U.S. patent application number 14/771252 was filed with the patent office on 2016-04-21 for chimeric single-stranded antisense polynucleotides and double-stranded antisense agent.
This patent application is currently assigned to NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL UNIVERSITY. The applicant listed for this patent is NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL UNIVERSITY. Invention is credited to Hidehiro MIZUSAWA, Kazutaka NISHINA, Takeshi WADA, Takanori YOKOTA.
Application Number | 20160108395 14/771252 |
Document ID | / |
Family ID | 51427945 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160108395 |
Kind Code |
A1 |
YOKOTA; Takanori ; et
al. |
April 21, 2016 |
CHIMERIC SINGLE-STRANDED ANTISENSE POLYNUCLEOTIDES AND
DOUBLE-STRANDED ANTISENSE AGENT
Abstract
Chimeric single-stranded polynucleotides and double-stranded
antisense agents useful for modifying the expression of a target
gene by means of an antisense effect are disclosed. The chimeric
single-stranded antisense polynucleotide and double-stranded
antisense agents comprise a central nucleotide region flanked by a
first 5'-wing region and a first 3'-wing region of modified
nucleotides, which are themselves flanked by a second 5'-wing
region and/or a second 3'-wing region of nucleotides that have a
low affinity for proteins and/or that have higher resistance to
DNase or RNase than a natural DNA or RNA and are missing in a cell
when the chimeric polynucleotide delivered. The double-stranded
antisense agent further comprises a complementary strand annealed
to the antisense strand. The polynucleotide can be used to modify
RNA transcription levels, miRNA activity, or protein levels in
cells.
Inventors: |
YOKOTA; Takanori;
(Bunkyo-ku, Tokyo, JP) ; NISHINA; Kazutaka;
(Bunkyo-ku, Tokyo, JP) ; MIZUSAWA; Hidehiro;
(Bunkyo-ku, Tokyo, JP) ; WADA; Takeshi;
(Bunkyo-ku, Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL
UNIVERSITY |
Bunkyo-ku, Tokyo |
|
JP |
|
|
Assignee: |
NATIONAL UNIVERSITY CORPORATION
TOKYO MEDICAL AND DENTAL UNIVERSITY
Bunkyo-ku, Tokyo
JP
|
Family ID: |
51427945 |
Appl. No.: |
14/771252 |
Filed: |
March 3, 2014 |
PCT Filed: |
March 3, 2014 |
PCT NO: |
PCT/JP2014/001159 |
371 Date: |
August 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61771115 |
Mar 1, 2013 |
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61806887 |
Mar 31, 2013 |
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61909179 |
Nov 26, 2013 |
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Current U.S.
Class: |
514/44A ;
435/375; 536/24.5 |
Current CPC
Class: |
C12N 2310/3231 20130101;
A61K 31/7125 20130101; C12N 2310/341 20130101; A61K 31/712
20130101; A61P 43/00 20180101; A61K 31/7088 20130101; C12N 2310/315
20130101; C12N 2310/53 20130101; A61K 31/713 20130101; C12N 2310/14
20130101; C12N 2310/11 20130101; C12N 2310/533 20130101; C12N
15/111 20130101; C12N 15/113 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. A chimeric antisense polynucleotide comprising: a center
nucleotide region comprising at least 5 nucleotides; a first
5'-wing region joined to the 5' end of the center nucleotide region
comprising 1-10 nucleotides wherein at least 1 is a nucleotide
analog; a first 3'-wing region joined to the 3' end of the center
nucleotide region comprising 1-10 nucleotides wherein at least 1 is
a nucleotide analog; and a second 5'-wing region and/or a second
3'-wing region, wherein: the second 5'-wing region is joined to the
5' end of the first 5'-wing region and comprises at least 1 low
protein-affinity nucleotide; and the second 3'-wing region is
joined to the 3' end of the first 3'-wing region and comprises at
least 1 low protein-affinity nucleotide; wherein the total number
of nucleotides, nucleotide analogs, and low protein-affinity
nucleotides is no more than 100 nucleotides.
2. A chimeric antisense polynucleotide comprising: a center
nucleotide region comprising at least 5 nucleotides; a first
5'-wing region joined to the 5' end of the center nucleotide region
comprising 1-10 nucleotides wherein at least 1 is a nucleotide
analog; a first 3'-wing region joined to the 3' end of the center
nucleotide region comprising 1-10 nucleotides wherein at least 1 is
a nucleotide analog; and a second 5'-wing region and/or a second
3'-wing region, wherein: the second 5'-wing region is joined to the
5' end of the first 5'-wing region, has higher resistance to DNase
or RNase than a natural DNA or RNA and is missing in a cell when
the chimeric polynucleotide delivered; and the second 3'-wing
region is joined to the 3' end of the first 3'-wing region, has
higher resistance to DNase or RNase than a natural DNA or RNA and
is missing in a cell when the chimeric polynucleotide delivered,
wherein the total number of nucleotides and nucleotide analogs is
no more than 100 nucleotides.
