U.S. patent number 8,741,869 [Application Number 13/759,922] was granted by the patent office on 2014-06-03 for oligodeoxynucleotide and its use to induce an immune response.
This patent grant is currently assigned to N/A, N/A, Uniformed Services University of the Health Sciences, an institution of higher learning within the Department of Defense, The United States of America as represented by the Secretary of the Department of Health and Human Services. The grantee listed for this patent is N/A, The United States of America as represented by the Secretary of the Department of Health and Human Services, The United States of America as represented by the Secretary of the Department of Health and Human Services. Invention is credited to Ken J. Ishii, Dennis M. Klinman, James J. Mond, Daniela Verthelyi.
United States Patent |
8,741,869 |
Klinman , et al. |
June 3, 2014 |
Oligodeoxynucleotide and its use to induce an immune response
Abstract
A substantially pure or isolated oligodeoxynucleotide of at
least 10 nucleotides is disclosed, wherein the oligodeoxynucleotide
comprised a sequence represented by either formula: 5'
N.sub.1N.sub.2N.sub.3T-CpG-WN.sub.4N.sub.5N.sub.6 3' wherein the
CpG motif is unmethylated, W is A or T, and N.sub.1, N.sub.2,
N.sub.3, N.sub.4, N.sub.5, and N.sub.6 are nucleotides, or the
formula: 5' RY-CpG-RY 3' wherein the central CpG motif is
unmethylated, R is A or G, and Y is C or T, as well as an
oligodeoxynucleotide delivery complex and a pharmacological
composition comprising the present inventive oligodeoxynucleotide,
and a method of inducing an immune response by administering the
present inventive oligodeoxynucleotide to a host. In some
embodiments, the oligodeoxynucleotide includes the nucleic acid
sequences set forth as SEQ ID NO: 137.
Inventors: |
Klinman; Dennis M. (Potomac,
MD), Ishii; Ken J. (Osaka, JP), Verthelyi;
Daniela (Potomac, MD), Mond; James J. (Silver Spring,
MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America as represented by the Secretary of the
Department of Health and Human Services
N/A |
Rockville |
MD |
US |
|
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Assignee: |
The United States of America as
represented by the Secretary of the Department of Health and Human
Services (Washington, DC)
N/A (Washington, DC)
Uniformed Services University of the Health Sciences, an
institution of higher learning within the Department of Defense
(N/A)
N/A (N/A)
|
Family
ID: |
22437528 |
Appl.
No.: |
13/759,922 |
Filed: |
February 5, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130136771 A1 |
May 30, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13220497 |
Aug 29, 2011 |
8389495 |
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11595211 |
Oct 4, 2011 |
8030285 |
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09958713 |
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PCT/US00/09839 |
Apr 12, 2000 |
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60128898 |
Apr 12, 1999 |
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Current U.S.
Class: |
514/44R;
536/23.1; 536/24.3; 536/24.5; 424/278.1; 536/25.6 |
Current CPC
Class: |
A61K
45/06 (20130101); C07H 21/00 (20130101); A61K
39/39 (20130101); A61K 39/008 (20130101); A61K
31/7088 (20130101); C12N 15/117 (20130101); C07H
21/04 (20130101); A61P 37/04 (20180101); A61K
39/39 (20130101); A61K 2300/00 (20130101); A61K
2039/55561 (20130101); Y02A 50/41 (20180101); A61K
38/00 (20130101); Y02A 50/30 (20180101); Y02A
50/423 (20180101); Y10T 428/2989 (20150115); C12N
2310/17 (20130101) |
Current International
Class: |
A61K
39/00 (20060101); A01N 43/04 (20060101); A61K
31/70 (20060101); A61K 47/00 (20060101); A61K
45/00 (20060101); A61K 39/38 (20060101); A61K
9/127 (20060101) |
References Cited
[Referenced By]
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Apr 2002 |
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EP |
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WO 92/18522 |
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Oct 1992 |
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WO |
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WO 94/19945 |
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Sep 1994 |
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WO |
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WO 96/02555 |
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Feb 1996 |
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WO |
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WO 97/28259 |
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Aug 1997 |
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WO |
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WO 98/18810 |
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May 1998 |
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WO |
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WO 98/37919 |
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Sep 1998 |
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WO |
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WO 99/11275 |
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Mar 1999 |
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WO |
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WO 00/06588 |
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Feb 2000 |
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WO |
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WO 00/20039 |
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Apr 2000 |
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WO |
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WO 00/21556 |
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Apr 2000 |
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WO |
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WO 00/61151 |
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Oct 2000 |
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WO |
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WO 00/62787 |
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Oct 2000 |
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WO |
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WO 00/67023 |
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Nov 2000 |
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WO |
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WO 01/00232 |
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Jan 2001 |
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WO |
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WO 01/02007 |
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Jan 2001 |
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WO |
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WO 01/12223 |
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Feb 2001 |
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WO |
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WO 01/22990 |
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Apr 2001 |
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WO |
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WO 01/51500 |
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Jul 2001 |
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WO |
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WO 01/55341 |
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Aug 2001 |
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WO |
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WO 01/68077 |
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Sep 2001 |
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WO |
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WO 01/68103 |
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Sep 2001 |
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WO |
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WO 01/68116 |
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Sep 2001 |
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WO |
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WO 01/68117 |
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Sep 2001 |
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WO |
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Primary Examiner: Minnifield; Nita M
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This is a continuation of U.S. patent application Ser. No.
13/220,497, filed on Aug. 29, 2011, issued as U.S. Pat. No.
8,389,495, which is a continuation of U.S. patent application Ser.
No. 11/595,211, filed Nov. 9, 2006, issued as U.S. Pat. No.
8,030,285, which is divisional of U.S. patent application Ser. No.
09/958,713, filed Oct. 7, 2002, abandoned, which is the .sctn.371
U.S. national stage of International Application No.
PCT/US00/09839, filed Apr. 12, 2000, which was published in English
under PCT Article 21(2), which in turn claims the benefit of U.S.
Provisional Application 60/128,898, filed Apr. 12, 1999. The prior
applications are incorporated by reference herein in their
entirety.
Claims
What is claimed is:
1. A substantially pure or isolated synthetic oligodeoxynucleotide,
wherein the oligodeoxynucleotide comprises the nucleic acid
sequence set forth as SEQ ID NO: 22, and wherein the
oligodeoxynucleotide is 100 nucleotides or less in length.
2. The synthetic oligodeoxynucleotide of claim 1, wherein the
oligodeoxynucleotide is modified to prevent degradation.
