U.S. patent application number 15/726805 was filed with the patent office on 2018-01-25 for immune regulatory oligonucleotide (iro) compounds to modulate toll-like receptor based immune response.
The applicant listed for this patent is IDERA PHARMACEUTICALS, INC.. Invention is credited to Sudhir Agrawal, Ekambar R. Kandimalla, Ireneusz Nowak, Daqing Wang.
Application Number | 20180023085 15/726805 |
Document ID | / |
Family ID | 46064566 |
Filed Date | 2018-01-25 |
United States Patent
Application |
20180023085 |
Kind Code |
A1 |
Kandimalla; Ekambar R. ; et
al. |
January 25, 2018 |
IMMUNE REGULATORY OLIGONUCLEOTIDE (IRO) COMPOUNDS TO MODULATE
TOLL-LIKE RECEPTOR BASED IMMUNE RESPONSE
Abstract
The invention provides novel immune regulatory oligonucleotides
(IRO) as antagonist of TLRs and methods of use thereof. These IROs
have unique sequences that inhibit or suppress TLR-mediated
signaling in response to a TLR ligand or TLR agonist. The methods
may have use in the prevention and treatment of cancer, an
autoimmune disorder, airway inflammation, inflammatory disorders,
infectious disease, skin disorders, allergy, asthma or a disease
caused by a pathogen.
Inventors: |
Kandimalla; Ekambar R.;
(Hopkinton, MA) ; Wang; Daqing; (Bedford, MA)
; Nowak; Ireneusz; (Allston, MA) ; Agrawal;
Sudhir; (Shrewsbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IDERA PHARMACEUTICALS, INC. |
CAMBRIDGE |
MA |
US |
|
|
Family ID: |
46064566 |
Appl. No.: |
15/726805 |
Filed: |
October 6, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15366509 |
Dec 1, 2016 |
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15726805 |
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14791749 |
Jul 6, 2015 |
9540651 |
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15366509 |
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13905242 |
May 30, 2013 |
9096858 |
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14791749 |
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13299555 |
Nov 18, 2011 |
8486908 |
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13905242 |
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61415494 |
Nov 19, 2010 |
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61511709 |
Jul 26, 2011 |
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Current U.S.
Class: |
424/130.1 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 19/08 20180101; A61P 1/16 20180101; C12N 15/117 20130101; C12N
2310/51 20130101; A61P 11/00 20180101; A61P 25/18 20180101; A61P
19/02 20180101; A61K 31/7125 20130101; C12N 2310/3521 20130101;
A61P 15/00 20180101; A61P 17/06 20180101; A61P 13/10 20180101; C12N
2310/321 20130101; C12N 2310/336 20130101; A61P 7/06 20180101; A61P
21/04 20180101; A61P 35/00 20180101; C12N 2310/3341 20130101; A61P
21/00 20180101; C12N 2310/31 20130101; A61P 43/00 20180101; A61P
5/14 20180101; C12N 2310/52 20130101; A61K 45/06 20130101; A61P
25/00 20180101; A61P 9/00 20180101; A61P 13/00 20180101; A61P 31/00
20180101; A61P 13/12 20180101; C12N 2310/317 20130101; Y02A 50/30
20180101; A61P 29/00 20180101; A61P 1/00 20180101; A61P 17/00
20180101; C12N 2310/17 20130101; A61P 37/02 20180101; A61K 31/7115
20130101; A61P 7/04 20180101; A61K 31/7115 20130101; A61K 2300/00
20130101 |
International
Class: |
C12N 15/117 20060101
C12N015/117; A61K 31/7125 20060101 A61K031/7125; A61K 31/7115
20060101 A61K031/7115; A61K 45/06 20060101 A61K045/06 |
Claims
1. An antagonist of TLR7 and/or TLR9 having the structure
5-N.sub.pN.sub.3N.sub.2N.sub.1C*G1N.sup.1N.sup.2N.sup.3N.sup.zN.sup.4N.su-
p.5-3', wherein C*G* is an oligonucleotide motif wherein C* is
5-Me-dC, and G1 is 7-deaza-dG; N.sub.1-N.sub.2, at each occurrence,
is independently a 2'-O-Me-ribonucleotide; N.sup.3, at each
occurrence, is independently a nucleotide; N.sup.1-N.sup.3, at each
occurrence, is independently a nucleotide; N.sub.m and N.sup.m, at
each occurrence, is independently a nucleotide; N.sup.4-N.sup.5, at
each occurrence, is independently a 2'-O-Me-ribonucleotide; p is 5;
and z is 3; provided that the compound contains less than 3
consecutive guanosines.
2. The antagonist according to claim 1 having the structure
5'-CTATCTGUC*G1TTCTCTGU-3' or 5'-CTATCTGAC*G1TTCTCTGU-3'.
3. A pharmaceutical composition comprising an antagonist according
to claim 1 or 2 and a pharmaceutically acceptable carrier.
4. An immune regulatory oligonucleotide (IRO) compound that is an
antagonist of TLR7 and/or TLR9, selected from compound number 1
through compound number 67 and compound number 69 through compound
number 111.
5. A pharmaceutical composition comprising an antagonist according
to claim 4 and a pharmaceutically acceptable carrier.
6. A method for inhibiting a TLR7- and/or TLR9-mediated immune
response in a mammal, the method comprising administering to the
mammal an antagonist according to any one of claim 1, 2 or 4 or a
composition thereof.
7. The method according to claim 6, wherein the route of
administration is parenteral, mucosal delivery, oral, sublingual,
transdermal, topical, inhalation, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form.
8. The method according to claim 6, wherein the mammal is a
human.
9. The method according to claim 6, wherein the antagonist is
administered in combination with one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonists, TLR antagonists, peptides,
proteins, gene therapy vectors, DNA vaccines, adjuvants, kinase
inhibitors or co-stimulatory molecules.
10. A method for inhibiting the activity of a TLR7 and/or TLR9
agonist comprising administering an antagonist according to any one
of claim 1, 2 or 4 or a composition thereof.
11. The method according to claim 10, comprising administering the
antagonist prior to or at the same time as the TLR7 and/or TLR9
agonist.
12. A method for therapeutically treating a mammal having an
autoimmune disease mediate by TLR7 and/or TLR9, wherein the
autoimmune disease is selected from psoriasis, rheumatoid
arthritis, alopecia universalis, acute disseminated
encephalomyelitis, Addison's disease, ankylosing spondylitis,
antiphospholipid antibody syndrome, autoimmune hemolytic anemia,
autoimmune hepatitis, Bullous pemphigoid, chagas disease, chronic
obstructive pulmonary disease, coeliac disease, dermatomyositis,
endometriosis, Goodpasture's syndrome, Graves' disease,
Guillain-Barre syndrome, Hashimoto's disease, hidradenitis
suppurativa, idiopathic thrombocytopenic purpura, interstitial
cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
pemphigus, pernicious anaemia, polymyositis, primary biliary
cirrhosis, schizophrenia, Sjogren's syndrome, temporal arteritis,
vasculitis, vitiligo, vulvodynia or Wegener's granulomatosis, such
method comprising administering to the mammal an antagonist
according to any one of claim 1, 2 or 4 or a composition
thereof.
13. The method according to claim 12, wherein the antagonist is
administered in combination with one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonists, TLR antagonists, peptides,
proteins, gene therapy vectors, DNA vaccines, adjuvants, kinase
inhibitors or co-stimulatory molecules.
14. The method according to claim 12, wherein the route of
administration is parenteral, mucosal delivery, oral, sublingual,
transdermal, topical, inhalation, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form.
15. The method according to claim 12, wherein the mammal is a
human.
16. A method for preventing an autoimmune disease mediate by a TLR7
and/or TLR9, wherein the autoimmune disease is selected from
psoriasis, rheumatoid arthritis, alopecia universalis, acute
disseminated encephalomyelitis, Addison's disease, ankylosing
spondylitis, antiphospholipid antibody syndrome, autoimmune
hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, chagas
disease, chronic obstructive pulmonary disease, coeliac disease,
dermatomyositis, endometriosis, Goodpasture's syndrome, Graves'
disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis
suppurativa, idiopathic thrombocytopenic purpura, interstitial
cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
pemphigus, pernicious anaemia, polymyositis, primary biliary
cirrhosis, schizophrenia, Sjogren's syndrome, temporal arteritis,
vasculitis, vitiligo, vulvodynia or Wegener's granulomatosis, such
method comprising administering to a mammal an antagonist according
to any one of claim 1, 2 or 4 or a composition thereof.
17. The method according to claim 16, wherein the antagonist is
administered in combination with one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonists, TLR antagonists, peptides,
proteins, gene therapy vectors, DNA vaccines, adjuvants or
co-stimulatory molecules.
18. The method according to claim 16, wherein the route of
administration is parenteral, mucosal delivery, oral, sublingual,
transdermal, topical, inhalation, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form.
19. The method according to claim 16, wherein the mammal is a
human.
20. The composition according to claim 3, further comprising one or
more vaccines, antigens, antibodies, cytotoxic agents, allergens,
antibiotics, antisense oligonucleotides, TLR agonists, TLR
antagonists, peptides, proteins, gene therapy vectors, DNA
vaccines, adjuvants or co-stimulatory molecules.
21. The composition according to claim 5, further comprising one or
more vaccines, antigens, antibodies, cytotoxic agents, allergens,
antibiotics, antisense oligonucleotides, TLR agonists, TLR
antagonists, peptides, proteins, gene therapy vectors, DNA
vaccines, adjuvants or co-stimulatory molecules.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/791,749, filed Jul. 6, 2015, which is a continuation of U.S.
application Ser. No. 13/905,242, filed May 30, 2013 (now U.S. Pat.
No. 9,096,858), which is a continuation of U.S. application Ser.
No. 13/299,555, filed Nov. 18, 2011 (now U.S. Pat. No. 8,486,908),
which claims the benefit of U.S. Provisional Application No.
61/415,494, filed on Nov. 19, 2010 and U.S. Provisional Application
No. 61/511,709, filed on Jul. 26, 2011. The entire teachings of the
above applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The invention generally relates to the field of immunology
and immunotherapy, and more specifically to immune regulatory
oligonucleotide (IRO) compositions and their use for inhibition
and/or suppression of Toll-like Receptor-mediated immune responses.
In particular, the invention relates to antagonists of Toll-Like
Receptors 7 (TLR7) and/or TLR9 that uniquely inhibit cytokines
normally produced through TLR7 and/or TLR9 stimulation.
Summary of the Related Art
[0003] Toll-like receptors (TLRs) are present on many cells of the
immune system and have been shown to be involved in the innate
immune response (Hornung, V. et al., (2002) J. Immunol.
168:4531-4537). In vertebrates, or mammals, this family consists of
ten proteins called TLR1 to TLR10, which are known to recognize
pathogen associated molecular patterns from bacteria, fungi,
parasites, and viruses (Poltorak, A. et al. (1998) Science
282:2085-2088; Underhill, D. M., et al. (1999) Nature 401:811-815;
Hayashi, F. et. al (2001) Nature 410:1099-1103; Zhang, D. et al.
(2004) Science 303:1522-1526; Meier, A. et al. (2003) Cell.
Microbiol. 5:561-570; Campos, M. A. et al. (2001) J. Immunol. 167:
416-423; Hoebe, K. et al. (2003) Nature 424: 743-748; Lund, J.
(2003) J. Exp. Med. 198:513-520; Heil, F. et al. (2004) Science
303:1526-1529; Diebold, S. S., et al. (2004) Science 303:1529-1531;
Hornung, V. et al. (2004) J. Immunol. 173:5935-5943). TLRs are a
key means by which mammals recognize and mount an immune response
to foreign molecules and also provide a means by which the innate
and adaptive immune responses are linked (Akira, S. et al. (2001)
Nature Immunol. 2:675-680; Medzhitov, R. (2001) Nature Rev.
Immunol. 1:135-145). TLRs have also been shown to play a role in
the pathogenesis of many diseases, including autoimmunity,
infectious disease, and inflammation (Cook, D. N. et al. (2004)
Nature Immunol. 5:975-979) and the regulation of TLR-mediated
activation using appropriate agents may provide a means for disease
intervention.
[0004] Some TLRs are located on the cell surface to detect and
initiate a response to extracellular pathogens and other TLRs are
located inside the cell to detect and initiate a response to
intracellular pathogens. Table 1 provides a representation of TLRs,
their cellular location, and the known agonists therefore (Diebold,
S. S. et al. (2004) Science 303:1529-1531; Liew, F. et al. (2005)
Nature 5:446-458; Hemmi H et al. (2002) Nat Immunol 3:196-200; Jurk
M et al., (2002) Nat Immunol 3:499; Lee J et al. (2003) Proc. Natl.
Acad. Sci. USA 100:6646-6651); (Alexopoulou, L. (2001) Nature
413:732-738).
TABLE-US-00001 TABLE 1 TLR Molecule Agonist Cell Surface TLRs: TLR2
bacterial lipopeptides TLR4 gram negative bacteria TLR5 motile
bacteria TLR6 gram positive bacteria Endosomal TLRs: TLR3 double
stranded RNA viruses TLR7 single stranded RNA viruses TLR8 single
stranded RNA viruses TLR9 unmethylated DNA
[0005] Certain unmethylated CpG motifs present in bacterial and
synthetic DNA have been shown to activate the immune system and
induce antitumor activity. (Tokunaga T et al., J. Natl. Cancer
Inst. (1984) 72:955-962; Shimada S, et al., Jpn. H cancer Res,
1986, 77, 808-816; Yamamoto S, et al., Jpn. J. Cancer Res., 1986,
79, 866-73). Other studies using antisense oligonucleotides
containing CpG dinucleotides have been shown to stimulate immune
responses (Zhao Q, et al. (1996) Biochem. Pharmacol. 26:173-182).
Subsequent studies demonstrated that TLR9 recognizes unmethylated
CpG motifs present in bacterial and synthetic DNA (Hemmi, H. et al.
(2000) Nature 408:740-745). Other modifications of CpG-containing
phosphorothioate oligonucleotides can also affect their ability to
act as modulators of immune response through TLR9 (see, e.g., Zhao
et al., Biochem. Pharmacol. (1996) 51:173-182; Zhao et al. (1996)
Biochem Pharmacol. 52:1537-1544; Zhao et al. (1997) Antisense
Nucleic Acid Drug Dev. 7:495-502; Zhao et al (1999) Bioorg. Med.
Chem. Lett. 9:3453-3458; Zhao et al. (2000) Bioorg. Med. Chem.
Lett. 10:1051-1054; Yu, D. et al. (2000) Bioorg. Med. Chem. Lett.
10:2585-2588; Yu, D. et al. (2001) Bioorg. Med. Chem. Lett.
11:2263-2267; and Kandimalla, E. et al. (2001) Bioorg. Med. Chem.