3. The chimeric antisense polynucleotide of claim 1, wherein the
center nucleotide region comprises nucleotides that, when
hybridized to an RNA polynucleotide, the center nucleotide
region/RNA polynucleotide duplex is recognized by RNase H.
4. The chimeric antisense polynucleotide of claim 3, wherein the
nucleotides of the center nucleotide region are independently
selected from DNA and phosphorothioate DNA nucleotides.
5. The chimeric antisense polynucleotide of claim 4, wherein the
center nucleotide region comprises 5-20 DNA nucleotides.
6. The chimeric antisense polynucleotide of claim 4, wherein the
center nucleotide region comprises 5-12 DNA nucleotides.
7. The chimeric antisense polynucleotide of claim 1, wherein the
center nucleotide region comprises nucleotides independently
selected from RNA nucleotides and nucleotide analogs.
8. The chimeric antisense polynucleotide of claim 7, wherein the
nucleotide analogs are independently selected from LNA nucleotides,
BNA nucleotides, 2'-O-Me RNA nucleotides, 2'-O-methoxyethyl RNA
nucleotides.
9. The chimeric antisense polynucleotide of claim 8, wherein the
center nucleotide region comprises nucleotides independently
selected from 2'-O-Me RNA nucleotides and 2'-O-methoxyethyl RNA
nucleotides.
10. The chimeric antisense polynucleotide of claim 1, wherein at
least one of the nucleotides in the center nucleotide region is
phosphorothioated.
11. The chimeric antisense polynucleotide of claim 10, wherein all
nucleotides in the center nucleotide region are
phosphorothioated.
12. The chimeric antisense polynucleotide of claim 1, wherein the
nucleotides in the first wing regions are bridged nucleotides.
13. The chimeric antisense polynucleotide of claim 1, wherein the
bridged nucleotides are independently selected from LNA, cEt BNA,
amideBNA, and cMOE ENA.
14. The chimeric antisense polynucleotides of claim 1, wherein at
least one of the nucleotide analogs in the center nucleotide region
and the first wing region(s) are phosphorothioated.
15. The chimeric antisense polynucleotide of claim 1, wherein the
low protein-affinity nucleotides are independently selected from
2'-O-methyl RNA nucleotides, 2'-O-methoxyethyl RNA nucleotides,
LNA, cMOE BNA, 2-fluoro RNA nucleotides, boranophosphate
nucleotides, methylphosphonate nucleotides, phosphoramidite
nucleotides, 5-methylcytosine, UNA and 5-propynyluridine.
16. The chimeric antisense polynucleotide of claim 1, wherein the
chimeric antisense polynucleotide includes the second 5'-wing
region.
17. The chimeric antisense polynucleotide of claim 1, wherein the
chimeric antisense polynucleotide includes the second 3'-wing
region.
18. The chimeric antisense polynucleotide of claim 1, wherein the
chimeric antisense polynucleotide includes the second 5'-wing
region and the second 3'-wing region.
19. The chimeric antisense polynucleotide of claim 1, wherein the
chimeric antisense polynucleotide further comprises a functional
moiety joined to the 3'-end and/or the 5'-end of the chimeric
antisense polynucleotide.
20. The chimeric antisense polynucleotide of claim 19, wherein the
functional moiety has a function selected from a labeling function,
a purification function, and a targeted delivery function.
21. The chimeric antisense polynucleotide of claim 19, wherein the
functional moiety is joined to the chimeric antisense
polynucleotide via a cleavable linker moiety.
22. The chimeric antisense polynucleotide of claim 19, wherein the
functional moiety is a molecule selected from a lipid, a peptide,
and a protein.
23. The chimeric antisense polynucleotides of claim 22, wherein the
lipid is selected from cholesterol, a fatty acid, a lipid-soluble
vitamin, a glycolipid, and a glyceride.
24. The chimeric antisense polynucleotides of claim 22, wherein the
lipid is selected from cholesterol, a tocopherol, and a
tocotrienol.
25. The chimeric antisense polynucleotides of claim 22, wherein the
peptide is selected from a receptor ligand fragment and an antibody
fragment.
26. The chimeric antisense polynucleotide of claim 22, wherein the
protein is selected from a receptor ligand and an antibody.
27. The chimeric antisense polynucleotide of claim 1, wherein the
chimeric antisense polynucleotide can hybridize to a cellular
transcription product in a 100 mM sodium chloride, 10 mM sodium
phosphate buffer, pH 7.2, at 25.degree. C.