3. The synthetic oligodeoxynucleotide of claim 1, wherein the
oligodeoxynucleotide has a phosphate backbone modification.
4. The synthetic oligodeoxynucleotide of claim 3, wherein the
phosphate backbone modification is a phosphorothioate backbone
modification.
5. The synthetic oligodeoxynucleotide of claim 1, wherein the
oligodeoxynucleotide is 20 nucleotides in length.
6. The synthetic oligodeoxynucleotide claim 1, wherein the
oligodeoxynucleotide is 50 nucleotides or less in length.
7. The synthetic oligodeoxynucleotide of claim 1, wherein the
oligodeoxynucleotide is 30 nucleotides or less in length.
8. An oligodeoxynucleotide delivery complex comprising the
synthetic oligodeoxynucleotide of claim 4 and a targeting
means.
9. The oligodeoxynucleotide delivery complex of claim 8, wherein
the targeting means is selected from the group consisting of
cholesterol, virosome, liposome, lipid, and a target cell specific
binding agent.
10. A composition comprising the synthetic oligodeoxynucleotide of
claim 1 and a pharmacologically acceptable carrier.
11. A method of inducing cytokine production by a peripheral blood
mononuclear cell, comprising contacting the peripheral blood
mononuclear cell with an effective amount of the synthetic
oligodexoynucleotide of claim 1, thereby inducing the production of
the cytokine, wherein the cytokine is interleukin (IL)-6 or
interferon .gamma..
12. The method of claim 11, wherein the synthetic
oligodeoxynucleotide is 30 nucleotides or less in length.
13. The method of claim 11, wherein the synthetic
oligodeoxynucleotide consists of the nucleic acid sequence set
forth as SEQ ID NO: 22.
14. The method of claim 11, wherein the synthetic
oligodeoxynucleotide is modified to prevent degradation.
15. The method of claim 11, wherein the synthetic
oligodeoxynucleotide has a phosphate backbone modification.
16. The method of claim 15, wherein the phosphate backbone
modification is a phosphorothioate backbone modification.
17. The method of claim 11, wherein the synthetic
oligodeoxynucleotide is 50 nucleotides or less in length.
18. The method of claim 11, wherein the peripheral blood
mononuclear cell is a B cell, and wherein the cytokine is IL-6.
19. The method of claim 11, wherein the cytokine is interferon
.gamma..
20. A substantially pure or isolated synthetic
oligodeoxynucleotide, wherein the oligodexoynucleotide consists of
the nucleic acid sequence set forth as SEQ ID NO: 22.
21. The synthetic oligodeoxynucleotide of claim 20, wherein the
oligodexoynucleotide is modified to prevent degradation.
22. An oligodeoxynucleotide delivery complex comprising the
synthetic oligodeoxynucleotide of claim 21 and a targeting
means.
23. A composition comprising the synthetic oligodeoxynucleotide of
claim 20 and a pharmacologically acceptable carrier.
Description
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to induction of an immune
response using specific oligodeoxynucleotides (ODNs).
BACKGROUND OF THE INVENTION
DNA is a complex macromolecule whose immunological activities are
influenced by its base composition and base modification, as well
as helical orientation. Certain unusual DNA structures (e.g.,
Z-DNA) can induce significant antibody responses when administered
to normal mice. In addition, bacterial DNA, as well as certain
synthetic ODNs containing unmethylated CpG sequences can induce
proliferation and immunoglobulin (Ig) production by murine B cells.
Unmethylated CpG dinucleotides are more frequent in the genomes of
bacteria and viruses than vertebrates. Recent studies suggest that
immune recognition of these motifs may contribute to the host's
innate immune response. D. M. Klinman et al., 93, Proc. Natl. Acad.
Sci. USA 2879, (1996); A.-K. Yi et al., 157, J. Immun. 5394,
(1996); Hua Liang et al., 98, J. Clin. Invest. 1119, (1996); A.M.
Krieg et al., 374, Nature 546, (1995).
In mice, CpG DNA induces proliferation in almost all (>95%) of B
cells and increases Ig secretion. This B-cell activation by CpG DNA
is T-cell independent and antigen non-specific. In addition to its
direct effects on B cells, CpG DNA also directly activates
monocytes, macrophages, and dendritic cells to secrete a variety of
cytokines. These cytokines stimulate natural killer (NK) cells to
secrete .gamma.-interferon (IFN-.gamma.) and have increased lytic
activity. Examples of which can be found in International Patent
Applications WO 95/26204, WO 96/02555, WO 98/11211, WO 98/18810, WO
98/37919, WO 98/40100, WO 98/52581, PCT/US98/047703, and
PCT/US99/07335; U.S. Pat. No. 5,663,153; and U.S. patent
applications Ser. Nos. 08/276,358, 08/386,063, 08/461,036,
08/462,799, 08/960,774, 08/738,652, 09/030,701, 09/082,649,
09/191,170, 09/136,138, 09/154,614, and 09/286,098.
Although bacterial DNA and certain ODNs can induce a murine immune
response, little is known about the immunostimulatory capacity of
these materials for the human immune system. Z. K. Ballas et al.,
157 J. Immun. 1840 (1996). Differences in the responsiveness of
human and murine B cells to certain stimuli render it impossible to
extrapolate results obtained from mouse to man.
In view of the above, there exists a need for ODNs that induce an
immune response in humans. In addition, there is a need for methods
utilizing ODNs in the treatment of human diseases. The present
invention provides such ODNs and methods of use. These and other
advantages of the present invention, as well as additional
inventive features, will be apparent from the description of the
invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a substantially pure or isolated ODN
of at least about 10 nucleotides comprising a sequence represented
by either the formula: 5'
N.sub.1N.sub.2N.sub.3T-CpG-WN.sub.4N.sub.5N.sub.6 3' wherein the
central CpG motif is unmethylated, W is A or T, and N.sub.1,
N.sub.2, N.sub.3, N.sub.4, N.sub.5, and N.sub.6 are any
nucleotides, or the formula: 5' RY-CpG-RY 3' wherein the central
CpG motif is unmethylated, R is A or G, and Y is C or T. The
present invention also provides an ODN delivery complex and
pharmacological composition comprising the present inventive ODN,
as well as a method of inducing an immune response by administering
the present inventive ODN to a host.
SEQUENCE LISTING
The Sequence Listing is submitted as an ASCII text file
4239-62001-04_Sequence_Listing.txt, Feb. 5, 2013, 27.3, KB], which
is incorporated by reference herein.
