttc
atcttgttaa ttctctcgct 1140 tatctgtaaa atatgtcatt tatggatcaa
gttgtttcca ccaattccag caccaaaaag 1200 taatatcaaa gatctctttg
taaccactaa ctatgagaaa gctgggtcca gtgagacgga 1260 aattgaagtc
atctgttata tagagaagcc tggagttgag accctggagg attctgtgtt 1320
ttgactgtca ctttggcatc ctctgatgaa ctcacacatg cctcagtgcc tcagtgaaaa
1380 gaacagggat gctggctctt ggctaagagg tgttcagaat ttaggcaaca
ctcaatttac 1440 ctgcgaagca atacacccag acacaccagt cttgtatctc
ttaaaagtat ggatgcttca 1500 tccaaatcgc ctcacctaca gcagggaagt
tgactcatcc aagcattttg ccatgttttt 1560 tctccccatg ccgtacaggg
tagcacctcc tcacctgcca atctttgcaa tttgcttgac 1620 tcacctcaga
cttttcattc acaacagaca gcttttaagg ctaacgtcca gctgtattta 1680
cttctggctg tgcccgtttg gctgtttaag ctgccaattg tagcactcag ctaccatctg
1740 aggaagaaag cattttgcat cagcctggag tgaatcatga acttggattc
aagactgtct 1800 tttctatagc aagtgagagc cacaaattcc tcacccccct
acattctaga atgatctttt 1860 tctaggtaga ttgtgtatgt gtgtgtatga
gagagagaga gagagagaga gagagagaga 1920 gagaaattat ctcaagctcc
agaggcctga tccaggatac atcatttgaa accaactaat 1980 ttaaaagcat
aatagagcta atatat 2006 177 20 DNA Artificial Sequence Description
of Artificial SequenceSynthetic 177 cctgagaaat gcggtggcca 20 178 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
178 gtgtctatgc tcgtggctgc 20 179 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 179 cgatcctctt
gttccgacca 20 180 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 180 atgcgccacg atgatcatat 20 181 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
181 gcagtatctc agtggccccc 20 182 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 182 tgctcttgat
caggatttgg 20 183 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 183 caggatggtc cgcacacttg 20 184 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
184 gggcatgaag ttcagcagaa 20 185 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 185 gccaggtgca
gtgaagggaa 20 186 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 186 ctccccagtg tgtctttgct 20 187 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
187 aagccagtca cgccctttgc 20 188 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 188 aaacagctga
tcaaagggcc 20 189 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 189 atggattgga aaagcagaca 20 190 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
190 tctgcacatg gagctcactg 20 191 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 191 aggttggctc
cactcactcc 20 192 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 192 tctgcacatg tagctcactg 20 193 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
193 tctgcacgtg taactcactg 20 194 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 194 tatgcacgtg
taactccctg 20 195 1998 DNA Homo sapiens 195 ccgctgcttc tcatcgcatg
gccaccgcat ttctcaggcc aggcacattg agcattggtc 60 ctgtgcctga
cgctatgcta gatgctgggg ttgcagccac gagcatagac acgacagaca 120
cggtcctcgc catcttctgt tgagtactgg tcggaacaag aggatcgtct gtagacaggc
180 tacagattgt tttagattga agtttcctgt catgttcact catctttaaa
tcctcatagt 240 aaaaaggata tgatcatcgt ggcgcatgta ttactcatcc
ttttgggggc cactgagata 300 ctgcaagctg acttacttcc tgatgaaaag
atttcacttc tcccacctgt caatttcacc 360 attaaagtta ctggtttggc
tcaagttctt ttacaatgga aaccaaatcc tgatcaagag 420 caaaggaatg
ttaatctaga atatcaagtg aaaataaacg ctccaaaaga agatgactat 480
gaaaccagaa tcactgaaag caaatgtgta accatcctcc acaaaggctt ttcagcaagt
540 gtgcggacca tcctgcagaa cgaccactca ctactggcca gcagctgggc
ttctgctgaa 600 cttcatgccc caccagggtc tcctggaacc tcaattgtga
atttaacttg caccacaaac 660 actacagaag acaattattc acgtttaagg
tcataccaag tttcccttca ctgcacctgg 720 cttgttggca cagatgcccc