9:807-813). In addition, structure activity relationship studies
have allowed identification of synthetic motifs and novel DNA-based
compounds that induce specific immune response profiles that are
distinct from those resulting from unmethylated CpG dinucleotides.
(Kandimalla, E. et al. (2005) Proc. Natl. Acad. Sci. USA
102:6925-6930. Kandimalla, E. et al. (2003) Proc. Nat. Acad. Sci.
USA 100:14303-14308; Cong, Y. et al. (2003) Biochem Biophys Res.
Commun. 310:1133-1139; Kandimalla, E. et al. (2003) Biochem.
Biophys. Res. Commun. 306:948-953; Kandimalla, E. et al. (2003)
Nucleic Acids Res. 31:2393-2400; Yu, D. et al. (2003) Bioorg. Med.
Chem. 11:459-464; Bhagat, L. et al. (2003) Biochem. Biophys. Res.
Commun. 300:853-861; Yu, D. et al. (2002) Nucleic Acids Res.
30:4460-4469; Yu, D. et al. (2002) J. Med. Chem. 45:4540-4548. Yu,
D. et al. (2002) Biochem. Biophys. Res. Commun. 297:83-90;
Kandimalla. E. et al. (2002) Bioconjug. Chem. 13:966-974; Yu, D. et
al. (2002) Nucleic Acids Res. 30:1613-1619; Yu, D. et al. (2001)
Bioorg. Med. Chem. 9:2803-2808; Yu, D. et al. (2001) Bioorg. Med.
Chem. Lett. 11:2263-2267; Kandimalla, E. et al. (2001) Bioorg. Med.
Chem. 9:807-813; Yu, D. et al. (2000) Bioorg. Med. Chem. Lett.
10:2585-2588; Putta, M. et al. (2006) Nucleic Acids Res.
34:3231-3238).
[0006] The selective localization of TLRs and the signaling
generated therefrom, provides some insight into their role in the
immune response. The immune response involves both an innate and an
adaptive response based upon the subset of cells involved in the
response. For example, the T helper (Th) cells involved in
classical cell-mediated functions such as delayed-type
hypersensitivity and activation of cytotoxic T lymphocytes (CTLs)
are Th1 cells. This response is the body's innate response to
antigen (e.g. viral infections, intracellular pathogens, and tumor
cells), and results in a secretion of IFN-gamma and a concomitant
activation of CTLs. Alternatively, the Th cells involved as helper
cells for B-cell activation are Th2 cells. Th2 cells have been
shown to be activated in response to bacteria and parasites and may
mediate the body's adaptive immune response (e.g. IgE production
and eosinophil activation) through the secretion of IL-4 and IL-5.
The type of immune response is influenced by the cytokines produced
in response to antigen exposure and the differences in the
cytokines secreted by Th1 and Th2 cells may be the result of the
different biological functions of these two sub sets.
[0007] While activation of TLRs is involved in mounting an immune
response, an uncontrolled stimulation of the immune system through
TLRs may exacerbate certain diseases in immune compromised
subjects. In recent years, several groups have shown the use of
synthetic oligodeoxyoligonucleotides (ODNs) as inhibitors of
inflammatory cytokines (Lenert, P. et al. (2003) DNA Cell Biol.
22(10):621-631).
[0008] Using certain synthetic ODNs, Lenert et al. report the
ability to produce inhibitory ODNs (Lenert, P. et al. (2003) DNA
Cell Biol. 22(10):621-631). These inhibitory ODN require two
triplet sequences, a proximal "CCT" triplet and a distal "GGG"
triplet. In addition to these triplet-containing inhibitory ODNs,
several groups have reported other specific DNA sequences that
could inhibit TLR-9-mediated activation by CpG-containing ODNs.
These "inhibitory" or "suppressive" motifs are rich in poly "G"
(e.g. "GGGG") or "GC" sequences, tend to be methylated, and are
present in the DNA of mammals and certain viruses (see e.g., Chen,
Y., et al., Gene Ther. 8: 1024-1032 (2001); Stunz, L. L., Eur. J.
Immunol. 32: 1212-1222 (2002). Duramad, O., et al., J. Immunol.,
174: 5193-5200 (2005) and Jurk et. al (US 2005/0239733), describe a
structure for inhibitory DNA oligonucleotides containing a GGGG
motif within the sequences. Patole et al. demonstrate that GGGG
containing ODNs will suppress systemic lupus (Patole, P. et al.
(2005) J. Am. Soc. Nephrol. 16:3273-3280). Additionally, Gursel,
I., et al., J. Immunol., 171: 1393-1400 (2003), describe repetitive
TTAGGG elements, which are present at high frequency in mammalian
telomeres, down-regulate CpG-induced immune activation. Shirota,
H., et al., J. Immunol., 173: 5002-5007 (2004), demonstrate that
synthetic oligonucleotides containing the TTAGGG element mimic this
activity and could be effective in the prevention/treatment of
certain Th1-dependent autoimmune diseases.
[0009] In contrast, some studies have called into question the view
that poly G containing ODNs are acting as antagonists of TLRs. For
example, U.S. Pat. No. 6,426,334, Agrawal et al., demonstrate that
administering CpG oligonucleotides containing GGGG strings have
potent antiviral and anticancer activity and that administration of
these compounds will cause an increase in serum IL-12
concentration. Further, CpG oligos containing polyG sequences are
known to induce immune responses through TLR9 activation (Verthelyi
D et al, J Immunol. 166, 2372, 2001; Gursel M et al, J Leukoc Biol,
71, 813, 2001, Krug A et al, Eur J Immunol, 31, 2154, 2001) and
show antitumor and antiviral activities (Ballas G K et al, J
Immunol, 167, 4878, 2001; Verthelyi D et al, J Immunol, 170, 4717,
2003). In addition, polyG oligonucleotides are known to inhibit HIV
and Rel A (McShan W M, et al, J Biol Chem., 267(8):5712-21, 1992;
Rando, R F et al., J Biol Chem, 270(4):1754-60, 1995; Benimetskaya
L, et al., Nucleic Acids Res., 25(13):2648-56, 1997); and ODNs
containing an immune stimulatory CpG motif and 4 consecutive G
nucleotides (known as class A ODNs) induce interferon-.gamma.
production and a Th1 shift in the immune response. Moreover, in
preclinical disease models, Class A ODNs have been shown to induce
a TLR-mediated immune response.
[0010] As an additional limitation, oligonucleotides containing
guanosine strings have been shown to form tetraplex structures, act
as aptamers, and inhibit thrombin activity (Bock L C et al.,
Nature, 355:564-6, 1992; Padmanabhan, K et al., J Biol Chem.,
268(24):17651-4, 1993). Thus, it is not clear whether
single-stranded or multiple-stranded structures are effective at
suppressing TLR9 activation.
[0011] Kandimalla et al. (Ser. No. 11/549,048) describe a novel
class of TLR antagonists that do not require a polyG sequence.
Kandimalla et al. also describes the application of these novel
compositions to treating and preventing various diseases and
disorders (Ser. Nos. 11/549,048; 11/743,876; 12/140,334;
12/140,338; 12/244,199). However a challenge remains to develop
additional TLR antagonists that do not require a polyG sequence and
thus do not present the problem of forming secondary structures.
This challenge may be solved through the design of new
oligonucleotide-based compounds and compositions that can act as
unique inhibitors of TLRs 7 and/or 9. Such new custom compounds and
compositions will find use in many clinically relevant
applications, including treating and preventing diseases and
disorders with an immune stimulatory component.
BRIEF SUMMARY OF THE INVENTION
[0012] The inventors have surprisingly discovered that uniquely
modifying the nucleic acid sequence on the 5'-side of a core immune
stimulatory dinucleotide, the nucleic acids within the core immune
stimulatory dinucleotide, the linkages between nucleotides or the
linkers connecting two or more oligonucleotides produces novel
antagonists of TLR7 and/or TLR9 that distinctly antagonize,
inhibit, suppress or prevent the in vitro and in vivo cytokine and
chemokine profiles normally generated through TLR7 and/or TLR9
stimulation. This ability to antagonize, inhibit suppress or
prevent the cytokine and chemokine response to a TLR7 and/or TLR9
agonist provides the ability to prevent and/or treat various
disease conditions in a disease-specific and even a
patient-specific manner.
[0013] Thus, the invention provides immune regulatory
oligonucleotides (IRO) compounds that act as distinct antagonists
of TLR7 and/or TLR9 and methods of using such compounds to
antagonize, inhibit, suppress or prevent TLR7- and/or TLR9-mediated
immune stimulation. These IRO compounds comprise an immune
stimulatory motif and would be immune stimulatory but for one or
more chemical modifications in the nucleic acid sequence on the
5'-side of the immune stimulatory motif and/or in the immune
stimulatory motif. The IRO compounds and compositions that
preferentially antagonize, inhibit, suppress or prevent the
activity of TLR7 and/or TLR9 have the structure
5-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3'
(SEQ ID NO: 70), wherein CG is an oligonucleotide motif selected
from CpG, C*pG, C*pG* or CpG* wherein C is cytosine, C* is a
cytosine analog or derivative, G is a guanine and G* is a guanine
analog or derivative; N.sub.1-N.sub.3, at each occurrence, is
independently a nucleotide or nucleotide derivative;
N.sup.1-N.sup.3, at each occurrence, is independently a nucleotide
or nucleotide derivative; N.sub.m and N.sup.m, at each occurrence,
is independently a nucleotide, nucleotide derivative or
non-nucleotide linker; provided that at least one of N.sub.1,
N.sub.2, and N.sub.3 and/or C and/or G of the oligonucleotide motif
is a nucleotide derivative that antagonizes, inhibits, suppresses
or prevents the activity of the oligonucleotide motif; and further
provided that the compound contains less than 4 consecutive
guanosine nucleotides and preferably less than 3 consecutive
guanosines, wherein the immune stimulatory activity of the
oligonucleotide motif is antagonized, inhibited, suppressed or
prevented by the nucleotide derivative; and wherein m is a number
from 0 to about 30.
[0014] In some embodiments, the IRO compounds may comprise at least
two oligonucleotides, wherein at least two oligonucleotides are
covalently linked via a direct nucleotide to nucleotide linkage at
their 3' ends through the 3' positions of the sugars or through a
modified sugar or modified nucleobase or via a non-nucleotide
linker at their 3' ends through the 3' positions of the sugars or
through a modified sugar or modified nucleobase. In preferred
aspects of this embodiment, at least one of oligonucleotides of the
IRO compound has the structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3'
(SEQ ID NO: 70), wherein N.sub.m, N.sub.1, N.sub.2, N.sub.3, C, G,
N.sup.1, N.sup.2, N.sup.3 and N.sub.m are as described above for
the general structure of the IRO compound. In more preferred
aspects of this embodiment, at least two of the oligonucleotides of
the IRO compound have the structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3'
(SEQ ID NO: 70), wherein N.sub.m, N.sub.1, N.sub.2, N.sub.3, C, G,
N.sup.1, N.sup.2, N.sup.3 and N.sub.m are as described above for
the general structure of the IRO compound. Such an IRO compound may
have the structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-
-3'-X-3'-N.sup.m-N.sup.3N.sup.2N.sup.1GCN.sub.1N.sub.2N.sub.3-N.sub.m-5'
(5'-SEQ ID NO: 70-3'-X-3'-SEQ ID NO: 70-5'), wherein X is a
nucleotide linkage or a non-nucleotide linker and N.sub.m, N.sub.1,
N.sub.2, N.sub.3, C, G, N.sup.1, N.sup.2, N.sup.3 and N.sub.m are
as described above for the general structure of the IRO
compound.
[0015] The IRO compounds and compositions according to the
invention preferentially inhibit TLR7 and/or TLR9-mediated immune
responses in various cell types and in various in vitro and in vivo
experimental models, with each compound or composition providing a
distinct immune inhibition profile.
[0016] The invention further provides for a pharmaceutical
composition comprising an IRO compound according to the invention
and a pharmaceutically acceptable carrier.
[0017] The invention further provides a method for inhibiting a
TLR-mediated immune response in a vertebrate, or mammal, the method
comprising administering to the mammal an IRO compound or
composition according to the invention in a pharmaceutically
effective amount. In some preferred embodiments, suppressing or
inhibiting TLR stimulation comprises administering an IRO compound
according to the invention, wherein the TLR is selected from TLR7
and TLR9.
[0018] The invention further provides a method for suppressing or
inhibiting the activity of a TLR agonist comprising administering
an IRO compound according to the invention, wherein the IRO
compound is administered at the same time, prior to or after the
TLR agonist. In preferred embodiments the TLR agonist is selected
from agonists of TLR7 and TLR9.
[0019] The invention further provides a method for therapeutically
treating a vertebrate, or mammal, having a disease mediated by TLR7
and/or TLR9, such method comprising administering to the mammal an
IRO compound according to the invention in a pharmaceutically
effective amount. In preferred embodiments, the disease is cancer,
an autoimmune disease or disorder, airway inflammation, an
inflammatory disease or disorder, infectious disease, malaria, Lyme
disease, ocular infections, conjunctivitis, skin disorders,
psoriasis, scleroderma, cardiovascular disease, atherosclerosis,
chronic fatigue syndrome, sarcoidosis, transplant rejection,
allergy, asthma or a disease caused by a pathogen. Preferred
autoimmune diseases and disorders include without limitation lupus
erythematosus, multiple sclerosis, type I diabetes mellitus,
irritable bowel syndrome, Chron's disease, rheumatoid arthritis,
septic shock, alopecia universalis, acute disseminated
encephalomyelitis, Addison's disease, ankylosing spondylitis,
antiphospholipid antibody syndrome, autoimmune hemolytic anemia,
autoimmune hepatitis, Bullous pemphigoid, chagas disease, chronic
obstructive pulmonary disease, coeliac disease, dermatomyositis,
endometriosis, Goodpasture's syndrome, Graves' disease,
Guillain-Barre syndrome, Hashimoto's disease, hidradenitis
suppurativa, idiopathic thrombocytopenic purpura, interstitial
cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
pemphigus, pernicious anaemia, polymyositis, primary biliary
cirrhosis, schizophrenia, Sjogren's syndrome, temporal arteritis
(also known as "giant cell arteritis"), vasculitis, vitiligo,
vulvodynia, and Wegener's granulomatosis. Preferred inflammatory
diseases and disorders include without limitation airway
inflammation, asthma, autoimmune diseases, chronic inflammation,
chronic prostatitis, glomerulonephritis, Behcet's disease,
hypersensitivities, inflammatory bowel disease, reperfusion injury,
rheumatoid arthritis, transplant rejection, ulcerative colitis,
uveitis, conjunctivitis, and vasculitis.