DETAILED DESCRIPTION OF THE INVENTION
Oligodeoxynucleotide
The present invention provides novel ODNs. These ODNs have at least
about 10 nucleotides and comprise a sequence represented by either
the formula: 5' N.sub.1N.sub.2N.sub.3T-CpG-WN.sub.4N.sub.5N.sub.6
3' wherein the central CpG motif is unmethylated, W is A or T, and
N.sub.1, N.sub.2, N.sub.3, N.sub.4, N.sub.5, and N.sub.6 are any
nucleotides, or the formula: 5' RY-CpG-RY 3' wherein the central
CpG motif is unmethylated, R is A or G, and Y is C or T. For
example, the ODN can be selected from the group consisting of SEQ
ID NO: 1 through SEQ ID NO: 99.
Preferably, the ODN of the present invention is substantially pure
or isolated. "Substantially pure" refers to an ODN that is
substantially free of other materials, particularly other nucleic
acids, proteins, lipids, carbohydrates, and other materials with
which it may be naturally associated, while "isolated" refers to an
ODN that is removed from its natural environment or state.
Preferably, the ODN of the present invention consists of about 100
nucleotides or less (e.g., about 10-75 nucleotides). More
preferably, the ODN consists of about 50 nucleotides or less (e.g.,
about 10-40 nucleotides). Even more preferably, the ODN consists of
about 30 nucleotides or less (e.g., about 10-20 nucleotides). Most
preferably the ODN consists of about 12 to about 16
nucleotides.
Any suitable modification can be used in the present invention to
render the ODN resistant to degradation in vivo (e.g., via an exo
or endonuclease). Preferably, the modification includes a
phosphorothioate modification. The phosphorothioate modifications
can occur at either termini, e.g., the last two or three 5' and/or
3' nucleotides can be liked with phosphorothioate bonds. The ODN
also can be modified to contain a secondary structure (e.g., stem
loop structure) such that it is resistant to degradation. Another
modification that renders the ODN less susceptible to degradation
is the inclusion of nontraditional bases such as inosine and
quesine, as well as acetyl-, thio- and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine. Other modified
nucleotides include nonionic DNA analogs, such as alkyl or aryl
phosphonates (i.e., the charged phosphonate oxygen is replaced with
an alkyl or aryl group, as set forth in U.S. Pat. No. 4,469,863),
phosphodiesters and alkylphosphotriesters (i.e., the charged oxygen
moiety is alkylated, as set forth in U.S. Pat. No. 5,023,243 and
European Patent No. 0 092 574). ODNs containing a diol, such as
tetraethyleneglycol or hexaethyleneglycol, at either or both
termini, have also been shown to be more resistant to
degradation.
Preferably, the ODNs inducing a humoral immune response, e.g., 5'
N.sub.1N.sub.2N.sub.3T-CpG-WN.sub.4N.sub.5N.sub.6 3', contain a
phosphate backbone modification, and more preferably, the phosphate
backbone modification is a phosphorothioate backbone modification
(i.e., one of the non-bridging oxygens is replaced with sulfur, as
set forth in International Patent Application WO 95/26204). For the
ODNs inducing a cell-mediated immune response and containing a
phosphodiester backbone, e.g., 5' RY-CpG-RY 3', the ODN preferably
has been modified to prevent degradation.
Oligodeoxynucleotide Delivery Complex
The present inventive oligodeoxynucleotide delivery complex
comprises the present inventive ODN and a targeting means. Any
suitable targeting means can be used within the context of the
present invention.
An ODN can be associated with (e.g., ionically or covalently bound
to, or encapsulated within) a targeting means (e.g., a molecule
that results in higher affinity binding to a target cell, such as a
B cell). A variety of coupling or cross-linking agents can be used
to form the delivery complex, such as protein A, carbodiamide, and
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Examples of
ODN delivery complexes include ODNs associated with a sterol (e.g.,
cholesterol), a lipid (e.g., a cationic lipid, virosome or
liposome), and a target cell specific binding agent (e.g., a ligand
recognized by target cell specific receptor). Preferred complexes
must be sufficiently stable in vivo to prevent significant
uncoupling prior to internalization by the target cell; however,
these complexes can be cleavable under appropriate circumstances
such that the ODN can be released in a functional form.
Pharmacological Composition
The present inventive pharmacological composition comprises the
present inventive ODN and a pharmacologically acceptable carrier.
Pharmacologically acceptable carriers (e.g., physiologically or
pharmaceutically acceptable carriers) are well known in the
art.
The present inventive pharmacological composition facilitates the
use of the present inventive ODN, both in vivo and ex vivo. Such a
composition can be suitable for delivery of the active ingredient
to any suitable host, such as a patient for medical application,
and can be manufactured in a manner that is itself known, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
Pharmacological compositions for use in accordance with the present
invention can be formulated in a conventional manner using one or
more pharmacologically (e.g., physiologically or pharmaceutically)
acceptable carriers comprising excipients, as well as optional
auxiliaries that facilitate processing of the active compounds into
preparations which can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen. Thus, for
injection, the active ingredient can be formulated in aqueous
solutions, preferably in physiologically compatible buffers. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art. For oral administration, the active
ingredient can be combined with carriers suitable for inclusion
into tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries, suspensions and the like. For administration by
inhalation, the active ingredient is conveniently delivered in the
form of an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant. The active
ingredient can be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion. Such
compositions can take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and can contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Other pharmacological excipients are known in the art.
Method of Inducing an Immune Response
The present inventive method of inducing an immune response
comprises administering the present inventive ODN to a host in
order to induce an immune response in the host.
Administration of the present inventive ODN can be by any suitable
method. For example, the ODN can be administered in vivo or ex
vivo. Preferably, the ODN is administered in vivo to a mammal,
particularly a human. Optionally, the ODN can be contained within
or conjugated with a protein, hydrocarbon or lipid. Once this
molecule is administered, the ODN sequence must be exposed on the
surface to induce an immune response. The ODN can also be
co-administered with a protein, hydrocarbon, or lipid.
Co-administration can be such that the ODN is administered before,
at substantially the same time as, or after the protein,
hydrocarbon, or lipid. Preferably, the ODN is administered at
substantially the same time as the protein, hydrocarbon, or
lipid.
After administration of the novel ODNs, while not intending to be
bound by any particular theory, it is thought that the ODNs
initially act on antigen presenting cells (e.g., macrophages and
dendritic cells). These cells then release cytokines, which
activate natural killer (NK) cells. Either a cell-mediated or
humoral immune response then occurs in the host.
The cell-mediated or local immune response is produced by T cells,
which are able to detect the presence of invading pathogens through
a recognition system referred to as the T-cell antigen receptor.