28. The chimeric antisense polynucleotide of claim 27, wherein the
center region is fully complementary to the cellular transcription
product to which the chimeric antisense polynucleotide can
hybridize.
29. The chimeric antisense polynucleotide of claim 27, wherein the
second 5'-wing region and/or the second 3'-wing region comprise at
least one mismatched base when the chimeric antisense
polynucleotide hybridizes to the cellular transcription product to
which the chimeric antisense polynucleotide can hybridize.
30. The chimeric antisense polynucleotide of claim 29, wherein all
bases of the second 5'-wing region and/or the second 3'-wing region
are mismatched.
31. A double-stranded antisense agent comprising the chimeric
antisense polynucleotide of claim 1 and a complementary strand
annealed to the chimeric antisense polynucleotide.
32. The double-stranded antisense agent comprising the chimeric
antisense polynucleotide of claim 31, wherein the complementary
strand further comprises a functional moiety joined to the 3'-end
and/or the 5'-end.
33. The double-stranded antisense agent of claim 32, wherein the
functional moiety has a function independently selected from a
labeling function, a purification function, and a targeted delivery
function.
34. The double-stranded antisense agent of claim 32, wherein the
functional moiety is independently joined to the chimeric antisense
polynucleotide and/or the complementary strand via a cleavable
linker moiety.
35. The double-stranded antisense agent of claim 32, wherein the
functional moiety is a molecule independently selected from a
lipid, a peptide, and a protein.
36. The double-stranded antisense agent of claim 35, wherein the
lipid is independently selected from cholesterol, a fatty acid, a
lipid-soluble vitamin, a glycolipid, and a glyceride.
37. The double-stranded antisense agent of claim 35, wherein the
lipid is independently selected from cholesterol, a tocopherol, and
a tocotrienol.
38. The double-stranded antisense agent of claim 35, wherein the
peptide is independently selected from a receptor ligand fragment
and an antibody fragment.
39. The double-stranded antisense agent of claim 35, wherein the
protein is independently selected from a receptor ligand and an
antibody.
40. The double-stranded antisense agent comprising the chimeric
antisense polynucleotide of claim 31, wherein the chimeric
antisense polynucleotide can hybridize to a cellular transcription
product in a 100 mM sodium chloride, 10 mM sodium phosphate buffer,
pH 7.2, at 25.degree. C.
41. A pharmaceutical composition comprising the chimeric antisense
polynucleotide of claim 1, and a pharmaceutically acceptable
carrier.
42. A method of modifying the function of a transcription product
in a cell comprising the step of administering to the cell a
composition comprising the chimeric antisense polynucleotide of
claim 1.
43. A method of changing the expressed level of a protein in a cell
comprising the step of administering to the cell a composition
comprising the chimeric antisense polynucleotide of claim 1.
44. A method of changing a protein structure in a cell comprising
the step of administering to the cell a composition comprising the
chimeric antisense polynucleotide of claim 1.
45. A method for treating a patient having a condition
characterized by changing expression level, function or editing of
a target gene, comprising: administering to said patient a
therapeutically effective amount of a pharmaceutical composition
comprising (a) at least one chimeric antisense polynucleotide of
claim 1; and (b) a pharmaceutically acceptable carrier.
Description
TECHNICAL FIELD
[0001] The present application relates to a chimeric
single-stranded polynucleotide and double-stranded antisense agent
useful for modifying the expression of a target gene by means of an
antisense effect. The chimeric single-stranded antisense
polynucleotide comprises a central nucleotide region flanked by a
first 5'-wing region and a first 3'-wing region of modified
nucleotides, which are themselves flanked by a second 5'-wing
region and/or a second 3'-wing region of nucleotides that have a
low affinity for proteins and/or that have higher resistance to
DNase or RNase than a natural DNA or RNA and are missing in a cell
when the chimeric polynucleotide delivered. The double-stranded
antisense agent comprises the chimeric single-stranded
polynucleotide. The double-stranded antisense agent further
comprises a complementary strand. The chimeric single-stranded
polynucleotide and double-stranded antisense agent can be used to
modify RNA transcription levels or protein levels in cells.
BACKGROUND ART
[0002] In recent years, oligonucleotides have been a subject of
interest in the on-going development of pharmaceutical products
called nucleic acid drugs, and particularly, from the viewpoints of
high selectivity of target gene and low toxicity, the development
of nucleic acid drugs utilizing an antisense method is actively
underway. The antisense method includes methods of selectively
modifying or inhibiting the expression of a protein that is encoded
by a target gene, by introducing into a cell an oligonucleotide
(antisense oligonucleotide (ASO) that is complementary to a partial
sequence of the mRNA (sense strand) of a target gene. Similarly,
antisense methods also target miRNA and operate to modify the
activity of such miRNA.