[0020] The invention further provides a method for preventing
cancer, autoimmune diseases or disorders, airway inflammation,
inflammatory diseases or disorders, infectious disease, malaria,
Lyme disease, ocular infections, conjunctivitis, skin disorders,
psoriasis, scleroderma, cardiovascular disease, atherosclerosis,
chronic fatigue syndrome, sarcoidosis, transplant rejection,
allergy, asthma or a disease caused by a pathogen in a vertebrate,
or mammal, such method comprising administering to the mammal an
IRO compound according to the invention in a pharmaceutically
effective amount. Preferred autoimmune diseases and disorders
include without limitation lupus erythematosus, multiple sclerosis,
type I diabetes mellitus, irritable bowel syndrome, Chron's
disease, rheumatoid arthritis, septic shock, alopecia universalis,
acute disseminated encephalomyelitis, Addison's disease, ankylosing
spondylitis, antiphospholipid antibody syndrome, autoimmune
hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, chagas
disease, chronic obstructive pulmonary disease, coeliac disease,
dermatomyositis, endometriosis, Goodpasture's syndrome, Graves'
disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis
suppurativa, idiopathic thrombocytopenic purpura, interstitial
cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
pemphigus, pernicious anaemia, polymyositis, primary biliary
cirrhosis, schizophrenia, Sjogren's syndrome, temporal arteritis
(also known as "giant cell arteritis"), vasculitis, vitiligo,
vulvodynia, and Wegener's granulomatosis. Preferred inflammatory
diseases and disorders include without limitation airway
inflammation, asthma, autoimmune diseases, chronic inflammation,
chronic prostatitis, glomerulonephritis, Behcet's disease,
hypersensitivities, inflammatory bowel disease, reperfusion injury,
rheumatoid arthritis, transplant rejection, ulcerative colitis,
uveitis, conjunctivitis, and vasculitis.
[0021] In some preferred embodiments, the IRO compound according to
the invention is administered in combination with one or more
vaccines, antigens, antibodies, cytotoxic agents, allergens,
antibiotics, antisense oligonucleotides, TLR agonists, TLR
antagonists, peptides, proteins, gene therapy vectors, DNA
vaccines, adjuvants, kinase inhibitors or co-stimulatory molecules
or combinations thereof. In some preferred embodiments, the route
of administration is parenteral, mucosal delivery, oral,
sublingual, transdermal, topical, inhalation, intranasal, aerosol,
intraocular, intratracheal, intrarectal, intragastric, vaginal, by
gene gun, dermal patch or in eye drop or mouthwash form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a synthetic scheme for the linear synthesis of
immune regulatory compounds of the invention.
DMTr=4,4'-dimethoxytrityl; CE=cyanoethyl.
[0023] FIG. 2 is a synthetic scheme for the parallel synthesis of
immune regulatory oligonucleotides of the invention.
DMTr=4,4'-dimethoxytrityl; CE=cyanoethyl.
[0024] FIGS. 3A-3C depicts the ability of TLR7/9 antagonists
according to the invention to inhibit TLR7-induced
cytokines/chemokines by TLR7/9 antagonists in mouse splenocytes
treated according to Example 3.
[0025] FIGS. 4A-4C depicts the ability of TLR7/9 antagonists
according to the invention to inhibit TLR9-induced
cytokines/chemokines by TLR7/9 antagonists in mouse splenocytes
treated according to Example 3.
[0026] FIG. 5 depicts the ability of TLR7/9 antagonists according
to the invention to inhibit TLR7-induced IL-12 in vivo in mice
treated according to Example 4.
[0027] FIG. 6 depicts the ability of TLR7/9 antagonists according
to the invention to inhibit TLR9-induced IL-12 in vivo in mice
treated according to Example 4.
[0028] FIG. 7 depicts the ability of TLR7/9 antagonists according
to the invention to inhibit TLR7-induced IL-12 in vivo over time in
mice treated according to Example 4.
[0029] FIG. 8 depicts the ability of TLR7/9 antagonists according
to the invention to inhibit TLR9-induced inhibition of IL-12 in
vivo over time in mice treated according to Example 4.
[0030] FIGS. 9A-9B depicts the ability of TLR7/9 antagonists
according to the invention to selectively inhibit mouse TLR7 &
9-induced cytokines in vivo in mice treated according to Example
4.
[0031] FIGS. 10A-10B demonstrates that TLR7/9 antagonists according
to the invention do not inhibit the activity of TLR3 or TLR5 in
vivo in mice treated according to Example 4.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] The present invention relates to the therapeutic use of
novel oligonucleotide-based compounds as immune modulatory agents
for immunotherapy applications. The invention provides novel
oligonucleotide-based compounds that provide distinct immune
inhibition profiles through their interaction with TLR7 and/or
TLR9. Specifically, the invention provides Immune Regulatory
Oligonucleotide (IRO) compounds as antagonists of toll-like
receptors (TLRs) to inhibit and/or suppress a TLR-mediated immune
response. These IROs have chemical modifications, and/or
internucleotide linkages, and/or linkers between oligonucleotides
that provide their inhibition or suppression of TLR7- and/or
TLR9-mediated signaling in response to endogenous and/or exogenous
TLR ligands or agonists. The references cited herein reflect the
level of knowledge in the field and are hereby incorporated by
reference in their entirety. Any conflicts between the teachings of
the cited references and this specification shall be resolved in
favor of the latter.
[0033] The invention further provides methods for suppressing an
immune response caused by TLRs and can be used for immunotherapy
applications such as, but not limited to, treatment of cancer,
autoimmune disorders, asthma, respiratory allergies, food
allergies, skin allergies, systemic lupus erythematosus (SLE),
arthritis, pleurisy, chronic infections, inflammatory diseases,
inflammatory bowel syndrome, sepsis, and bacteria, parasitic, and
viral infections in adult and pediatric human and veterinary
applications. Thus, the invention provides IRO compounds having
optimal levels of immune modulatory effect for immunotherapy and
methods for making and using such compounds. In addition, IRO
compounds of the invention are useful in combination with, for
example, vaccines, antigens, antibodies, allergens,
chemotherapeutic agents (both chemotherapy and targeted therapies),
and/or antisense oligonucleotides for prevention and treatment of
diseases.
Definitions
[0034] The term "oligonucleotide" generally refers to a
polynucleoside comprising a plurality of linked nucleoside units.
Such oligonucleotides can be obtained from existing nucleic acid
sources, including genomic or cDNA, but are preferably produced by
synthetic methods. In preferred embodiments each nucleoside unit
can encompass various chemical modifications and substitutions as
compared to wild-type oligonucleotides, including but not limited
to modified nucleoside base and/or modified sugar unit. Examples of
chemical modifications are known to the person skilled in the art
and are described, for example, in Uhlmann E et al. (1990) Chem.
Rev. 90:543; "Protocols for Oligonucleotides and Analogs" Synthesis
and Properties & Synthesis and Analytical Techniques, S.
Agrawal, Ed, Humana Press, Totowa, USA 1993; and Hunziker, J. et
al. (1995) Mod. Syn. Methods 7:331-417; and Crooke, S. et al.
(1996) Ann. Rev. Pharm. Tox. 36:107-129. The nucleoside residues
can be coupled to each other by any of the numerous known
internucleoside linkages. Such internucleoside linkages include,
without limitation, phosphodiester, phosphorothioate,
phosphorodithioate, alkylphosphonate, alkylphosphonothioate,
phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy,
acetamidate, carbamate, morpholino, borano, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphorothioate, and sulfone internucleoside linkages. The term
"oligonucleotide" also encompasses polynucleosides having one or
more stereospecific internucleoside linkage (e.g., (R.sub.P)- or
(S.sub.P)-phosphorothioate, alkylphosphonate, or phosphotriester
linkages). As used herein, the terms "oligonucleotide" and
"dinucleotide" are expressly intended to include polynucleosides
and dinucleosides having any such internucleoside linkage, whether
or not the linkage comprises a phosphate group. In certain
preferred embodiments, these internucleoside linkages may be
phosphodiester, phosphorothioate or phosphorodithioate linkages or
combinations thereof.
[0035] The term "2'-substituted ribonucleoside" or "2'-substituted
arabinoside" generally includes ribonucleosides or
arabinonucleosides in which the hydroxyl group at the 2' position
of the pentose moiety is substituted to produce a 2'-substituted or
2'-O-substituted ribonucleoside. In certain embodiments, such
substitution is with a lower hydrocarbyl group containing 1-6
saturated or unsaturated carbon atoms, with a halogen atom, or with
an aryl group having 6-10 carbon atoms, wherein such hydrocarbyl,
or aryl group may be unsubstituted or may be substituted, for
example, with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl,
acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examples
of 2'-O-substituted ribonucleosides or
2'-O-substituted-arabinosides include, without limitation 2'-amino,
2'-fluoro, 2'-allyl, 2'-O-alkyl and 2'-propargyl ribonucleosides or
arabinosides, 2'-O-methylribonucleosides or 2'-O-methylarabinosides
and 2'-O-methoxyethoxyribonucleosides or
2'-O-methoxyethoxyarabinosides.
[0036] The term "3'", when used directionally, generally refers to
a region or position in a polynucleotide or oligonucleotide 3'
(downstream) from another region or position in the same
polynucleotide or oligonucleotide.
[0037] The term "5'", when used directionally, generally refers to
a region or position in a polynucleotide or oligonucleotide 5'
(upstream) from another region or position in the same
polynucleotide or oligonucleotide.
[0038] The term "about" generally means that the exact number is
not critical. Thus, the number of nucleoside residues in the
oligonucleotides is not critical, and oligonucleotides having one
or two fewer nucleoside residues, or from one to several additional
nucleoside residues are contemplated as equivalents of each of the
embodiments described above.
[0039] The term "agonist" generally refers to a substance that
binds to a receptor of a cell and induces a response. An agonist
often mimics the action of a naturally occurring substance such as
a ligand.
[0040] The term "antagonist" generally refers to a substance that
attenuates, inhibits or suppresses the effects of an agonist or
ligand.
[0041] The term "adjuvant" generally refers to a substance which,
when added to an immunogenic agent such as vaccine or antigen,
enhances or potentiates an immune response to the agent in the
recipient host upon exposure to the mixture.
[0042] The term "airway inflammation" generally includes, without
limitation, asthma.
[0043] The term "allergen" generally refers to an antigen or
antigenic portion of a molecule, usually a protein, which elicits
an allergic response upon exposure to a subject. Typically the
subject is allergic to the allergen as indicated, for instance, by
the wheal and flare test or any method known in the art. A molecule
is said to be an allergen even if only a small subset of subjects
exhibit an allergic immune response upon exposure to the
molecule.
[0044] The term "allergy" generally refers to an inappropriate
immune response characterized by inflammation and includes, without
limitation, food allergies and respiratory allergies.
[0045] The term "antigen" generally refers to a substance that is
recognized and selectively bound by an antibody or by a T cell
antigen receptor, resulting in induction of an immune response.
Antigens may include but are not limited to peptides, proteins,
nucleosides, nucleotides, and combinations thereof. Antigens may be
natural or synthetic and generally induce an immune response that
is specific for that antigen.
[0046] The terms "autoimmune disease" and autoimmune disorder"
generally refer to diseases or disorders in which "self" components
undergo attack by the immune system.
[0047] The term "TLR-mediated disease" or TLR-mediated disorder"
generally means any pathological condition for which activation of
one or more TLRs is a contributing factor. Such conditions include
but are not limited, cancer, autoimmune diseases or disorders,
airway inflammation, inflammatory diseases or disorders, infectious
diseases, skin disorders, allergy, asthma or diseases caused by a
pathogen.
[0048] The term "physiologically acceptable" generally refers to a
material that does not interfere with the effectiveness of an IRO
compound or composition according to the invention and that is
compatible with a biological system such as a cell, cell culture,
tissue or organism. Preferably, the biological system is a living
organism, such as a vertebrate, or mammal.
[0049] The term "carrier" generally encompasses any excipient,
diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid,
lipid containing vesicle, microspheres, liposomal encapsulation or
other material well known in the art for use in pharmaceutical
formulations. It will be understood that the characteristics of the
carrier, excipient or diluent will depend on the route of
administration for a particular application. The preparation of
pharmaceutically acceptable formulations containing these materials
is described in, for example, Remington's Pharmaceutical Sciences,
18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa.,
1990.
[0050] The term "co-administration" generally refers to the
administration of at least two different substances sufficiently
close in time to modulate, suppress or inhibit an immune response.
Co-administration refers to simultaneous administration, as well as
temporally spaced order of up to several days apart, of at least
two different substances in any order, either in a single dose or
separate doses.
[0051] The term "complementary" generally means having the ability
to hybridize to a nucleic acid. Such hybridization is ordinarily
the result of hydrogen bonding between complementary strands,
preferably to form Watson-Crick or Hoogsteen base pairs, although
other modes of hydrogen bonding, as well as base stacking can also
lead to hybridization.
[0052] The term an "effective amount" or a "sufficient amount"
generally refers to an amount sufficient to affect a desired
biological effect, such as beneficial results. Thus, an "effective
amount" or "sufficient amount" will depend upon the context in
which it is being administered. In the context of administering a
compound or composition that modulates an immune response to a
co-administered antigen, an effective amount of an IRO compound or
composition according to the invention and antigen is an amount
sufficient to achieve the desired modulation, inhibition or
suppression as compared to the immune response obtained when the
antigen is administered alone. An effective amount may be
administered in one or more administrations.
[0053] The term "in combination with" generally means in the course
of treating a disease or disorder in a patient, administering an
IRO compound or composition according to the invention and an agent
useful for treating the disease or disorder that does not diminish
the immune inhibitory effect of the IRO compound or composition
according to the invention. Such combination treatment may also
include more than a single administration of an IRO compound or
composition according to the invention and/or independently an
agent. The administration of the IRO compound or composition
according to the invention and/or the agent may be by the same or
different routes.
[0054] The term "individual" or "subject" or "vertebrate" generally
refers to a mammal. Mammals generally include, but are not limited
to, humans, non-human primates, rats, mice, cats, dogs, horses,
cattle, cows, pigs, sheep, and rabbits.
[0055] The term "kinase inhibitor" generally refers to molecules
that antagonize or inhibit phosphorylation-dependent cell signaling
and/or growth pathways in a cell. Kinase inhibitors may be
naturally occurring or synthetic and include small molecules that
have the potential to be administered as oral therapeutics. Kinase
inhibitors have the ability to rapidly and specifically inhibit the
activation of the target kinase molecules. Protein kinases are
attractive drug targets, in part because they regulate a wide
variety of signaling and growth pathways and include many different
proteins. As such, they have great potential in the treatment of
diseases involving kinase signaling, including cancer,
cardiovascular disease, inflammatory disorders, diabetes, macular
degeneration and neurological disorders. Examples of kinase
inhibitors include sorafenib (NEXAVAR.RTM.), SUTENT.RTM.,
dasatinib, DASATINIB.TM., ZACTIMA.TM., TYKERB.TM. and STI571.