Upon detection of an antigen, T cells direct the release of
multiple T-cell cytokines, including IL-2, IL-3, IFN-.gamma.,
TNF-.beta., GM-CSF and high levels of TNF-.alpha., and chemokines
MIP-1.alpha., MIP-1.beta., and RANTES. IL-2, is a T-cell growth
factor that promotes the production of additional T cells sensitive
to the particular antigen. This production constitutes a clone of
the T cells. The sensitized T cells attach to cells containing the
antigen. T cells carry out a variety of regulatory and defense
functions and play a central role in immunologic responses. When
stimulated to produce a cell-mediated immune response, some T cells
respond by acting as killer cells, killing the host's own cells
when these cells are infected or cancerous and therefore recognized
as foreign. Some T cells respond by stimulating B cells, while
other T cells respond by suppressing immune response. Preferably,
if a cell-mediated immune response is induced, non-B cells are
activated, more preferably, cytokines are produced, and most
preferably, IFN-.gamma. is produced.
The humoral or systemic immune response depends on the ability of
the B cells to recognize specific antigens. The mechanism by which
B cells recognize antigens is through specific receptors on the
surface of the B cells. When an antigen attaches to the receptor
site of a B cell, the B cell is stimulated to divide. The daughter
cells become plasma cells that manufacture antibodies complementary
to the attached antigen. Each plasma cell produces thousands of
antibody molecules per minute, which are released into the
bloodstream. Many B cells appear to be regulated by the helper T
cells and suppressor T cells and produce various cytokines, e.g.,
IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels
of TNF-.alpha.. Helper T cells stimulate B cells to produce
antibodies against antigens, while suppressor T cells inhibit
antibody production. Some B cells, however, are T-cell independent
and require no stimulation by the T cells. Preferably, if a humoral
immune response is induced, B cells are activated, more preferably,
IL-6, is produced, and most preferably, antibodies are
produced.
In addition, induction of one type of immune response may allow for
immune regulation because up regulation of one type of immune
response may down regulate the other type of immune response. This
immune regulation allows for customizing or tailoring of the type
of immune response when administering an ODN.
The present inventive method can be used to treat, prevent, or
ameliorate any suitable allergic reaction in combination with any
suitable anti-allergenic agent. An allergy, in the context of the
present invention, refers to an acquired hypersensitivity to a
substance (i.e., an allergen). Allergic conditions include eczema,
allergic rhinitis or coryza, hay fever, bronchial asthma, uticaria
(hives), food allergies, and other atopic conditions. The list of
allergens is extensive and includes pollens, insect venoms, animal
dander, dust, fungal spores, and drugs (e.g., penicillin). Examples
of natural, animal, and plant allergens can be found in
International Patent Application WO 98/18810. Preferably, the
present inventive method is used to treat allergic asthma. Suitable
anti-allergenic agents include those substances given in treatment
of the various allergic conditions described above, examples of
which can be found in the Physicians' Desk Reference (1998).
The present inventive method can be used to treat any suitable
cancer in combination with any suitable anti-cancer agent. Suitable
cancers include cancers of the brain, lung (e.g., small cell and
non-small cell), ovary, breast, prostate, and colon, as well as
carcinomas and sarcomas. Preferably, the present inventive method
is used to treat a solid tumor cancer. Suitable anti-cancer agents
include those substances given in treatment of the various
conditions described above, examples of which can be found in the
Physicians' Desk Reference (1998).
The present inventive method can be used to improve the efficacy of
any suitable vaccine. Suitable vaccines include those directed
against Hepatitis A, B, and C, examples of which can be found in
the Physicians' Desk Reference (1998), and DNA vaccines directed
against HIV and malaria. See generally D. Klinman et al., CpG
Motifs as Immune Adjuvants, 17 Vaccine 19 (1999); M. J. McCluskie
and H. L. Davis, CpG DNA is a Potent Enhancer of Systemic &
Mucosal Immune Response Against Hepatitis B Surface Antigen with
Intra-Nasal Administration to Mice, 161 J. Immun. 4463 (1998).
The present inventive method can be used to treat, prevent, or
ameliorate any suitable disease associated with the immune system.
Preferred diseases associated with the immune system are autoimmune
disorders and immune system deficiencies, e.g., lupus
erythematosus, and autoimmune diseases such as rheumatoid arthritis
and multiple sclerosis. Immune system deficiencies include those
diseases or disorders in which the immune system is not functioning
at normal capacity, or in which it would be useful to boost the
immune system response.
The present inventive method can be used with any suitable
antisense therapy. Suitable antisense agents are those that bind
either with DNA or RNA and block their function by inhibiting
expression of the sequence to which the antisense agents are bound.
See generally H. Lonnberg et al., Towards Genomic Drug Therapy with
Antisense Oligonucleotides, 28 Ann. Med. 511 (1996); A. Alama et
al., Antisense Oligonucleotides as Therapeutic Agents, 36
Pharmacol. Res. 171 (1997); K. J. Scanlon et al.,
Oligonucleotide-Mediated Modulation of Mammalian Gene Expression, 9
FASEB J. 1288 (1995); R. Oberbauer, Not Non-Sense but
Antisense--Applications of Antisense Oligonucleotides in Different
Fields of Medicine, 109 Wien Klin Wochenschr 40 (1997).
The present inventive method can be used to treat, prevent, or
ameliorate any suitable infection in combination with any suitable
anti-infectious agent. Examples include francisella,
schistosomiasis, tuberculosis, AIDS, malaria, and leishmania.
Examples of suitable infectious viruses, bacteria, fungi, and other
organisms (e.g., protists) can be found in International Patent
Application WO 98/18810. Suitable anti-infectious agents include
those substances given in treatment of the various conditions
described elsewhere, examples of which can be found in the
Physicians' Desk Reference (1998).
The present inventive method can be used to treat, prevent, or
ameliorate the symptoms resulting from exposure to a bio-warfare
agent. Suitable bio-warfare agents include those naturally
occurring biological agents that have been specifically modified in
the laboratory. Often, modification of these agents has altered
them such that there is no known treatment. Examples include Ebola,
Anthrax, and Listeria. In the course of ameliorating the symptoms
after exposure, use of the present inventive ODNs may not cure the
patient, but rather can extend the patient's life sufficiently such
that some other treatment can then be applied.
The present invention is further described in the following
examples. These examples are intended only to illustrate the
invention and are not intended to limit the scope of the invention
in any way.
EXAMPLES
Example 1
The following example demonstrates induction of an immune response
by various ODNs. Induction was measured by production of the
cytokines IL-6 and TNF-.gamma., and cell proliferation.