tgaggacacg cagtattttc tctactatag gtatggctct 780 tggactgaag
aatgccaaga atacagcaaa gacacactgg ggagaaatat cgcatgctgg 840
tttcccagga cttttatcct cagcaaaggg cgtgactggc tttcggtgct tgttaacggc
900 tccagcaagc actctgctat caggcccttt gatcagctgt ttgcccttca
cgccattgat 960 caaataaatc ctccactgaa tgtcacagca gagattgaag
gaactcgtct ctctatccaa 1020 tgggagaaac cagtgtctgc ttttccaatc
cattgctttg attatgaagt aaaaatacac 1080 aatacaagga atggatattt
gcagatagaa aaattgatga ccaatgcatt catctcaata 1140 attgatgatc
tttctaagta cgatgttcaa gtgagagcag cagtgagctc catgtgcaga 1200
gaggcagggc tctggagtga gtggagccaa cctatttatg tgggaaatga tgaacacaag
1260 cccttgagag agtggtttgt cattgtgatt atggcaacca tctgcttcat
cttgttaatt 1320 ctctcgctta tctgtaaaat atgtcattta tggatcaagt
tgtttccacc aattccagca 1380 ccaaaaagta atatcaaaga tctctttgta
accactaact atgagaaagc tgggtccagt 1440 gagacggaaa ttgaagtcat
ctgttatata gagaagcctg gagttgagac cctggaggat 1500 tctgtgtttt
gactgtcact ttggcatcct ctgatgaact cacacatgcc tcagtgcctc 1560
agtgaaaaga acagggatgc tggctcttgg ctaagaggtg ttcagaattt aggcaacact
1620 caatttacct gcgaagcaat acacccagac acaccagtct tgtatctctt
aaaagtatgg 1680 atgcttcatc caaatcgcct cacctacagc agggaagttg
actcatccaa gcattttgcc 1740 atgttttttc tccccatgcc gtacagggta
gcacctcctc acctgccaat ctttgcaatt 1800 tgcttgactc acctcagact
ttcattcaca acagacagct tttaaggcta acgtccagct 1860 gtatttactt
ctggctgtgc cgtttggctg tttaagctgc caattgtagc actcagctac 1920
catctgagga agaaagcatt ttgcatcagc ctggagtgaa ccatgaactt ggattcaaga
1980 ctgtcttttc tatagcaa 1998 196 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 196 acccagcttt
ctgcaaaaca 20 197 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 197 tcaacattac ctcatagtta 20 198 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
198 taaatgacat ctgaaaacag 20 199 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 199 gaacacttac
attttacaga 20 200 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 200 tcatcatttc ctggtggaaa 20 201 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
201 tcatcattta ctggtggaaa 20 202 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 202 tcagcattta
ctggtgtaaa 20 203 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 203 tcagcagtta cttgtgtaaa 20 204 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
204 agcggcagag cattgagaac 20 205 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 205 agcggcagag
cattgagaac 20 206 20 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 206 gaagcagcgg cagagcattg 20 207 20
DNA Artificial Sequence Description of Artificial SequenceSynthetic
207 gaagcagcgg cagagcattg 20 208 612 DNA Homo sapiens 208
ggtaccagac ctgctcacaa agcagagaag agctaaggcg gttctctaag ggcagagaat
60 tgctgctatt
gcctagtgag tggggagagg gtactcctca ggccttactt cctatcaaat 120
catgtgtcag tgttgcctag gagacagagg cacagtaact actgtagcca aacaaggcac
180 ataaacaaaa cagaaatgca acgctttaga gtacccacgg aaaacttgtt
taccttgtca 240 ccatgagtaa aagttaattc ccactcctga agagagcaaa
ccaactctga aagagagtga 300 aaatgcagac aagacagtta tcagataatg
gctatctgga cgagagattc tttcgtttga 360 cagcagtttg gttgttggga
gttccagttc agctcctgca cagttgctct gtacaaatcc 420 tcctccatat
ttgcttagag aaaacgtgtt gccatcccat catgaaggaa gctgcctgag 480
agtttttaac cattacagcc gtgatgatga aagagtgaag aaccgcctct aagttaaaaa
540 gtgcacccag agataaggtt cgttctcaat gctctgccgc tgcttctcat
cgcatggcca 600 ccgcatttct ca 612 209 15 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 209 tctaccaagg acttc 15
210 15 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 210 tcaacctaga acttc 15 70
* * * * *