[0056] The term "nucleoside" generally refers to compounds
consisting of a sugar, usually ribose or deoxyribose, and a purine
or pyrimidine base.
[0057] The term "nucleotide" generally refers to a nucleoside
comprising a phosphate group attached to the sugar.
[0058] As used herein, the term "pyrimidine nucleoside" refers to a
nucleoside wherein the base component of the nucleoside is a
pyrimidine base (e.g., cytosine (C) or thymine (T) or Uracil (U)).
Similarly, the term "purine nucleoside" refers to a nucleoside
wherein the base component of the nucleoside is a purine base
(e.g., adenine (A) or guanine (G)).
[0059] The terms "analog" or "derivative" can be used
interchangeable to generally refer to any purine and/or pyrimidine
nucleotide or nucleoside that has a modified base and/or sugar. A
modified base is a base that is not guanine, cytosine, adenine,
thymine or uracil. A modified sugar is any sugar that is not ribose
or 2'deoxyribose and can be used in the backbone for an
oligonucleotide.
[0060] The term "inhibiting" or "suppressing" generally refers to a
decrease in or a prevention of a response or qualitative difference
in a response, which could otherwise arise from eliciting and/or
stimulation of a response.
[0061] The term "non-nucleotide linker" generally refers to any
linkage or moiety that can link or be linked to the
oligonucleotides other than through a phosphorous-containing
linkage. Preferably such linker is from about 2 angstroms to about
200 angstroms in length.
[0062] The term "nucleotide linkage" generally refers to a direct
3'-5' linkage that directly connects the 3' and 5' hydroxyl groups
of two nucleosides through a phosphorous-containing linkage.
[0063] The terms "oligonucleotide motif" means an oligonucleotide
sequence, including a dinucleotide selected from CpG, C*pG, C*pG*
or CpG*. The terms CpG, C*pG, C*pG* and CpG* refer to
oligonucleotide motifs that are immune stimulatory wherein C is
cytosine, C* is a cytosine analog or derivative, G is a guanine and
G* is a guanine analog or derivative.
[0064] An "oligonucleotide motif that would be immune stimulatory,
but for one or more modifications" means an oligonucleotide motif
which is immune stimulatory in a parent oligonucleotide, but not in
a derivative oligonucleotide, wherein the derivative
oligonucleotide is based upon the parent oligonucleotide, but has
one or more modifications. In other words, an "oligonucleotide
motif that would be immune stimulatory, but for one or more
modifications" refers to a TLR9-inducing moiety that would have
TLR9 agonistic activity but for that fact that it has been
functionally blocked or inhibited from inducing TLR9 mediated
immune response through modification(s) of the TLR9-inducing moiety
itself and/or by one or more chemical modification within the
oligonucleotide based compound. Modifications that inhibit the
activity of a TLR9-inducing moiety include, but not limited to,
2'-OMe-ribonucleosides, 3'-OMe-ribonucleosides, 3-nitropyrrole,
5-nitroindole, dU, .beta.-L-deoxynucleosides,
.alpha.-deoxynucleosides, abasic nucleoside, propanediol linker,
amino linker, isopropoxyl, glycerol linker, 2'-5'-DNA, 2'-5' RNA,
and P-Me DNA.
[0065] The term "treatment" generally refers to an approach
intended to obtain a beneficial or desired results, which may
include alleviation of symptoms and/or delaying and/or ameliorating
the progression of a disease or disorder.
[0066] Certain IROs according to the invention are shown in Table
2. In this table, the IRO compounds have all phosphorothioate (PS)
linkages, except where indicated with `o`. Except where indicated,
all nucleotides are deoxyribonucleotides.
TABLE-US-00002 TABLE 2 IRO compound # Sequence/Structure/SEQ ID NO
1 5'-UGUCG1TTCT-X1-TCTTG1CUGU-5'; 5'-SEQ ID NO 1-3'-X1-3'-SED ID NO
1-5' 2 5'-UGUCG1TTC-X1-CTTG1CUGU-5'; 5'-SEQ ID NO 2-3'-X1-3'-SED ID
NO 2-5' 3 5'-UGUCG1TT-X1-TTG1CUGU-5'; 5'-SEQ ID NO 3-3'-X1-3'-SED
ID NO 3-5' 4 5'-UGUCoG1TTCTo-Z-oTCTTG1oCUGU-5'; 5'-SEQ ID NO
4-3'-Z-3'-SED ID NO 4-5' 5 5'-GUCG1TTCTT-Z-TTCTTG1CUG-5'; 5'-SEQ ID
NO 5-3'-Z-3'-SED ID NO 5-5' 6 5'-UGUCG2TTCT-Z-TCTTG2CUGU-5'; 5'-SEQ
ID NO 6-3'-Z-3'-SED ID NO 6-5' 7 5'-UGUCG1TTCT-X4-TCTTG1CUGU-5';
5'-SEQ ID NO 7-3'-X4-3'-SED ID NO 7-5' 8
5'-UGUCG1TTC-X4-CTTG1CUGU-5'; 5'-SEQ ID NO 8-3'-X4-3'-SED ID NO
8-5' 9 5'-UGUCoG1TTCTo-X4-oTCTTG1oCUGU-5'; 5'-SEQ ID NO
9-3'-X4-3'-SED ID NO 9-5' 10 5'-GUCG1TTCTT-X4-TTCTTG1CUG-5'; 5'-SEQ
ID NO 10-3'-X4-3'-SED ID NO 10-5' 11 5'-UGUCG1TT-X4-TTG1CUGU-5';
5'-SEQ ID NO 11-3'-X4-3'-SED ID NO 11-5' 12
5'-UGUCG1TTC-X5-CTTG1CUGU-5'; 5'-SEQ ID NO 12-3'-X5-3'-SED ID NO
12-5' 13 5'-UGUCG2TTC-X5-CTTG2CUGU-5'; 5'-SEQ ID NO 13-3'-X5-3'-SED
ID NO 13-5' 14 5'-UGUCG1TTC-X6-CTTG1CUGU-5'; 5'-SEQ ID NO
14-3'-X6-3'-SED ID NO 14-5' 15 5'-UGUCG2TTC-X6-CTTG2CUGU-5'; 5'-SEQ
ID NO 15-3'-X6-3'-SED ID NO 15-5' 16
5'-UGUCG1TTCT-X7-TCTTG1CUGU-5'; 5'-SEQ ID NO 16-3'-X7-3'-SED ID NO
16-5' 17 5'-UGUCG2TTCT-X7-TCTTG2CUGU-5'; 5'-SEQ ID NO
17-3'-X7-3'-SED ID NO 17-5' 18 5'-UGUCG1TTC-X7-CTTG1CUGU-5'; 5'-SEQ
ID NO 18-3'-X7-3'-SED ID NO 18-5' 19 5'-TGUCG1TTCT-X-TCTTG1CUGT-5';
5'-SEQ ID NO 19-3'-X-3'-SED ID NO 19-5' 20
5'-CTTGUCG1TTCT-X-TCTTG1CUGTTC-5'; 5'-SEQ ID NO 20-3'-X-3'-SED ID
NO 20-5' 21 5'-TTGUCG1TTC-X-CTTG1CUGTT-5'; 5'-SEQ ID NO
21-3'-X-3'-SED ID NO 21-5' 22 5'-CTTTGUCG1TTC-X-CTTG1CUGTTTC-5';
5'-SEQ ID NO 22-3'-X-3'-SED ID NO 22-5' 23
5'-TGUCG1TTCT-X7-TCTTG1CUGT-5'; 5'-SEQ ID NO 23-3'-X7-3'-SED ID NO
23-5' 24 5'-TTGUCG1TTC-X7-CTTG1CUGTT-5'; 5'-SEQ ID NO
24-3'-X7-3'-SED ID NO 24-5' 25 5'-GUCG1TTCTT-Z-TTCTTG1CUG-5';
5'-SEQ ID NO 25-3'-Z-3'-SED ID NO 25-5' 26
5'-TGUCG1TTCA-X-ACTTG1CUGT-5'; 5'-SEQ ID NO 26-3'-X-3'-SED ID NO
26-5' 27 5'-TCTGACG1TTCT-X-TCTTG1CAGTCT-5'; 5'-SEQ ID NO
27-3'-X1-3'-SED ID NO 27-5' 28 5'-TCTGACG2TTCT-X-TCTTG2CAGTCT-5';
5'-SEQ ID NO 28-3'-X-3'-SED ID NO 28-5' 29
5'-TTGUCG1TTA-X-ATTG1CUGTT-5'; 5'-SEQ ID NO 29-3'-X-3'-SED ID NO
29-5' 30 5'-CTCTGUCG1TTA-X-ATTG1CUGTCTC-5'; 5'-SEQ ID NO
30-3'-X-3'-SED ID NO 30-5' 31 5'-TGTC*GTTCT-X-TCTTGC*TGT-5'; 5'-SEQ
ID NO 31-3'-X-3'-SED ID NO 31-5' 32 5'-TGTCGTTCT-X-TCTTGCTGT-5';
5'-SEQ ID NO 32-3'-X-3'-SED ID NO 32-5' 33
5'-TGTC*GTTCT-X-TCTTGC*TGT-5'; 5'-SEQ ID NO 33-3'-X-3'-SED ID NO
33-5' 34 5'-TGTCGTTCT-X-TCTTGCTGT-5'; 5'-SEQ ID NO 34-3'-X-3'-SED
ID NO 34-5' 35 5'-UGUCG1ACAT-X-TACAG1CUGU-5'; 5'-SEQ ID NO
35-3'-X-3'-SED ID NO 35-5' 36 5'-UGUCG1TTC-X-CTTG1CUGU-5'; 5'-SEQ
ID NO 36-3'-X-3'-SED ID NO 36-5' 37 5'-UGUCG1TT-X-TTG1CUGU-5';
5'-SEQ ID NO 37-3'-X-3'-SED ID NO 37-5' 38
5'-UoGUCG1TToCTo-X-oTCoTTG1CUGoU-5'; 5'-SEQ ID NO 38-3'-X-3'-SED ID
NO 38-5' 39 5'-UoGoUCG1TTCTo-X-oTCTTG1CUoGoU-5'; 5'-SEQ ID NO
39-3'-X-3'-SED ID NO 39-5' 40 5'-UGACG1TTCT-X-TCTTG1CAGU-5'; 5'-SEQ
ID NO 40-3'-X-3'-SED ID NO 40-5' 41 5'-UGUCG1ACAT-Z-TACAG1CUGU-5';
5'-SEQ ID NO 41-3'-Z-3'-SED ID NO 41-5' 42
5'-UGUCG1TTCT-Z-TCTTG1CUGU-5'; 5'-SEQ ID NO 42-3'-Z-3'-SED ID NO
42-5' 43 5'-UGUCG1TTC-Z-CTTG1CUGU-5'; 5'-SEQ ID NO 43-3'-Z-3'-SED
ID NO 43-5' 44 5'-UGUCG1TT-Z-TTG1CUGU-5'; 5'-SEQ ID NO
44-3'-Z-3'-SED ID NO 44-5' 45 5'-UGUC*GTTCT-X-TCTTGC*UGU-5'; 5'-SEQ
ID NO 45-3'-X-3'-SED ID NO 45-5' 46 5'-T{circumflex over ( )}G T
C*GTTCT-X-TCTTGC*T G T -5'; 5'-SEQ ID NO 46-3'-X-3'-SED ID NO 46-5'
47 5'-UGUC*GTTCT-X-TCTTGC*UGU-5'; 5'-SEQ ID NO 47-3'-X-3'-SED ID NO
47-5' 48 5'-T{circumflex over ( )}G{circumflex over (
)}T{circumflex over ( )}C*GTTCT-X-TCTTGC*T{circumflex over (
)}G{circumflex over ( )}T{circumflex over ( )}-5'; 5'-SEQ ID NO
48-3'-X-3'-SED ID NO 48-5' 49 5'-UGUCG1ACAT-X1-TACAG1CUGU-5';
5'-SEQ ID NO 49-3'-X1-3'-SED ID NO 49-5' 50
5'-UGACG2TTCT-X-TCTTG2CAGU-5'; 5'-SEQ ID NO 50-3'-X-3'-SED ID NO
50-5' 51 5'-TCTGUCG1TTCT-X-TCTTG1CUGTCT-5'; 5'-SEQ ID NO
51-3'-X-3'-SED ID NO 51-5' 52 5'-TCTGUCG2TTCT-X-TCTTG2CUGTCT-5';
5'-SEQ ID NO 52-3'-X-3'-SED ID NO 52-5' 53
5'-UGUCG2TTCT-X-TCTTG2CUGU-5'; 5'-SEQ ID NO 53-3'-X-3'-SED ID NO
53-5' 54 5'-UGUCG2TT-Z-TTG2CUGU-5'; 5'-SEQ ID NO 54-3'-Z-3'-SED ID
NO 54-5' 55 5'-TUGUCG1TTC-Z-CTTG1CUGUT-5'; 5'-SEQ ID NO
55-3'-Z-3'-SED ID NO 55-5' 56 5'-CTUGUCG1TT-Z-TTG1CUGUTC-5'; 5'-SEQ
ID NO 56-3'-Z-3'-SED ID NO 56-5' 57 5'-UCG1TTCTTC-Z-CTTCTTG1CU-5';
5'-SEQ ID NO 57-3'-Z-3'-SED ID NO 57-5' 58
5'-CTATCTGAC*GTTCTCTGT-3'; 5'-SEQ ID NO 58-3' 59
5'-CTATCTGACGTTCTCTGT-3'; 5'-SEQ ID NO 59-3' 60
5'-CTATCTGAC*GTTCTCTGT-3'; 5'-SEQ ID NO 60-3' 61
5'-CTATCTGACGTTCTCTGT-3'; 5'-SEQ ID NO 61-3' 62
5'-CTATCTG{circumflex over ( )}A{circumflex over ( )}CGTTCTCTGT-3';
5'-SEQ ID NO 62-3' 63 5'-CTATCTGUC*GTTCTCTGT-3'; 5'-SEQ ID NO 63-3'
64 5'-CTATCTGUCGTTCTCTGT-3'; 5'-SEQ ID NO 64-3' 65
5'-CTATCTGUC*GTTCTCTGT-3'; 5'-SEQ ID NO 65-3' 66
5'-CTATCTGUCGTTCTCTGT-3'; 5'-SEQ ID NO 66-3' 67
5'-CTTGUC*G1TTCT-X-TCTTG1C*UGTTC-5' 5'-SEQ ID NO 67-3'-X-3'-SED ID
NO 67-5' 68 5'-CTATCTGUC*G1TTCTCTGU-3' 5'-SEQ ID NO 68-3' 69
5'-UGUCG1TTCT-X-TCTTG1CUGU-5' 5'-SEQ ID NO 69-3'-X-3'-SED ID NO
69-5' 70 5'-TGUC*G1TTCT-X-TCTTG1C*UGT-5' 5'-SEQ ID NO
70-3'-X-3'-SED ID NO 70-5' 71 5'-CTTTGUC*G1TTC-X-CTTG1C*UGTTTC-5'
5'-SEQ ID NO 71-3'-X-3'-SED ID NO 71-5' 72
5'-GUC*G1TTCTT-X-TTCTTG1C*UG-5' 5'-SEQ ID NO 72-3'-X-3'-SED ID NO
72-5' 73 5'-TGUC*G1TTCA-X-ACTTG1C*UGT-5' 5'-SEQ ID NO
73-3'-X-3'-SED ID NO 73-5' 74 5'-CTTGUC*G1TTCT-X1-TCTTG1C*UGTTC-5'
5'-SEQ ID NO 74-3'-X1-3'-SED ID NO 74-5' 75
5'-CTTGUC*G2TTCT-X-TCTTG2C*UGTTC-5' 5'-SEQ ID NO 75-3'-X-3'-SED ID
NO 75-5' 76 5'-CTTGUC*G1TTC-X5-CTTG1C*UGTTC-5' 5'-SEQ ID NO
76-3'-X5-3'-SED ID NO 76-5' 77 5'-CTTGUC*G1TTCT-X7-TCTTG1C*UGTTC-5'
5'-SEQ ID NO 77-3'-X7-3'-SED ID NO 77-5' 78