Human peripheral blood mononuclear cells (PBMC) were isolated, as
described elsewhere (Z. K. Ballas et al., 85 J. Allergy Clin.
Immunol. 453 (1990); Z. K. Ballas and W. Rasmussen, 45 J. Immunol.
1039 (1990); Z. K. Ballas and W. Rasmussen, 150 J. Immunol. 17
(1993)). ODNs were synthesized on a DNA synthesizer (Applied
Biosystems Inc., Foster City, Calif.), as described elsewhere
(Beacage and Caruthers, Deoxynucleoside Phosphoramidites--A New
Class of Key Intermediates for Deoxypolynucleotide Synthesis, 22
Tetrahedron Letters 1859 (1981)). In some ODNs, the normal DNA
backbone phosphodiesterase linkages were replaced with
phosphorothioate linkages, as described elsewhere (Agrawal et al.,
94 Proc. Natl. Acad. Sci. USA 2620 (1997); Agrawal 14 TIB TECH 376
(1996)). To reduce degradation of the ODNs, those that did not have
an entire phosphorothioate backbone contained phosphorothioate
linkages at the 5' and 3' ends. Cells were incubated for
approximately 72 hrs with the various ODNs. IL-6 and TNF-.gamma.
levels were determined by ELISA using anti-IL-6 and
anti-TNF-.gamma. antibodies, as described elsewhere (Maniatis et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York, 1989). Cell proliferation was
determined by [.sup.3H] thymidine incorporation, as described
elsewhere (Liang et al., 98 J. Clin. Invest. at 1121).
IL-6 levels and cell proliferation are set forth in Table 1:
Induction of a Humoral Immune Response In Vitro. These data
demonstrate that a sequence containing 5'
N.sub.1N.sub.2N.sub.3T-CpG-WN.sub.4N.sub.5N.sub.6 3', wherein the
central CpG motif is unmethylated, W is A or T, and N.sub.1,
N.sub.2, N.sub.3, N.sub.4, N.sub.5, and N.sub.6 are any
nucleotides, is desirable to induce a humoral immune response. In
addition, maximum induction was observed for ODNs that contained a
phosphorothioate backbone. IFN-.gamma. levels and cell
proliferation are set forth in Table 2: Induction of a
Cell-Mediated Immune Response In Vitro. These data demonstrate that
a sequence containing 5' RY-CpG-RY 3', wherein the central CpG
motif is unmethylated, R is A or G and Y is C or T, is desirable to
induce a cell-mediated immune response. Maximum induction occurred
with ODNs containing phosphodiesterase linkages.
TABLE-US-00001 TABLE 1 Induction of a Humoral Immune Response In
Vitro. IL-6 Levels Cell Proliferation (ELISA) (.sup.3H Thymidine
Incorporation) SEQ ID NO: 1 65 52 SEQ ID NO: 2 85 44 SEQ ID NO: 3
54 50 SEQ ID NO: 4 48 61 SEQ ID NO: 5 42 100 SEQ ID NO: 6 55 23 SEQ
ID NO: 7 35 69 SEQ ID NO: 8 28 38 SEQ ID NO: 9 41 20 SEQ ID NO: 10
42 16 SEQ ID NO: 11 33 77 SEQ ID NO: 12 25 13 SEQ ID NO: 13 28 13
SEQ ID NO: 14 35 67 SEQ ID NO: 15 28 54 SEQ ID NO: 16 39 50 SEQ ID
NO: 17 50 32 SEQ ID NO: 18 26 1 SEQ ID NO: 19 12 2 SEQ ID NO: 20 55
92 SEQ ID NO: 21 53 26 SEQ ID NO: 22 8 2 SEQ ID NO: 23 12 1 SEQ ID
NO: 24 14 0 SEQ ID NO: 25 30 42 SEQ ID NO: 26 43 60 SEQ ID NO: 27
17 15 SEQ ID NO: 28 14 0 SEQ ID NO: 29 10 1 SEQ ID NO: 30 28 23 SEQ
ID NO: 31 16 17
TABLE-US-00002 TABLE 2 Induction of a Cell-Mediated Immune Response
In Vitro. IFN-.gamma. Levels Cell Proliferation (ELISA) (.sup.3H
Thymidine Incorporation) SEQ ID NO: 32 78 1 SEQ ID NO: 33 100 2 SEQ
ID NO: 34 73 2 SEQ ID NO: 35 88 4 SEQ ID NO: 36 81 5 SEQ ID NO: 37
45 4 SEQ ID NO: 38 78 0 SEQ ID NO: 39 33 5 SEQ ID NO: 40 68 2 SEQ
ID NO: 41 54 2 SEQ ID NO: 42 54 1 SEQ ID NO: 43 74 4 SEQ ID NO: 44
53 4 SEQ ID NO: 45 32 9 SEQ ID NO: 46 24 1 SEQ ID NO: 47 23 8 SEQ
ID NO: 48 22 25 SEQ ID NO: 49 34 26 SEQ ID NO: 50 36 8 SEQ ID NO:
51 24 17 SEQ ID NO: 52 21 9 SEQ ID NO: 53 19 2 SEQ ID NO: 54 12 8
SEQ ID NO: 55 15 5 SEQ ID NO: 56 22 6 SEQ ID NO: 57 18 3 SEQ ID NO:
58 18 6 SEQ ID NO: 59 12 21 SEQ ID NO: 60 13 4 SEQ ID NO: 61 -- 2
SEQ ID NO: 62 12 23 SEQ ID NO: 63 16 1 SEQ ID NO: 64 16 4 SEQ ID
NO: 65 19 2 SEQ ID NO: 66 16 4 SEQ ID NO: 67 14 2 SEQ ID NO: 68 13
1 SEQ ID NO: 69 12 2 SEQ ID NO: 70 19 2 SEQ ID NO: 71 13 1 SEQ ID
NO: 72 14 46 SEQ ID NO: 73 -- 4 SEQ ID NO: 74 16 1 SEQ ID NO: 75 24
1 SEQ ID NO: 76 13 1 SEQ ID NO: 77 12 1 SEQ ID NO: 78 13 1 SEQ ID
NO: 79 13 1 SEQ ID NO: 80 12 1 SEQ ID NO: 81 14 20 SEQ ID NO: 82 14
43 SEQ ID NO: 83 14 1 SEQ ID NO: 84 12 1 SEQ ID NO: 85 15 2 SEQ ID
NO: 86 13 1 SEQ ID NO: 87 12 0 SEQ ID NO: 88 -- 3 SEQ ID NO: 89 15
1 SEQ ID NO: 90 18 2 SEQ ID NO: 91 13 2 SEQ ID NO: 92 12 1 SEQ ID
NO: 93 14 2 SEQ ID NO: 94 14 1 SEQ ID NO: 95 44 3 SEQ ID NO: 96 24
1 SEQ ID NO: 97 21 6 SEQ ID NO: 98 36 38 SEQ ID NO: 99 21 26
The foregoing data demonstrates the induction of an immune response
in human cells, as exemplified by PBMC, and as measured by the
production of the cytokines IFN-.gamma. and IL-6, and cell
proliferation, occurs upon the administration of various ODNs.
Example 2
The following example demonstrates induction of an immune response
ex vivo by various ODNs. Induction was measured by production of
the cytokine IL-6.
A human B cell line (RPMI 8226) was maintained according to the
manufacturers recommendations. ODNs were synthesized as described
in Example 1. In some ODNs, the normal DNA phosphodiesterase
linkages were replaced with phosphorothioate linkages, as described
in Example 1. To reduce degradation of the ODNs, those that did not
have an entire phosphorothioate backbone contained phosphorothioate
linkages at the ends. The cells were incubated with various ODNs
for 14 hrs. IL-6 production was determined by ELISA using anti-IL-6
antibodies, as described in Example 1.
IL-6 levels are set forth in Table 3:, Induction of a Humoral
Immune Response Ex Vivo. These data confirm that a sequence
containing 5' N.sub.1N.sub.2N.sub.3T-CpG-WN.sub.4N.sub.5N.sub.6 3',
which are linked by phosphorothioate bonds and wherein the central
CpG motif is unmethylated, W is A or T, and N.sub.1, N.sub.2,
N.sub.3, N.sub.4, N.sub.5, and N.sub.6 are any nucleotides, is
desirable to induce a humoral immune response.
TABLE-US-00003 TABLE 3 Induction of a Humoral Immune Response Ex
Vivo. IL-6 Levels (ELISA) SEQ ID NO: 1 100 SEQ ID NO: 2 89 SEQ ID
NO: 3 85 SEQ ID NO: 4 82 SEQ ID NO: 5 82 SEQ ID NO: 6 78 SEQ ID NO:
7 78 SEQ ID NO: 8 78 SEQ ID NO: 9 73 SEQ ID NO: 10 65 SEQ ID NO: 11
62 SEQ ID NO: 12 58 SEQ ID NO: 13 57 SEQ ID NO: 14 56 SEQ ID NO: 15
50 SEQ ID NO: 16 48 SEQ ID NO: 17 47 SEQ ID NO: 18 45 SEQ ID NO: 19
40 SEQ ID NO: 20 39 SEQ ID NO: 21 33 SEQ ID NO: 22 25 SEQ ID NO: 23
23 SEQ ID NO: 24 21 SEQ ID NO: 25 18 SEQ ID NO: 26 17 SEQ ID NO: 27
17 SEQ ID NO: 28 16 SEQ ID NO: 29 16 SEQ ID NO: 30 13 SEQ ID NO: 31
13
The foregoing data demonstrates the induction of an immune response
in human cells, as exemplified by the human B cell line RPMI 8226,
and as measured by production of the cytokine IL-6, occurs upon
administration of various ODNs.
The following table lists additional ODNs which fall within the
scope of the present invention.
TABLE-US-00004 TABLE 4 SEQ ID NO: 100 SEQ ID NO: 101 SEQ ID NO: 102
SEQ ID NO: 103 SEQ ID NO: 104 SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID
NO: 107 SEQ ID NO: 108 SEQ ID NO: 109 SEQ ID NO: 110 SEQ ID NO: 111
SEQ ID NO: 112 SEQ ID NO: 113 SEQ ID NO: 114 SEQ ID NO: 115 SEQ ID
NO: 116 SEQ ID NO: 117 SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 120
SEQ ID NO: 121 SEQ ID NO: 122 SEQ ID NO: 123 SEQ ID NO: 124 SEQ ID
NO: 125 SEQ ID NO: 126 SEQ ID NO: 127 SEQ ID NO: 128 SEQ ID NO: 129
SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 132 SEQ ID NO: 133 SEQ ID
NO: 134 SEQ ID NO: 135 SEQ ID NO: 136 SEQ ID NO: 137 SEQ ID NO: 138
SEQ ID NO: 139 SEQ ID NO: 140 SEQ ID NO: 141 SEQ ID NO: 142 SEQ ID
NO: 143
All of the references cited herein, including patents, patent
applications, and publications, are hereby incorporated in their
entireties by reference.
While this invention has been described with an emphasis upon
preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments may
be used and it is intended that the invention may be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the following claims.