5'-CTTTGUC*oG1TTC-X-CTTG1oC*UGTTTC-5' 5'-SEQ ID NO 78-3'-X-3'-SED
ID NO 78-5' 79 5'-CTTTGoUC*oG1TTC-X-CTTG1oC*UoGTTTC-5' 5'-SEQ ID NO
79-3'-X-3'-SED ID NO 79-5' 80 5'-CTTGUC*oG1TTCT-X-TCTTG1oC*UGTTC-5'
5'-SEQ ID NO 80-3'-X-3'-SED ID NO 80-5' 81
5'-CTTGoUC*oG1TTCT-X-TCTTG1oC*UoGTTC-5' 5'-SEQ ID NO 81-3'-X-3'-SED
ID NO 81-5'
82 5'-CTGUC*oG1TTCTT-X-TTCTTG1oC*UGTC-5' 5'-SEQ ID NO
82-3'-X-3'-SED ID NO 82-5' 83
5'-CTGoUC*oG1TTCTT-X-TTCTTG1oC*UoGTC-5' 5'-SEQ ID NO 83-3'-X-3'-SED
ID NO 83-5' 84 5'-UGUC*G1TTCT-X1-TCTTG1C*UGU-5' 5'-SEQ ID NO
84-3'-X1-3'-SED ID NO 84-5' 85 5'-UGUC*G2TTCT-X-TCTTG2C*UGU-5'
5'-SEQ ID NO 85-3'-X-3'-SED ID NO 85-5' 86
5'-UGUC*G1TTC-X5-CTTG1C*UGU-5' 5'-SEQ ID NO 86-3'-X5-3'-SED ID NO
86-5' 87 5'-UGUC*G1TTCT-X7-TCTTG1C*UGU-5' 5'-SEQ ID NO
87-3'-X7-3'-SED ID NO 87-5' 88 5'-CTATCTGUC*G1TTCTCTGT-3' 5'-SEQ ID
NO 88-3' 89 5'-CTATCTGUC*G1TTCTCTGT-3' 5'-SEQ ID NO 89-3' 90
5'-TGAC*G1TTCT-X-TCTTG1C*AGT-5' 5'-SEQ ID NO 90-3'-X-3'-SEQ ID NO
90-5' 91 5'-CTTGAC*G1TTCT-X-TCTTG1C*AGTTC-5' 5'-SEQ ID NO
91-3'-X-3'-SED ID NO 91-5' 92 5'-CTTTGAC*G1TTC-X-CTTG1C*AGTTTC-5'
5'-SEQ ID NO 92-3'-X-3'-SED ID NO 92-5' 93
5'-GAC*G1TTCTT-X-TTCTTG1C*AG-5' 5'-SEQ ID NO 93-3'-X-3'-SED ID NO
93-5' 94 5'-TGAC*G1TTCA-X-ACTTG1C*AGT-5' 5'-SEQ ID NO
94-3'-X-3'-SED ID NO 94-5' 95 5'-CTTGAC*G1TTCT-X1-TCTTG1C*AGTTC-5'
5'-SEQ ID NO 95-3'-X1-3'-SED ID NO 95-5' 96
5'-CTTGAC*G2TTCT-X-TCTTG2C*AGTTC-5' 5'-SEQ ID NO 96-3'-X-3'-SED ID
NO 96-5' 97 5'-CTTGAC*G1TTC-X5-CTTG1C*AGTTC-5' 5'-SEQ ID NO
97-3'-X5-3'-SED ID NO 97-5' 98 5'-CTTGAC*G1TTCT-X7-TCTTG1C*AGTTC-5'
5'-SEQ ID NO 98-3'-X7-3'-SED ID NO 98-5' 99
5'-CTTTGAC*oG1TTC-X-CTTG1oC*AGTTTC-5' 5'-SEQ ID NO 99-3'-X-3'-SED
ID NO 99-5' 100 5'-CTTTGoAC*oG1TTC-X-CTTG1oC*AoGTTTC-5' 5'-SEQ ID
NO 100-3'-X-3'-SED ID NO 100-5' 101
5'-CTTGAC*oG1TTCT-X-TCTTG1oC*AGTTC-5' 5'-SEQ ID NO 101-3'-X-3'-SED
ID NO 101-5' 102 5'-CTTGoAC*oG1TTCT-X-TCTTG1oC*AoGTTC-5' 5'-SEQ ID
NO 102-3'-X-3'-SED ID NO 102-5' 103
5'-CTGAC*oG1TTCTT-X-TTCTTG1oC*AGTC-5' 5'-SEQ ID NO 103-3'-X-3'-SED
ID NO 103-5' 104 5'-CTGoAC*oG1TTCTT-X-TTCTTG1oC*AoGTC-5' 5'-SEQ ID
NO 104-3'-X-3'-SED ID NO 104-5' 105 5'-UGAC*G1TTCT-X-TCTTG1C*AGU-5'
5'-SEQ ID NO 105-3'-X-3'-SED ID NO 105-5' 106
5'-UGAC*G1TTCT-X1-TCTTG1C*AGU-5' 5'-SEQ ID NO 106-3'-X1-3'-SED ID
NO 106-5' 107 5'-UGAC*G2TTCT-X-TCTTG2C*AGU-5' 5'-SEQ ID NO
107-3'-X-3'-SED ID NO 107-5' 108 5'-UGAC*G1TTC-X5-CTTG1C*AGU-5'
5'-SEQ ID NO 108-3'-X5-3'-SED ID NO 108-5' 109
5'-UGAC*G1TTCT-X7-TCTTG1C*AGU-5' 5'-SEQ ID NO 109-3'-X7-3'-SED ID
NO 109-5' 110 5'-CTATCTGAC*G1TTCTCTGT-3' 5'-SEQ ID NO 110-3' 111
5'-CTATCTGAC*G1TTCTCTGT-3' 5'-SEQ ID NO 111-3' 112
5'-CTATCTGAC*G1TTCTCTGU-3' 5'-SEQ ID NO 112-3'
G1=7-deaza-dG; G2=AraG; C*=5-Me-dC; C*=2'-O-Me-5-Me-C;
AA/GA/TA/GA=2'-O-(2-methoxyethyl)-ribonucelotides; X=Glycerol
Linker (also known as 1,2,3-Propanetriol Linker);
X1=1,2,4-Butanetriol Linker; Z=1,3,5-Pentanetriol Linker;
X4=3-Trimethylamino-1, 2-propanediol Linker;
X5=Bis-1,5-O-(3'-thymidyl)-1,3,5-pentanetriol Linker;
X6=Bis-1,5-O-[3'-(1,2-dideoxy-D-ribosyl)]-1,3-5-pentanetriol
Linker; X7=3-(2-Hydroxyethyl)-1,5-pentanediol Linker;
G/U/A/C=2'-O-Me-ribonucleotides; o=Phosphodiester linkage.
[0067] In a first aspect, the invention provides immune regulatory
oligonucleotide (IRO) compounds. The term "IRO" refers to an immune
regulatory oligonucleotide-based compound that is an antagonist for
TLR7- and/or TLR9, wherein the compound comprises an
oligonucleotide motif and at least one modification, wherein the
oligonucleotide motif would be immune stimulatory but for the one
or more modifications that functionally block or inhibit the
activity of the oligonucleotide motif, provided that the compound
contains less than 4 consecutive guanosine nucleotides and
preferably less than 3 consecutive guanosine nucleotides. Such
modifications may be in the oligonucleotide 5' terminus, in the 5'
sequence flanking the oligonucleotide motif, and/or within the
immune stimulatory oligonucleotide motif. These modifications
result in an IRO compound that antagonize, inhibit, suppresses or
prevent TLR7- and/or TLR9-mediated immune stimulation. Such
modifications can be to the bases, sugar residues and/or the
phosphate backbone of the nucleotides/nucleosides flanking the
immune stimulatory oligonucleotide motif or within such
oligonucleotide motif.
[0068] The general structure of the IRO compound has the structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3'
(SEQ ID NO: 70) wherein CG is an oligonucleotide motif selected
from CpG, C*pG, C*pG* or CpG* wherein C is cytosine, C* is a
cytosine analog or derivative, G is a guanine and G* is a guanine
analog or derivative; N.sub.1-N.sub.3, at each occurrence, is
independently a nucleotide or nucleotide derivative;
N.sup.1-N.sup.3, at each occurrence, is independently a nucleotide
or nucleotide derivative; N.sub.m and N.sup.m, at each occurrence,
is independently a nucleotide, nucleotide derivative or
non-nucleotide linker; provided that at least one of N.sub.1,
N.sub.2, and N.sub.3 and/or C and/or G of the oligonucleotide motif
is a nucleotide derivative that functionally blocks or inhibits the
activity of the oligonucleotide motif; and further provided that
the compound contains less than 4 consecutive guanosine nucleotides
and preferably less than 3 consecutive guanosines, wherein the
immune stimulatory activity of the oligonucleotide motif is
antagonized, inhibited, suppressed or prevented by the nucleotide
derivative; and wherein m is a number from 0 to about 30.
[0069] In preferred embodiments, N.sub.1 is a nucleotide derivative
that functionally blocks or inhibits the activity of the
oligonucleotide motif. In preferred embodiments N.sub.1 and
N.sub.2, or N.sub.1 and N.sub.3, or N.sub.2 and N.sub.3, or
N.sub.1, N.sub.2 and N.sub.3 are nucleotide derivatives that
functionally blocks or inhibits the activity of the oligonucleotide
motif.
[0070] In preferred embodiments the IRO compound is not an
antisense oligonucleotide.
[0071] In certain embodiments of the invention, the IRO compound
may comprise at least two oligonucleotides (for example 2, 3, 4, 5
or 6 oligonucleotides), wherein at least two oligonucleotides are
covalently linked via a direct nucleotide to nucleotide linkage at
their 3' ends through the 3' positions of the sugars or through a
modified sugar or modified nucleobase or via a non-nucleotide
linker at their 3' ends through the 3' positions of the sugars or
through a modified sugar or modified nucleobase. In preferred
aspects of this embodiment, at least one of oligonucleotides of the
IRO compound has the structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3'
(SEQ ID NO: 70), wherein N.sub.m, N.sub.1, N.sub.2, N.sub.3, C, G,
N.sup.1, N.sup.2, N.sup.3 and N.sub.m are as described above for
the general structure of the IRO compound. In more preferred
aspects of this embodiment, at least two of the oligonucleotides of
the IRO compound have the structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3'
(SEQ ID NO: 70), wherein N.sub.m, N.sub.1, N.sub.2, N.sub.3, C, G,
N.sup.1, N.sup.2, N.sup.3 and N.sup.m are as described above for
the general structure of the IRO compound. Such an IRO compound may
have the structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-
-3'-X-3'-N.sup.m-N.sup.3N.sup.2N.sup.1GCN.sub.1N.sub.2N.sub.3-N.sub.m-5'
(5'-SEQ ID NO: 70-3'-X-3'-SEQ ID NO: 70-5'), wherein X is a
nucleotide linkage or a non-nucleotide linker and N.sub.m, N.sub.1,
N.sub.2, N.sub.3, C, G, N.sup.1, N.sup.2, N.sup.3 and N.sub.m are
as described above for the general structure of the IRO
compound.
[0072] In certain embodiments of the invention, the IRO compound
that is an antagonist of TLR7 and/or TLR9 has the structure
5-N.sub.pN.sub.3N.sub.2N.sub.1C*G*N.sup.1N.sup.2N.sup.3N.sup.zN.sup.4N.su-
p.5-3 ` (SEQ ID NO: 71), wherein C*G* is an oligonucleotide motif
wherein C* is 5-Me-dC, and G* is 7-deaza-dG; N.sub.1-N.sub.2, at
each occurrence, is independently a 2`-O-Me-ribonucleotide;
N.sub.3, at each occurrence, is independently a nucleotide or
nucleotide derivative; N.sup.1-N.sup.3, at each occurrence, is
independently a nucleotide or nucleotide derivative; N.sub.p and
N.sup.z, at each occurrence, is independently a nucleotide or
nucleotide derivative; N.sup.4-N.sup.5, at each occurrence, is
independently a 2'-O-Me-ribonucleotide; p is a number from 0 to
about 30 and z is a number from 0 to about 30; provided that the
compound contains less than 3 consecutive guanosines. In certain
embodiments p and z are independently a number from 1 to about 20.
In certain embodiments p and z are independently a number from 2 to
about 15. In certain embodiments p and z are independently a number
from 3 to about 10.
[0073] In certain embodiments of the invention, the IRO compound
that is an antagonist of TLR7 and/or TLR9 has the structure
5-N.sub.pN.sub.3N.sub.2N.sub.1C*G*N.sup.1N.sup.2N.sup.3N.sup.zN.sup.4N.su-
p.5-3' (SEQ ID NO: 72), wherein C*G* is an oligonucleotide motif
wherein C* is 5-Me-dC, and G* is 7-deaza-dG; N.sub.1-N.sub.2, at
each occurrence, is independently a 2'-O-Me-ribonucleotide;
N.sub.3, at each occurrence, is independently a nucleotide;
N.sup.1-N.sup.3, at each occurrence, is independently a nucleotide;
N.sub.p and N.sup.z, at each occurrence, is independently a
nucleotide; N.sup.4-N.sup.5 at each occurrence, is independently a
2'-O-Me-ribonucleotide; p is 5 and z is 3; provided that the
compound contains less than 3 consecutive guanosines.