SEQUENCE LISTINGS
1
143112DNAArtificial SequenceSynthetic Oligonucleotide 1tcgagcgttc
tc 12219DNAArtificial SequenceSynthetic Oligonucleotide 2atcgactctc
gagcgttct 19324DNAArtificial SequenceSynthetic Oligonucleotide
3tcgtcgtttt gtcgttttgc tgtt 24414DNAArtificial SequenceSynthetic
Oligonucleotide 4tctcgagcgt tctc 14519DNAArtificial
SequenceSynthetic Oligonucleotide 5tcgactctcg agcgttctc
19620DNAArtificial SequenceSynthetic Oligonucleotide 6atcgactagc
gttcgttctc 20716DNAArtificial SequenceSynthetic Oligonucleotide
7actctcgagc gttctc 16815DNAArtificial SequenceSynthetic
Oligonucleotide 8ctctcgagcg ttctc 15912DNAArtificial
SequenceSynthetic Oligonucleotide 9gtcgacgttg ac
121012DNAArtificial SequenceSynthetic Oligonucleotide 10gtcggcgttg
ac 121118DNAArtificial SequenceSynthetic Oligonucleotide
11cgactctcga gcgttctc 181212DNAArtificial SequenceSynthetic
Oligonucleotide 12gtcgacgctg ac 121312DNAArtificial
SequenceSynthetic Oligonucleotide 13gtcagcgttg ac
121417DNAArtificial SequenceSynthetic Oligonucleotide 14gactctcgag
cgttctc 171512DNAArtificial SequenceSynthetic Oligonucleotide
15gtcgtcgatg ac 121620DNAArtificial SequenceSynthetic
Oligonucleotide 16atgcactctc gagcgttctc 201713DNAArtificial
SequenceSynthetic Oligonucleotide 17ctcgagcgtt ctc
131812DNAArtificial SequenceSynthetic Oligonucleotide 18tgcagcgttc
tc 121912DNAArtificial SequenceSynthetic Oligonucleotide
19tttggcgttt tt 122020DNAArtificial SequenceSynthetic
Oligonucleotide 20atcgactctc gagcgttctc 202120DNAArtificial
SequenceSynthetic Oligonucleotide 21agcgtttctc gatcgacctc
202219DNAArtificial SequenceSynthetic Oligonucleotide 22ggtgcaccga
tgcaggggg 192314DNAArtificial SequenceSynthetic Oligonucleotide
23gtcgtcgacg acgg 142412DNAArtificial SequenceSynthetic
Oligonucleotide 24gggggcgttg gg 122520DNAArtificial
SequenceSynthetic Oligonucleotide 25atgcactctg cagcgttctc
202620DNAArtificial SequenceSynthetic Oligonucleotide 26atcgactctc
gaggcttctc 202717DNAArtificial SequenceSynthetic Oligonucleotide
27ggtgcatcga tgcaggg 172825DNAArtificial SequenceSynthetic
Oligonucleotide 28gggtcgtcgt tttgtcgttt cgttg 252912DNAArtificial
SequenceSynthetic Oligonucleotide 29aaaggcgtta aa
123012DNAArtificial SequenceSynthetic Oligonucleotide 30cccggcgttc
cc 123112DNAArtificial SequenceSynthetic Oligonucleotide
31gtcatcgatg ca 123220DNAArtificial SequenceSynthetic
Oligonucleotide 32ggtgcatcga tgcagggggg 203320DNAArtificial
SequenceSynthetic Oligonucleotide 33ggggtcatcg atgaaaaaaa
203420DNAArtificial SequenceSynthetic Oligonucleotide 34ggtgcatcga
tgcagggggg 203520DNAArtificial SequenceSynthetic Oligonucleotide
35aaggtcaacg ttgaaaaaaa 203620DNAArtificial SequenceSynthetic
Oligonucleotide 36aaggtcatcg atgggggggg 203720DNAArtificial
SequenceSynthetic Oligonucleotide 37ggtgcatcga tgcagggggg
203820DNAArtificial SequenceSynthetic Oligonucleotide 38ggtgcatcga
tgcagggggg 203920DNAArtificial SequenceSynthetic Oligonucleotide
39ggtgcgtcga cgcagggggg 204020DNAArtificial SequenceSynthetic
Oligonucleotide 40ggtgcgtcga tgcagggggg 204120DNAArtificial
SequenceSynthetic Oligonucleotide 41ggtgcgtcga cgcagggggg
204220DNAArtificial SequenceSynthetic Oligonucleotide 42ggtgcaccgg
tgcagggggg 204320DNAArtificial SequenceSynthetic Oligonucleotide
43ggtgcatcga tgcagggggg 204412DNAArtificial SequenceSynthetic
Oligonucleotide 44gtcaacgtcg ac 124512DNAArtificial
SequenceSynthetic Oligonucleotide 45gtcggcgtcg ac
124619DNAArtificial SequenceSynthetic Oligonucleotide 46ggggtcaacg
ttgaggggg 194712DNAArtificial SequenceSynthetic Oligonucleotide
47gtcggcgctg ac 124820DNAArtificial SequenceSynthetic
Oligonucleotide 48atgcactctc gaggcttctc 204917DNAArtificial
SequenceSynthetic Oligonucleotide 49aatgcatcga tgcaaaa
175012DNAArtificial SequenceSynthetic Oligonucleotide 50gtcagcgtcg
ac 125112DNAArtificial SequenceSynthetic Oligonucleotide
51gtcaacgttg ac 125212DNAArtificial SequenceSynthetic
Oligonucleotide 52tgcatcgatg ca 125319DNAArtificial
SequenceSynthetic Oligonucleotide 53ggtgcatcga tgcaggggg
195412DNAArtificial SequenceSynthetic Oligonucleotide 54gtcgacgtcg
ac 125512DNAArtificial SequenceSynthetic Oligonucleotide
55gtcgacgccg ac 125612DNAArtificial SequenceSynthetic
Oligonucleotide 56cccaacgttc cc 125712DNAArtificial
SequenceSynthetic Oligonucleotide 57gtcaacgctg ac
125810DNAArtificial SequenceSynthetic Oligonucleotide 58gagcgttctc
105912DNAArtificial SequenceSynthetic Oligonucleotide 59gggaacgttg
gg 126012DNAArtificial SequenceSynthetic Oligonucleotide
60gtcagcgctg ac 126116DNAArtificial SequenceSynthetic
Oligonucleotide 61gggggaacgt tcgggg 166212DNAArtificial
SequenceSynthetic Oligonucleotide 62gtcggcgccg ac
126316DNAArtificial SequenceSynthetic Oligonucleotide 63ggggtaacgt
tagggg 166412DNAArtificial SequenceSynthetic Oligonucleotide
64gtcaacgccg ac 126512DNAArtificial SequenceSynthetic
Oligonucleotide 65tgcctcgagg ca 126612DNAArtificial
SequenceSynthetic Oligonucleotide 66tttaacgttt tt
126712DNAArtificial SequenceSynthetic Oligonucleotide 67aaaaacgtta
aa 126816DNAArtificial SequenceSynthetic Oligonucleotide
68gggggaagct tcgggg 166912DNAArtificial SequenceSynthetic
Oligonucleotide 69gtcagcgccg ac 127011DNAArtificial
SequenceSynthetic Oligonucleotide 70cgagcgttct c
117116DNAArtificial SequenceSynthetic Oligonucleotide 71ggtgcatcga
tgcagg 167220DNAArtificial SequenceSynthetic Oligonucleotide