Alternatively, SEQ ID NO: 72 can be written as
5'-NNNNNN.sub.3N.sub.2N.sub.1C*G*N.sup.1N.sup.2N.sup.3NNNN.sup.4N.sup.5-3-
'.
[0074] In preferred embodiments, two oligonucleotides having the
structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3'
(SEQ ID NO: 70) are covalently linked via a direct nucleotide to
nucleotide linkage at their 3' ends through the 3' positions of the
sugars or through a modified sugar or modified nucleobase or via a
non-nucleotide linker at their 3' ends through the 3' positions of
the sugars or through a modified sugar or modified nucleobase. In
preferred aspects of this embodiment, the IRO compound has the
structure
5'-N.sub.m-N.sub.3N.sub.2N.sub.1CGN.sup.1N.sup.2N.sup.3-N.sup.m-3'-X-3'-N-
.sup.m-N.sup.3N.sup.2N.sup.1GCN.sub.1N.sub.2N.sub.3-N.sub.m-5'
(5'-SEQ ID NO: 70-3'-X-3'-SEQ ID NO: 70-5'), wherein X is a
nucleotide linkage or a non-nucleotide linker and N.sub.m, N.sub.1,
N.sub.2, N.sub.3, C, G, N.sup.1, N.sup.2, N.sup.3 and N.sup.m are
as described above for the general structure of the IRO compound.
In preferred embodiments, the two oligonucleotides are covalently
linked directly via a nucleotide linkage. In more preferred
embodiments, the two oligonucleotides are covalently linked via a
non-nucleotide linker.
[0075] As a non-limiting example, the non-nucleotide linker
covalently linking the two oligonucleotides may be attached to the
3'-hydroxyl of the sugar. In such embodiments, the linker comprises
a functional group, which is attached to the 3'-hydroxyl by means
of a phosphate-based linkage like, for example, phosphodiester,
phosphorothioate, phosphorodithioate, methylphosphonate, or by a
non-phosphate-based linkage. Possible sites of conjugation for the
linker to the 3' end of the oligonucleotide are indicated in
Formula I, below, wherein B represents a heterocyclic base and
wherein the arrow pointing to P indicates any attachment to
phosphorous.
##STR00001##
[0076] In certain embodiments according to this aspect of the
invention, the non-nucleotide linker is a small molecule,
macromolecule or biomolecule, including, without limitation,
polypeptides, antibodies, lipids, antigens, allergens, and
oligosaccharides. In certain other embodiments, the non-nucleotide
linker is a small molecule. For purposes of the invention, a small
molecule is an organic moiety having a molecular weight of less
than 1,000 Da. In some embodiments, the small molecule has a
molecular weight of less than 750 Da.
[0077] In some embodiments, the small molecule is an aliphatic or
aromatic hydrocarbon, either of which optionally can include,
either in the linear chain connecting the oligonucleotides or
appended to it, one or more functional groups including, but not
limited to, hydroxy, amino, thiol, thioether, ether, amide,
thioamide, ester, urea, or thiourea. The small molecule can be
cyclic or acyclic. Examples of small molecule linkers include, but
are not limited to, amino acids, carbohydrates, cyclodextrins,
adamantane, cholesterol, haptens, and antibiotics. However, for
purposes of describing the non-nucleotide linker, the term "small
molecule" is not intended to include a nucleoside.
[0078] In some embodiments, the non-nucleotide linker is an alkyl
linker or amino linker. The alkyl linker may be branched or
unbranched, cyclic or acyclic, substituted or unsubstituted,
saturated or unsaturated, chiral, achiral or racemic mixture. The
alkyl linkers can have from about 2 to about 18 carbon atoms. In
some embodiments such alkyl linkers have from about 2 to about 9
carbon atoms. In other embodiments, the alkyl linker has less than
3 carbon atoms. In further embodiments, the alkyl linker has at
least 3 carbon atoms and preferentially more than three carbon
atoms. Some alkyl linkers include one or more functional groups
including, but not limited to, hydroxy, amino, thiol, thioether,
ether, amide, thioamide, ester, urea, and thioether. Such alkyl
linkers can include, but are not limited to, 1,2 propanediol, 1,2,3
propanetriol, 1,3 propanediol, 1,2,4-Butanetriol,
1,3,5-Pentanetriol, 3-trimethylamino-1,2-propanediol,
Bis-1,5-O-(3'thymidyl(-1,3,5-pentanetriol,
Bis-1,5-O-[3'-(1,2-dideoxy-D-robosyl)]-1,3,5-pentanetriol,
3-(2-Hydroxyethyl)-1,5-pentanediol, triethylene glycol hexaethylene
glycol, polyethylene glycol linkers (e.g. [--O--CH2-CH2-].sub.n
(n=1-9)), methyl linkers, ethyl linkers, propyl linkers, butyl
linkers or hexyl linkers. In some embodiments, such alkyl linkers
may include peptides or amino acids.
[0079] In some embodiments, the non-nucleotide linker may include,
but are not limited to, those listed in Table 3.
TABLE-US-00003 TABLE 3 Representative Non-Nucleotidic Linkers
##STR00002## ##STR00003## ##STR00004## ##STR00005## ##STR00006##
##STR00007## ##STR00008##
[0080] In some embodiments, the small molecule linker is glycerol
or a glycerol homolog of the formula
HO--(CH.sub.2).sub.o--CH(OH)--(CH.sub.2).sub.p--OH, wherein o and p
independently are integers from 1 to about 6, from 1 to about 4, or
from 1 to about 3. In some other embodiments, the small molecule
linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such
derivatives have the formula
HO--(CH.sub.2).sub.m--C(O)NH--CH.sub.2--CH(OH)--CH.sub.2--NHC(O)--(CH.sub-
.2).sub.m--OH, wherein m is an integer from 0 to about 10, from 0
to about 6, from 2 to about 6, or from 2 to about 4.
[0081] Some non-nucleotide linkers according to the invention
permit attachment of more than two oligonucleotides. For example,
the small molecule linker glycerol has three hydroxyl groups to
which oligonucleotides may be covalently attached. Some IROs
according to the invention, therefore, comprise two or more
oligonucleotides linked to a nucleotide or a non-nucleotide linker.
Such IROs are referred to as being "branched".
[0082] IRO compounds also may comprise at least two
oligonucleotides non-covalently linked, such as by electrostatic
interactions, hydrophobic interactions, .pi.-stacking interactions,
hydrogen bonding and combinations thereof. Non-limiting examples of
such non-covalent linkage includes Watson-Crick base pairing,
Hoogsteen base pairing and base stacking.
[0083] In preferred embodiments one of the oligonucleotides of the
IRO compound is not an antisense oligonucleotide. In more preferred
embodiments neither of the oligonucleotides of the IRO compound is
an antisense oligonucleotide.
[0084] In certain embodiments, pyrimidine nucleosides in the immune
regulatory oligonucleotides used in the compositions and methods
according to the invention have the structure (II):
##STR00009##
wherein:
[0085] D is a hydrogen bond donor;
[0086] D' is selected from the group consisting of hydrogen,
hydrogen bond donor, hydrogen bond acceptor, hydrophilic group,
hydrophobic group, electron withdrawing group and electron donating
group;
[0087] A is a hydrogen bond acceptor or a hydrophilic group;
[0088] A' is selected from the group consisting of hydrogen bond
acceptor, hydrophilic group, hydrophobic group, electron
withdrawing group and electron donating group;
[0089] X is carbon or nitrogen; and
[0090] S' is a pentose or hexose sugar ring, or a sugar analog.
[0091] In certain embodiments, the sugar ring is derivatized with a
phosphate moiety, modified phosphate moiety, or other linker moiety
suitable for linking the pyrimidine nucleoside to another
nucleoside or nucleoside analog.
[0092] In some embodiments hydrogen bond donors include, without
limitation, --NH--, --NH.sub.2, --SH and --OH. Preferred hydrogen
bond acceptors include, without limitation, C.dbd.O, C.dbd.S, and
the ring nitrogen atoms of an aromatic heterocycle, e.g., N3 of
cytosine.
[0093] In some embodiments, structure (II) is a pyrimidine
nucleoside derivative. Examples of pyrimidine nucleoside
derivatives include, without limitation, 5-hydroxycytosine,
5-hydroxymethylcytosine, N4-alkylcytosine, or N4-ethylcytosine,
araC, 5-OH-dC, N3-Me-dC, 2'-O-Me-C, 2'-O-Me-U, 2'-O-Me-T, and
4-thiouracil. Chemical modified derivatives also include, but are
not limited to, thymine or uracil analogues. In some embodiments,
the sugar moiety S' in (II) is a sugar derivative. Suitable sugar
derivatives include, but are not limited to, trehalose or trehalose
derivatives, hexose or hexose derivatives, arabinose or arabinose
derivatives.
[0094] In some embodiments, the purine nucleosides in immune
regulatory oligonucleotides used in the compositions and methods
according to the invention have the structure (III):
##STR00010##
[0095] wherein:
[0096] D is a hydrogen bond donor;
[0097] D' is selected from the group consisting of hydrogen,
hydrogen bond donor, and hydrophilic group;
[0098] A is a hydrogen bond acceptor or a hydrophilic group;
[0099] X is carbon or nitrogen;
[0100] each L is independently selected from the group consisting
of C, O, N and S; and
[0101] S' is a pentose or hexose sugar ring, or a sugar analog.
[0102] In certain embodiments, the sugar ring is derivatized with a
phosphate moiety, modified phosphate moiety, or other linker moiety
suitable for linking the pyrimidine nucleoside to another
nucleoside or nucleoside analog.
[0103] In certain embodiments hydrogen bond donors include, without
limitation, --NH--, --NH.sub.2, --SH and --OH. In certain
embodiments hydrogen bond acceptors include, without limitation,
C.dbd.O, C.dbd.S, --NO.sub.2 and the ring nitrogen atoms of an
aromatic heterocycle, e.g., N1 of guanine.
[0104] In some embodiments, structure (III) is a purine nucleoside
derivative. Examples of purine nucleoside derivatives include,
without limitation, guanine analogues such as 7-deaza-G,
7-deaza-dG, ara-G, 6-thio-G, Inosine, Iso-G, loxoribine,
TOG(7-thio-8-oxo)-G, 8-bromo-G, 8-hydroxy-G, 5-aminoformycin B,
Oxoformycin, 7-methyl-G, 9-p-chlorophenyl-8-aza-G, 9-phenyl-G,
9-hexyl-guanine, 7-deaza-9-benzyl-G, 6-Chloro-7-deazaguanine,
6-methoxy-7-deazaguanine, 8-Aza-7-deaza-G(PPG),
2-(Dimethylamino)guanosine, 7-Methyl-6-thioguanosine,
8-Benzyloxyguanosine, 9-Deazaguanosine,
1-(B-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
2'-O-methyl-G, and N1-Me-dG. Chemically modified derivatives also
include, but are not limited to, adenine analogues such as
9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine, 2-Amino-N2-O--,
methyladenosine, 8-Aza-7-deaza-A, 7-deaza-A, Vidarabine,
2-Aminoadenosine, N1-Methyladenosine, 8-Azaadenosine,
5-Iodotubercidin, and 2'-O-Me-A. In some embodiments, the sugar
moiety S' in (III) is a sugar derivative as defined for Formula
II.
[0105] In certain embodiments of the invention, the immune
regulatory nucleic acid comprises a nucleic acid sequence
containing at least one B-L-deoxy nucleoside or 3'-deoxy
nucleoside.
[0106] In certain embodiments of the invention, the immune
regulatory oligonucleotide comprises a nucleic acid sequence
containing at least one dinucleotide selected from CpG, C*pG, C*pG*
and CpG*, wherein C is cytosine or 2'-deoxycytidine, G is guanosine
or 2'-deoxyguanosine, C* is 2'-deoxythymidine,
1-(2'-deoxy-B-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
5-Me-dC, 2'-dideoxy-5-halocytosine, 2'-dideoxy-5-nitrocytosine,
arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine,
2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine,
2'-O-substituted ribonucleotides (including, but not limited to,
2'-O-Me-5-Me-C, 2'-O-(2-methoxyethyl)-ribonucelotides or
2'-O-Me-ribonucleotides) or other pyrimidine nucleoside analogs or
derivative, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'-deoxyinosine,
2'-0-substituted ribonucleotides (including, but not limited to,
2'-O-(2-methoxyethyl)-ribonucelotides; or 2'-O-Me-ribonucleotides)
or other purine nucleoside analogs or derivative, and p is an
internucleoside linkage selected from the group consisting of
phosphodiester, phosphorothioate, and phosphorodithioate, and
wherein the activity of the at least one dinucleotide is regulated
by the flanking sequence.
[0107] In some embodiments, the oligonucleotides of the IRO
compound each have from about 6 to about 35 nucleoside residues,
preferably from about 9 to about 30 nucleoside residues, more
preferably from about 11 to about 23 nucleoside residues. In some
embodiments, the oligonucleotides have from about 6 to about 18
nucleotide residues.
[0108] In some embodiments, the IRO compounds can be combined with
one or more vaccines, antigens, antibodies, cytotoxic agents,
allergens, antibiotics, antisense oligonucleotides, TLR agonist,
TLR antagonist, peptides, proteins, gene therapy vectors, DNA
vaccines, adjuvants or kinase inhibitors to enhance the specificity
or magnitude of the immune response, or co-stimulatory molecules
such as cytokines, chemokines, protein ligands, trans-activating
factors, peptides and peptides comprising modified amino acids.
[0109] In a second aspect, the invention provides a pharmaceutical
composition comprising an IRO compound according to the invention
and a physiologically acceptable carrier.
[0110] In embodiments of this aspect of the invention, the
composition can further comprise one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonist, TLR antagonist, peptides, proteins,
gene therapy vectors, DNA vaccines, adjuvants or kinase inhibitors
to enhance the specificity or magnitude of the immune response, or
co-stimulatory molecules such as cytokines, chemokines, protein
ligands, trans-activating factors, peptides and peptides comprising
modified amino acids.
[0111] In a third aspect, the invention provides methods for
inhibiting or suppressing TLR-mediated induction of an immune
response in a mammal, such methods comprising administering to the
mammal an IRO compound according to the invention. In some
embodiments, the mammal is a human. In preferred embodiments, the
IRO compound is administered to a mammal in need of immune
suppression.
[0112] According to this aspect of the invention, an IRO compound
is capable of suppressing a TLR-based immune response to a further
TLR ligand or TLR agonist. As discussed further in the Examples
below, the activation of a TLR-based immune response by a TLR
agonist or TLR ligand (for example, an immune stimulatory
oligonucleotide) can be antagonized, inhibited, suppressed or
prevented by the simultaneous, pre- or post-administration of an
IRO compound, and such antagonism, inhibition, suppression or
prevention may be maintained for an extended period of time (for
example, days) after administration. This beneficial property of
the current invention has a unique advantage for the prevention
and/or treatment of a disease or disorder. For example, application
of certain TLR-agonists in the course of treating the disease may
cause unwanted immune stimulation that an IRO compound could
antagonize, suppress, inhibit or prevent. Administration of the IRO
simultaneously, pre and/or post administration of the TLR-agonist
may allow therapeutic benefits from the TLR-agonist while
antagonizing, suppressing, inhibiting or preventing the unwanted
side effect(s). Additionally, pre-administration of an IRO compound
according to the invention could antagonize, suppress, inhibit or
prevent an immune response (for example, an allergic reaction) to a
subsequent or later challenge by a TLR-agonist. Preferably a TLR7
and/or TLR9 agonist.
[0113] In the methods according to this aspect of the invention,
administration of IRO compound according to the invention can be by
any suitable route, including, without limitation, parenteral,
mucosal delivery, oral, sublingual, transdermal, topical,
inhalation, intragastric, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form. Administration of the therapeutic
compositions of IRO compound can be carried out using known
procedures at dosages and for periods of time effective to reduce
symptoms or surrogate markers of the disease. When administered
systemically, the therapeutic composition is preferably
administered at a sufficient dosage to attain a blood concentration
of IRO compound from about 0.0001 micromolar to about 100
micromolar. More preferably, systemic administration would be at a
sufficient dosage to attain a blood concentration of the IRO
compound from about 0.001 micromolar to about 10 micromolar. For
localized administration, much lower concentrations than this may
be effective, and much higher concentrations may be tolerated.
Preferably, a total dosage of IRO compound ranges from about 0.001
mg per patient per day to about 200 mg per kg body weight per day.
It may be desirable to administer the IRO compound according to the
invention daily, every second day, every third day, every fourth
day, every fifth day, every sixth day or weekly. It may be
desirable to administer simultaneously, or sequentially, a
therapeutically effective amount of one or more of the IRO
containing therapeutic compositions of the invention to an
individual as a single treatment episode.
[0114] The IRO compound may optionally be linked to one or more
allergens and/or antigens (self or foreign), an immunogenic
protein, such as keyhole limpet hemocyanin (KLH), cholera toxin B
subunit, or any other immunogenic carrier protein. IRO can also be
used in combination with other compounds (for example, adjuvants)
including, without limitation, TLR agonists (e.g. TLR2 agonists,
TLR4 agonists, and TLR9 agonists), Freund's incomplete adjuvant,
KLH, monophosphoryl lipid A (MPL), alum, Merck alum adjuvant (MAA),
and saponins, including QS-21 and imiquimod, or combinations
thereof.
[0115] The methods according to this aspect of the invention are
useful for model studies of the immune system. The methods are also
useful for the prophylactic or therapeutic treatment of human or
animal disease. For example, the methods are useful for pediatric,
adult, and veterinary vaccine applications.
[0116] In a fourth aspect, the invention provides methods for
therapeutically treating a patient having a disease or disorder,
such methods comprising administering to the patient a IRO compound
according to the invention. In various embodiments, the disease or
disorder to be treated is cancer, an autoimmune disorder, airway
inflammation, inflammatory disorders, infectious disease, malaria,
Lyme disease, ocular infections, conjunctivitis, skin disorders,
psoriasis, scleroderma, cardiovascular disease, atherosclerosis,
chronic fatigue syndrome, sarcoidosis, transplant rejection,
allergy, asthma or a disease caused by a pathogen. Preferred
autoimmune disorders include without limitation lupus
erythematosus, multiple sclerosis, type I diabetes mellitus,
irritable bowel syndrome, Chron's disease, rheumatoid arthritis,
septic shock, alopecia universalis, acute disseminated
encephalomyelitis, Addison's disease, ankylosing spondylitis,
antiphospholipid antibody syndrome, autoimmune hemolytic anemia,
autoimmune hepatitis, Bullous pemphigoid, chagas disease, chronic
obstructive pulmonary disease, coeliac disease, dermatomyositis,
endometriosis, Goodpasture's syndrome, Graves' disease,
Guillain-Barre syndrome, Hashimoto's disease, hidradenitis
suppurativa, idiopathic thrombocytopenic purpura, interstitial
cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
pemphigus, pernicious anaemia, polymyositis, primary biliary
cirrhosis, schizophrenia, Sjogren's syndrome, temporal arteritis
("giant cell arteritis"), vasculitis, vitiligo, vulvodynia and
Wegener's granulomatosis. Preferred inflammatory disorders include
without limitation airway inflammation, asthma, autoimmune
diseases, chronic inflammation, chronic prostatitis,
glomerulonephritis, Behcet's disease, hypersensitivities,
inflammatory bowel disease, reperfusion injury, rheumatoid
arthritis, transplant rejection, ulcerative colitis, uveitis,
conjunctivitis and vasculitis. Pathogens include bacteria,
parasites, fungi, viruses, viroids, and prions. Administration is
carried out as described for the third aspect of the invention.
[0117] In a fifth aspect, the invention provides methods for
preventing a disease or disorder, such methods comprising
administering to the patient IRO compound according to the
invention. In various embodiments, the disease or disorder to be
prevented is cancer, an autoimmune disorder, airway inflammation,
inflammatory disorders, infectious disease, malaria, Lyme disease,
ocular infections, conjunctivitis, skin disorders, psoriasis,
scleroderma, cardiovascular disease, atherosclerosis, chronic
fatigue syndrome, sarcoidosis, transplant rejection, allergy,
asthma or a disease caused by a pathogen. Preferred autoimmune
disorders include without limitation lupus erythematosus, multiple
sclerosis, type I diabetes mellitus, irritable bowel syndrome,
Chron's disease, rheumatoid arthritis, septic shock, alopecia
universalis, acute disseminated encephalomyelitis, Addison's
disease, ankylosing spondylitis, antiphospholipid antibody
syndrome, autoimmune hemolytic anemia, autoimmune hepatitis,
Bullous pemphigoid, chagas disease, chronic obstructive pulmonary
disease, coeliac disease, dermatomyositis, endometriosis,
Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome,
Hashimoto's disease, hidradenitis suppurativa, idiopathic
thrombocytopenic purpura, interstitial cystitis, morphea,
myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious
anaemia, polymyositis, primary biliary cirrhosis, schizophrenia,
Sjogren's syndrome, temporal arteritis ("giant cell arteritis"),
vasculitis, vitiligo, vulvodynia and Wegener's granulomatosis.
Preferred inflammatory disorders include without limitation airway
inflammation, asthma, autoimmune diseases, chronic inflammation,
chronic prostatitis, glomerulonephritis, Behcet's disease,
hypersensitivities, inflammatory bowel disease, reperfusion injury,
rheumatoid arthritis, transplant rejection, ulcerative colitis,
uveitis, conjunctivitis and vasculitis. Pathogens include bacteria,
parasites, fungi, viruses, viroids, and prions. Administration is
carried out as described for the third aspect of the invention.
[0118] In any of the methods according to the third, fourth or
fifth aspect of the invention, the IRO compound can be administered
in combination with any other agent useful for treating or
preventing the disease or condition that does not abolish the
immune antagonist, inhibitory, suppression or prevention effect or
activity of the IRO compound. In any of the methods according to
the invention, the agent useful for treating or preventing the
disease or condition includes, but is not limited to, one or more
vaccines, antigens, antibodies, cytotoxic agents, allergens,
antibiotics, antisense oligonucleotides, TLR agonist, TLR
antagonist, peptides, proteins, gene therapy vectors, DNA vaccines,
adjuvants or kinase inhibitors to enhance the specificity or
magnitude of the immune response, or co-stimulatory molecules such
as cytokines, chemokines, protein ligands, trans-activating
factors, peptides and peptides comprising modified amino acids. For
example, in the treatment of cancer, it is contemplated that the
IRO compound may be administered in combination with one or more
chemotherapeutic compound, targeted therapeutic agent and/or
monoclonal antibody; And in preventing a disease, it is
contemplated that the IRO compound may be administered in
combination with one or more vaccine. Alternatively, the agent can
include DNA vectors encoding for antigen or allergen. In these
embodiments, the IRO compounds of the invention can variously act
as adjuvants and/or produce direct immune modulatory effects.
[0119] The following examples are intended to further illustrate
certain exemplary embodiments of the invention and are not intended
to limit the scope of the invention. For example, representative
TLR-ligands are shown in the following examples, but do not limit
the scope of ligands to which the IROs of the invention act as
antagonists.
Example 1
Synthesis of Oligonucleotides Containing Immune Regulatory
Moieties
[0120] All IRO compounds of the invention were synthesized
according to standard procedures (see e.g. U.S. Patent Publication
No. 20040097719).
[0121] Oligonucleotides were synthesized on a 1 .mu.M scale using
an automated DNA synthesizer (Expedite 8909; PerSeptive Biosystems,
Framingham, Mass.), following standard linear synthesis or parallel
synthesis procedures (see e.g. FIGS. 5 and 6 of U.S. Patent
Publication No. 20040097719).
[0122] Deoxyribonucleoside phosphoramidites were obtained from
(Aldrich-Sigma, St Louis, Mo.). 1',2'-dideoxyribose
phosphoramidite, propyl-1-phosphoramidite, 2-deoxyuridine
phosphoramidite,
1,3-bis-[5-(4,4'-dimethoxytrityl)pentylamidyl]-2-propanol
phosphoramidite and methyl phosponamidite were obtained from Glen
Research (Sterling, Va.). .beta.-L-2'-deoxyribonucleoside
phosphoramidite, .alpha.-2'-deoxyribonucleoside phosphoramidite,
mono-DMT-glycerol phosphoramidite and di-DMT-glycerol
phosphoramidite were obtained from ChemGenes (Willmington, Mass.).
(4-Aminobutyl)-1,3-propanediol phosphoramidite was obtained from
Clontech (Palo Alto, Calif.). Arabinoguanosine, was obtained from
Reliable Pharmaceutical (St. Louis, Mo.). Arabinoguanosine
phosphoramidite was synthesized at Idera Pharmaceuticals, Inc.
(Cambridge, Mass.) (Noronha et al. (2000) Biochem.,
39:7050-7062).
[0123] All nucleoside phosphoramidites were characterized by
.sup.31P and .sup.1H NMR spectra. Modified nucleosides were
incorporated at specific sites using normal coupling cycles. After
synthesis, oligonucleotides were deprotected using concentrated
ammonium hydroxide and purified by reverse phase HPLC, followed by
dialysis. Purified oligonucleotides as sodium salt form were
lyophilized prior to use. Purity was tested by CGE and MALDI-TOF
MS.
Example 2
Inhibition of TLR7 and TLR9 Stimulation
[0124] C57BL/6 mice were injected s.c. at left underarm with 5
mg/kg of an IRO compound at 0 hours and 0.25 mg/kg TLR9 agonist or
10 mg/kg TLR7 agonist at 24 hours. Serum samples were taken at 2
hours after injection of the TLR9 or TLR7 agonist and IL-12
concentration was determined by ELISA. For IRO number 40, the TLR7
and TLR9 agonists were administered 72 hours after administration
of the IRO. The results for all IROs are shown in Tables 4-11.
These results demonstrate that an IRO compounds according to the
invention can inhibit TLR7 and/or TLR9 activity in vivo, and more
generally that IRO compounds according to the invention can inhibit
TLR activation.
TABLE-US-00004 TABLE 4 Antagonist Activity in vivo in mice %
Inhibition of % Inhibition of Oligo TLR9 agonist TLR7 agonist No.
Sequences and Modification induced IL-12 induced IL-12 1
5'-UGUCG1TTCT-X1-TCTTG1CUGU-5' 44.8 94.0 2
5'-UGUCG1TTC-X1-CTTG1CUGU-5' 69.8 91.6 3 5'-UGUCG1TT-X1-TTG1CUGU-5'
63.7 89.8 4 5'-UGUCoG1TTCTo-Z-oTCTTG1oCUGU-5' 27.6 53.7 5
5'-GUCG1TTCTT-Z-TTCTTG1CUG-5' 75.9 97.1 6
5'-UGUCG2TTCT-Z-TCTTG2CUGU-5' 70.9 99.0 7
5'-UGUCG1TTCT-X4-TCTTG1CUGU-5' 83.2 92.6 8
5'-UGUCG1TTC-X4-CTTG1CUGU-5' 68.7 78.5 9
5'-UGUCoG1TTCTo-X4-oTCTTG1oCUGU- 76.5 18.6 5' 10
5'-GUCG1TTCTT-X4-TTCTTG1CUG-5' 87.8 100 11
5'-UGUCG1TT-X4-TTG1CUGU-5' 31.1 56.8 12
5'-UGUCG1TTC-X5-CTTG1CUGU-5' 7.3 80.4 13
5'-UGUCG2TTC-X5-CTTG2CUGU-5' 48.6 98.1 14
5'-UGUCG1TTC-X6-CTTG1CUGU-5' 64.6 92.2 15
5'-UGUCG2TTC-X6-CTTG2CUGU-5' 57.1 99.9 16
5'-UGUCG1TTCT-X7-TCTTG1CUGU-5' 96.5 98.5 17
5'-UGUCG2TTCT-X7-TCTTG2CUGU-5' 86.2 97.3 18
5'-UGUCG1TTC-X7-CTTG1CUGU-5' 94.0 98.1
TABLE-US-00005 TABLE 5 Antagonist Activity in vivo in mice
%Inhibition of % Inhibition of Oligo TLR9 agonist TLR7 agonist No.