72ggtgcatcga tgcagggggg 207319DNAArtificial SequenceSynthetic
Oligonucleotide 73ggtgcatcga tgcaggggg 197413DNAArtificial
SequenceSynthetic Oligonucleotide 74ggcgtcgacg ggg
137519DNAArtificial SequenceSynthetic Oligonucleotide 75ggtgcgtcgt
tgcaggggg 197619DNAArtificial SequenceSynthetic Oligonucleotide
76ggtgcgccga tgcaggggg 197716DNAArtificial SequenceSynthetic
Oligonucleotide 77gggggatcga tcgggg 167813DNAArtificial
SequenceSynthetic Oligonucleotide 78ggggtcgaca ggg
137919DNAArtificial SequenceSynthetic Oligonucleotide 79ggtgcgtcgg
tgcaggggg 198016DNAArtificial SequenceSynthetic Oligonucleotide
80gggggatgca tcgggg 168120DNAArtificial SequenceSynthetic
Oligonucleotide 81ggtgcgtcga tgcagggggg 208220DNAArtificial
SequenceSynthetic Oligonucleotide 82ggtgcgtcga tgcagggggg
208319DNAArtificial SequenceSynthetic Oligonucleotide 83ggtgcgtcga
tgcaggggg 198419DNAArtificial SequenceSynthetic Oligonucleotide
84ggtgcctcga ggcaggggg 198516DNAArtificial SequenceSynthetic
Oligonucleotide 85gggggctcga gagggg 168616DNAArtificial
SequenceSynthetic Oligonucleotide 86ggggtatcga tagggg
168719DNAArtificial SequenceSynthetic Oligonucleotide 87ggtgcatcga
tgcgagaga 198819DNAArtificial SequenceSynthetic Oligonucleotide
88ggtgcatcga cgcaggggg 198920DNAArtificial SequenceSynthetic
Oligonucleotide 89ggggtcaacg ttgagggggg 209020DNAArtificial
SequenceSynthetic Oligonucleotide 90ggtgcatgca tgcagggggg
209120DNAArtificial SequenceSynthetic Oligonucleotide 91ggggtcaagc
ttgagggggg 209216DNAArtificial SequenceSynthetic Oligonucleotide
92ggggtaagct tagggg 169317DNAArtificial SequenceSynthetic
Oligonucleotide 93ggtgcatgca tgcaggg 179420DNAArtificial
SequenceSynthetic Oligonucleotide 94ggtgcataaa tgcagggggg
209517DNAArtificial SequenceSynthetic Oligonucleotide 95aatgcatgca
tgcaaaa 179620DNAArtificial SequenceSynthetic Oligonucleotide
96ggtgcatgca tgcagggggg 209720DNAArtificial SequenceSynthetic
Oligonucleotide 97atcgactctg caggcttctc 209812DNAArtificial
SequenceSynthetic Oligonucleotide 98tcgaggcttc tc
129920DNAArtificial SequenceSynthetic Oligonucleotide 99atgcactctg
caggcttctc 2010012DNAArtificial SequenceSynthetic Oligonucleotide
100tgcaggcttc tc 1210112DNAArtificial SequenceSynthetic
Oligonucleotide 101tcgtttgttc tc 1210212DNAArtificial
SequenceSynthetic Oligonucleotide 102acgagggttc tc
1210313DNAArtificial SequenceSynthetic Oligonucleotide
103ttccttcgag ctc 1310412DNAArtificial SequenceSynthetic
Oligonucleotide 104tcgatgcttc tc 1210512DNAArtificial
SequenceSynthetic Oligonucleotide 105gcgaggcttc tc
1210612DNAArtificial SequenceSynthetic Oligonucleotide
106ccgaggcttc tc 1210712DNAArtificial SequenceSynthetic
Oligonucleotide 107tgcaggcttc tc 1210812DNAArtificial
SequenceSynthetic Oligonucleotide 108tcgttcgttc tc
1210912DNAArtificial SequenceSynthetic Oligonucleotide
109tcgccgcttc tc 1211012DNAArtificial SequenceSynthetic
Oligonucleotide 110tcgaatgttc tc 1211112DNAArtificial
SequenceSynthetic Oligonucleotide 111tcgagtgttc tc
1211212DNAArtificial SequenceSynthetic Oligonucleotide
112tcgtatgttc tc 1211312DNAArtificial SequenceSynthetic
Oligonucleotide 113tcggatgttc tc 1211412DNAArtificial
SequenceSynthetic Oligonucleotide 114tcgcatgttc tc
1211512DNAArtificial SequenceSynthetic Oligonucleotide
115tcgactgttc tc 1211612DNAArtificial SequenceSynthetic
Oligonucleotide 116tcgcctgttc tc 1211712DNAArtificial
SequenceSynthetic Oligonucleotide 117tcggctgttc tc
1211812DNAArtificial SequenceSynthetic Oligonucleotide
118tcgtctgttc tc 1211912DNAArtificial SequenceSynthetic
Oligonucleotide 119tcgtgtgttc tc 1212012DNAArtificial
SequenceSynthetic Oligonucleotide 120tcgcttgttc tc
1212112DNAArtificial SequenceSynthetic Oligonucleotide
121ttgttcgaac tc 1212212DNAArtificial SequenceSynthetic
Oligonucleotide 122ttgttcgctc tc 1212312DNAArtificial
SequenceSynthetic Oligonucleotide 123ttgttcgccc tc
1212412DNAArtificial SequenceSynthetic Oligonucleotide
124ttgttcgggc tc 1212512DNAArtificial SequenceSynthetic
Oligonucleotide 125ttgttcgttc tc 1212612DNAArtificial
SequenceSynthetic Oligonucleotide 126ttgttcgtac tc
1212712DNAArtificial SequenceSynthetic Oligonucleotide
127tcgagttcgc tc 1212812DNAArtificial SequenceSynthetic
Oligonucleotide 128tcgagttcgt tc 1212912DNAArtificial
SequenceSynthetic Oligonucleotide 129tcgagttcga gc
1213012DNAArtificial SequenceSynthetic Oligonucleotide
130ctcgtttgtt ct 1213112DNAArtificial SequenceSynthetic
Oligonucleotide 131ttcgtttgtt ct 1213212DNAArtificial
SequenceSynthetic Oligonucleotide 132cccgtttgtt ct
1213312DNAArtificial SequenceSynthetic Oligonucleotide
133tcggttgttc tc 1213411DNAArtificial SequenceSynthetic
Oligonucleotide 134tgcgcaaggg g 1113512DNAArtificial
SequenceSynthetic Oligonucleotide 135tcgccctttc tc
1213620DNAArtificial SequenceSynthetic Oligonucleotide
136ggtatatcga tatagggggg 2013720DNAArtificial SequenceSynthetic
Oligonucleotide 137ggtggatcga tccagggggg 2013820DNAArtificial
SequenceSynthetic Oligonucleotide 138ggtccatcga tccagggggg
2013920DNAArtificial SequenceSynthetic Oligonucleotide
139ggtggatcga tggagggggg 2014011DNAArtificial SequenceSynthetic
Oligonucleotide 140agcgctaggg g 1114120DNAArtificial
SequenceSynthetic Oligonucleotide 141ggtgcatgta tgcagggggg
2014220DNAArtificial SequenceSynthetic Oligonucleotide
142ggtgcacgcg tgcagggggg 2014313DNAArtificial SequenceSynthetic
Oligonucleotide 143cgttctcggg ggg 13
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