Sequences and Modification induced IL-12 induced IL12 19
5'-TGUCG1TTCT-X-TCTTG1CUGT-5' 45.2 89.8 20
5'-CTTGUCG1TTCT-X-TCTTG1CUGTTC-5' 74.5 77.9 21
5'-TTGUCG1TTC-X-CTTG1CUGTT-5' 66.5 86.8 22
5'-CTTTGUCG1TTC-X-CTTG1CUGTTTC-5' 47.5 88.9 23
5'-TGUCG1TTCT-X7-TCTTG1CUGT-5' 45.4 83.6 24
5'-TTGUCG1TTC-X7-CTTG1CUGTT-5' 42.5 88.3 25
5'-GUCG1TTCTT-Z-TTCTTG1CUG-5' 80.4 92.3 26
5'-TGUCG1TTCA-X-ACTTG1CUGT-5' 65.8 93.2
TABLE-US-00006 TABLE 6 Antagonist Activity in vivo in mice Oligo %
Inhibition of TLR9 No. Sequences and Modification agonist induced
IL-12 27 5'-TCTGACG1TTCT-X-TCTTG1CAGTCT-5' 95.8 28
5'-TCTGACG2TTCT-X-TCTTG2CAGTCT-5' 97.4
TABLE-US-00007 TABLE 7 Antagonist Activity in vivo in mice Oligo %
Inhibition of TLR9 % Inhibition of TLR7 No. Sequences and
Modification agonist induced IL-12 agonist induced IL12 29
5'-TTGUCG1TTA-X-ATTG1CUGTT-5' 36.6 95.3 30
5'-CTCTGUCG1TTA-X-ATTG1CUGTCTC- 22.6 91.6 5' 31
5'-TGTC*GTTCT-X-TCTTGC*TGT-5' 78.9 32 5'-TGTCGTTCT-X-TCTTGCTGT-5'
73.4 33 5'-TGTC*GTTCT-X-TCTTGC*TGT-5' 75.5 34
5'-TGTCGTTCT-X-TCTTGCTGT-5' 85.8
TABLE-US-00008 TABLE 8 Antagonist Activity in vivo in mice Oligo %
Inhibition of TLR9 No. Sequences and Modification agonist induced
IL-12 35 5'-UGUCG1ACAT-X-TACAG1CUGU-5' 65.1 36
5'-UGUCG1TTC-X-CTTG1CUGU-5' 35.7 37 5'-UGUCG1TT-X-TTG1CUGU-5' 26.5
38 5'-UoGUCG1TToCTo-X-oTCoTTG1CUGoU-5' 6.9 39
5'-UoGoUCG1TTCTo-X-oTCTTG1CUoGoU-5' 16.8
TABLE-US-00009 Table 9 Antagonist Activity in vivo in mice Oligo %
Inhibition of TLR9 No. Sequences and Modification agonist induced
IL-12 40 5'-UGACG1TTCT-X-TCTTG1CAGU-5' 54.9 Table 10 Antagonist
Activity in vivo in mice Oligo % Inhibition of TLR9 No. Sequences
and Modification agonist induced IL-12 41
5'-UGUCG1ACAT-Z-TACAG1CUGU-5' 86.9 42 5'-UGUCG1TTCT-Z-TCTTG1CUGU-5'
69.6 43 5'-UGUCG1TTC-Z-CTTG1CUGU-5' 60.8 44
5'-UGUCG1TT-Z-TTG1CUGU-5' 43.8
TABLE-US-00010 TABLE 11 Antagonist Activity in vivo in mice oligo %
Inhibition of TLR9 % Inhibition of TLR7 No. Sequences and
Modification agonist induced IL-12 agonist induced IL12 58
5'-CTATCTGAC*GTTCTCTGT-3' 72.6 79.8 62 5'-CTATCTG{circumflex over (
)}A{circumflex over ( )}CGTTCTCTGT-3' 68.1 30.9
TABLE-US-00011 TABLE 12 Antagonist Activity in vivo in mice oligo %
Inhibition of IL-12 No. Sequence TLR9 TLR7 71
5'-CTTTGUC*G1TTC-X-CTTG1C*UGTTTC-5' 55.5 94.9 77
5'-CTTGUC*G1TTCT-X-TCTTG1C*UGTTC-5' 71.5 92.8 78
5'-CTTTGUC*oG1TTC-X-CTTG1oC*UGTTTC-5' 25.6 83.4 80
5'-CTTGUC*oG1TTCT-X-TCTTG1oC*UGTTC-5' 6.7 68.2 82
5'-CTGUC*oG1TTCTT-X-TTCTTG1oC*UGTC-5' 9.0 78.4 88
5'-CTATCTGUC*G1TTCTCTGT-3' 63.2 77.7 89 5'-CTATCTGUC*G1TTCTCTGT-3'
36.4 40.5
Example 3
TLR7/TLR9 In Vitro Antagonist Study
[0125] C57BL/6 mice were used in this study. Mouse splenocytes were
cultured for 24 hrs (at 37.degree. C., 5% CO.sub.2) with TLR7/TLR9
antagonists over a dose range 0.3, 1, 5, 10 mg/ml or at a single
dose, 10 mg/ml in the presence of a TLR7 agonist (200 mg/ml) or in
the presence of a TLR9 agonist (1 mg/ml) or in the presence of PBS.
Supernatants were collected and cytokine/chemokine responses were
then evaluated in supernatants by multiplex assays using the
Luminex xMAP system. Samples were assayed in duplicate (.+-.SD).
Results are shown in FIGS. 3 and 4.
Example 4
TLR7/TLR9 In Vivo Antagonist Study
[0126] Female C57BL/6 mice (2/group) were s.c injected with 1, 5 or
15 mg/kg antagonist compound at 0 hr in the right flank. The mice
were then injected with TLR7 agonist (10 mg/kg) or with TLR9
agonist (0.25 mg/kg) at 24 hrs in the left flank. Blood was
collected by orbital bleeding 2 hrs post the agonist
administration. The serum samples were then analyzed by IL-12
ELISA. Results are shown in FIGS. 5 and 6.
[0127] Additionally, female C57BL/6 mice (2/group) were s.c
injected with 5 mg/kg antagonist compound at day 0 in the right
flank. The mice were then injected with TLR7 (10 mg/kg) agonist at
days 1, 2, 4, 9 and 11 or with TLR9 (0.25 mg/kg) agonist at days 1,
2, 4, 7, 9 and 11 in the left flank. Blood was collected by orbital
bleeding 2 hrs post the agonist administration. The serum samples
were then analyzed by IL-12 ELISA. Results are shown in FIGS. 7 and
8.
[0128] Female C57BL/6 mice (2/group) were also s.c injected with 5
mg/kg antagonist compound at 0 hr in the right flank. The mice were
then injected with TLR9 (0.25 mg/kg) or TLR7 (10 mg/kg) agonists at
24 hrs in the left flank. Blood was collected by orbital bleeding 2
hrs post the agonist administration. Cytokine/chemokine responses
were then evaluated in serum samples by multiplex assays using the
Luminex xMAP system. Results are shown in FIG. 9.
[0129] Finally, female C57BL/6 mice (2/group) were s.c injected
with 5 mg/kg antagonist compound at 0 hr in the right flank. The
mice were then injected with TLR3 (10 mg/kg) or TLR5 (0.25 mg/kg)
agonists at 24 hrs in the left flank. Blood was collected by
orbital bleeding 2 hrs post the agonist administration.
Cytokine/chemokine responses were then evaluated in serum samples
by multiplex assays using the Luminex xMAP system. Results are
shown in FIG. 10.
[0130] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
7219DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)7-deaza-dG 1ugucgttct 9
28DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-G-A, 2'-O-Me-A, 2'-O-Me-C, 2'-O-Me-U, or
2'-O-Me-G-C, 2'- O-Me-A, 2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-G-U, or
2'-O-Me-A, 2'-O-Me-C, 2'-O-Me-U, or
2'-O-Me-G-Gmodified_base(5)..(5)7-deaza-dG 2ugucgttc 8
37DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)7-deaza-dG 3ugucgtt 7
49DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(2)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(4)..(4)7-deaza-dG 4gucgttctt 9
59DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)AraG 5ugucgttct 9
68DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)AraG 6ugucgttc 8
79DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(2)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)7-deaza-dG 7tgucgttct 9
811DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(7)..(7)7-deaza-dG 8cttgucgttc
t 1199DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(3)..(4)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(6)..(6)7-deaza-dG 9ttgucgttc 9
1011DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(5)..(6)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(8)..(8)7-deaza-dG 10ctttgucgtt
c 11119DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(2)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)7-deaza-dG 11tgucgttca
9 1211DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(7)..(7)7-deaza-dG 12tctgacgttc
t 111311DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(7)..(7)AraG 13tctgacgttc t
11149DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(3)..(4)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(6)..(6)7-deaza-dG 14ttgucgtta
9 1511DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(5)..(6)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(8)..(8)7-deaza-dG 15ctctgucgtt
a 11169DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(4)5-Me-dC 16tgtcgttct 9
179DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(4)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-G 17tgtcgttct 9 189DNAArtificial
SequenceSynthetic oligonucleotidemodified_base(5)..(5)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-G 18tgtcgttct 9 199DNAArtificial
SequenceSynthetic oligonucleotidemodified_base(1)..(3)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)7-deaza-dG
19ugucgacat 9 209DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)7-deaza-dG 20ugacgttct
9 219DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(4)..(4)5-Me-dC 21ugucgttct 9
229DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-(2-methoxyethyl)modified_base(4)-
..(4)5-Me-dC 22tgtcgttct 9 239DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(4)..(4)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-G-5-Me-C 23ugucgttct 9 249DNAArtificial
SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-(2-methoxyethyl)modified_base(4)-
..(4)2'-O-Me-A, 2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-G-5-Me-C
24tgtcgttct 9 259DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)AraG 25ugacgttct 9
2611DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(7)..(7)7-deaza-dG 26tctgucgttc
t 112711DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(7)..(7)AraG 27tctgucgttc t
11287DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(5)..(5)AraG 28ugucgtt 7
299DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(2)..(4)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(6)..(6)7-deaza-dG 29tugucgttc
9 309DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(3)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(7)..(7)7-deaza-dG 30ctugucgtt
9 319DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(1)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(3)..(3)7-deaza-dG 31ucgttcttc
9 3218DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(9)..(9)5-Me-dC 32ctatctgacg
ttctctgt 183318DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(9)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-G 33ctatctgacg ttctctgt 183418DNAArtificial
SequenceSynthetic oligonucleotidemodified_base(7)..(8)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-Gmodified_base(9)..(9)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-G-5-Me-C 34ctatctgacg ttctctgt
183518DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(10)..(10)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-G 35ctatctgacg ttctctgt 183618DNAArtificial
SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-(2-methoxyethyl)
36ctatctgacg ttctctgt 183718DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(9)..(9)5-Me-dC 37ctatctgucg
ttctctgt 183818DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(9)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-G 38ctatctgucg ttctctgt 183918DNAArtificial
SequenceSynthetic oligonucleotidemodified_base(7)..(8)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-Gmodified_base(9)..(9)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-G-5-Me-C 39ctatctgucg ttctctgt
184018DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-Gmodified_base(10)..(10)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-G 40ctatctgucg ttctctgt 184111DNAArtificial
SequenceSynthetic oligonucleotidemodified_base(4)..(5)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or
2'-O-Me-Gmodified_base(6)..(6)5-Me-dCmodified_base(7)..(7)7-deaza-dG
41cttgucgttc t 114218DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(9)..(9)5-Me-dCmodified_base(10)..(10)7-deaza-dGmod-
ified_base(17)..(18)2'-O-Me-A, 2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-G
42ctatctgucg ttctctgu 18439DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(2)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)7-deaza-dG
43tgucgttct 9 4411DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(5)..(6)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(7)..(7)5-Me-dCmodified_base(8)..(8)7-deaza-dG
44ctttgucgtt c 11459DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(2)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(3)..(3)5-Me-dCmodified_base(4)..(4)7-deaza-dG
45gucgttctt 9 469DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(2)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)7-deaza-dG
46tgucgttca 9 4711DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(6)..(6)5-Me-dCmodified_base(7)..(7)AraG
47cttgucgttc t 114810DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(6)..(6)5-Me-dCmodified_base(7)..(7)7-deaza-dG
48cttgucgttc 104911DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(3)..(4)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(5)..(5)5-Me-dCmodified_base(6)..(6)7-deaza-dG
49ctgucgttct t 11509DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)7-deaza-dG
50ugucgttct 9 519DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)AraG
51ugucgttct 9 528DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)7-deaza-dG
52ugucgttc 8 5318DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(9)..(9)5-Me-dCmodified_base(10)..(10)7-deaza-dG
53ctatctgucg ttctctgt 185418DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(8)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(9)..(9)5-Me-dCmodified_base(10)..(10)7-deaza-dG
54ctatctgucg ttctctgt 18559DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(2)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)7-deaza-dG
55tgacgttct 9 5611DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(6)..(6)5-Me-dCmodified_base(7)..(7)7-deaza-dG
56cttgacgttc t 115711DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(5)..(6)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(7)..(7)5-Me-dCmodified_base(8)..(8)7-deaza-dG
57ctttgacgtt c 11589DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(2)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(3)..(3)5-Me-dCmodified_base(4)..(4)7-deaza-dG
58gacgttctt 9 599DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(2)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)7-deaza-dG
59tgacgttca 9 6011DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(6)..(6)5-Me-dCmodified_base(7)..(7)AraG
60cttgacgttc t 116110DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(5)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(6)..(6)5-Me-dCmodified_base(7)..(7)7-deaza-dG
61cttgacgttc 106211DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(3)..(4)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(5)..(5)5-Me-dCmodified_base(6)..(6)7-deaza-dG
62ctgacgttct t 11639DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)7-deaza-dG
63ugacgttct 9 649DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)AraG
64ugacgttct 9 658DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(1)..(3)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(4)..(4)5-Me-dCmodified_base(5)..(5)7-deaza-dG
65ugacgttc 8 6618DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(9)..(9)5-Me-dCmodified_base(10)..(10)7-deaza-dG
66ctatctgacg ttctctgt 186718DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(8)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(9)..(9)5-Me-dCmodified_base(10)..(10)7-deaza-dG
67ctatctgacg ttctctgt 186818DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(7)..(8)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or
2'-O-Me-Gmodified_base(9)..(9)5-Me-dCmodified_base(10)..(10)7-deaza-dGmod-
ified_base(17)..(18)2'-O-Me-A, 2'-O-Me-C, 2'-O-Me-U, or
2'-O-Me-G
68ctatctgacg ttctctgu 18699DNAArtificial SequenceSynthetic
oligonucleotidemodified_base(4)..(4)2'-O-Me-A, 2'-O-Me-C,
2'-O-Me-U, or 2'-O-Me-G-5-Me-C 69tgtcgttct 9 7010DNAArtificial
SequenceSynthetic oligonucleotidemisc_featurem =
0-30misc_feature(1)..(4)n is a, c, g, or tmisc_feature(7)..(10)n is
a, c, g, or t 70nnnncgnnnn 107112DNAArtificial SequenceSynthetic
oligonucleotidemisc_featurez/p = 0-30misc_feature(1)..(2)n is a, c,
g, or tmisc_feature(3)..(4)2'-O-Me-A, 2'-O-Me-C, 2'-O-Me-U, or
2'-O-Me-Gmisc_feature(5)..(5)5-Me-dCmisc_feature(6)..(6)7-deaza-dGmisc_fe-
ature(7)..(10)n is a, c, g, or tmisc_feature(11)..(12)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-G 71nnnncgnnnn nn
127218DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(1)..(6)n is a, c, g, or
tmisc_feature(7)..(8)2'-O-Me-A, 2'-O-Me-C, 2'-O-Me-U, or
2'-O-Me-Gmisc_feature(9)..(9)5-Me-dCmisc_feature(10)..(10)7-deaza-dGmisc_-
feature(11)..(16)n is a, c, g, or tmisc_feature(17)..(18)2'-O-Me-A,
2'-O-Me-C, 2'-O-Me-U, or 2'-O-Me-G 72nnnnnnnncg nnnnnnnn 18
* * * * *