[0003] As illustrated in FIG. 1 (upper portion), when an
oligonucleotide comprising RNA is introduced into a cell as an ASO,
the ASO binds to a transcription product (mRNA) of the target gene,
and a partial double strand is formed. It is known that this double
strand plays a role as a cover to prevent translation by a
ribosome, and thus the expression of the protein encoded by the
target gene is inhibited.
[0004] On the other hand, when an oligonucleotide comprising a DNA
is introduced into a cell as an ASO, a partial DNA-RNA
hetero-duplex is formed. Because this structure is recognized by
RNase H, and the mRNA of the target gene is thereby decomposed, the
expression of the protein encoded by the target gene is inhibited.
(FIG. 1, lower portion). In many cases, the gene expression
suppressing effect is higher when DNA is used as an ASO (RNase
H-dependent route), as compared with the case of using an RNA
ASO.
[0005] When utilizing an oligonucleotide as a nucleic acid drug,
various nucleic acid analogs such as Locked Nucleic Acid (LNA)
(registered trademark), other bridged nucleic acids (BNA), and the
like have been developed to enhance binding affinity to target RNA
and stability in vivo.
[0006] As illustrated in FIG. 2, since the sugar moiety of a
natural nucleic acid (RNA or DNA) has a five-membered ring with
four carbon atoms and one oxygen atom, the sugar moiety has two
kinds of conformations, an N-form and an S-form. It is known that
these conformations swing from one to the other, and thereby, the
helical structure of the nucleic acid also adopts different forms,
an A-form and a B-form. Since the mRNA that serves as the target of
the aforementioned ASO adopts a helical structure in the A-form,
with the sugar moiety being mainly in the N-form, it is important
for the sugar moiety of the ASO to adopt the N-form from the
viewpoint of increasing the affinity to RNA. A product that has
been developed under this concept is a modified nucleic acid such
as a LNA (2'-0,4'-C-methylene-bridged nucleic acid (2',4'-BNA)).
For example, in the LNA, as the oxygen at the 2'-position and the
carbon at the 4'-position are bridged by a methylene group, the
conformation is fixed to the N-form, and there is no more
fluctuation between the conformations. Therefore, an
oligonucleotide synthesized by incorporating several units of LNA
has very high affinity to RNA and very high sequence specificity,
and also exhibits excellent heat resistance and nuclease
resistance, as compared with oligonucleotides synthesized with
conventional natural nucleic acids (see Patent Document 1). Since
other artificial nucleic acids also have such characteristics, much
attention has been paid to artificial nucleic acids in connection
with the utilization of an antisense method and the like (see
Patent Documents 1 to 9).
[0007] Nonetheless, the designs of antisense oligonucleotides using
even these high-performance modified nucleic acids still lack
suitable efficiency, potency, and/or safety for use as therapeutic
agents.
[0008] It is known that the length of an ASO dramatically affects
the performance of ASO's, but with two contrasting results.
(Non-Patent Documents 5-7.) A longer probe length (e.g., 16-22
nucleotides) provides higher binding strength (higher Tm) and
greater specificity for the targeted RNA sequence. The greater
sequence specificity generally results in fewer side effects and
little or no toxicity. However, the potency of longer probes is
low. To compensate for such low activity, large doses are
required.
[0009] In contrast, shorter probe lengths (e.g., 12-15 nucleotides)
are the most potent--this length range generally provides the
highest degree of suppression or inhibition of gene expression
levels. The shorter probes, though, have lower sequence specificity
and thus generally cause toxic side reactions.
[0010] Accordingly, there exists a need for polynucleotides that
provide the efficiency and potency of a shorter ASO yet also have
the safety of a longer ASO.
[0011] Furthermore, when an antisense oligonucleotide is used as a
drug, it is important that the relevant oligonucleotide can be
delivered to the target site with high specificity and high
efficiency. Methods for delivering an oligonucleotide include using
lipids such as cholesterol and vitamin E (Non-Patent Documents 1
and 2), using a receptor-specific peptide such as RVG-9R
(Non-Patent Document 3), or using an antibody specific to the
target site (Non-Patent Document 4).
CITATION LIST
Patent Literature
[0012] {PTL 1} JP 10-304889 A
[0013] {PTL 2} WO 2005/021570
[0014] {PTL 3} JP 10-195098 A
[0015] {PTL 4} JP 2002-521310 W
[0016] {PTL 5} WO 2007/143315
[0017] {PTL 6} WO 2008/043753
[0018] {PTL 7} WO 2008/029619
[0019] {PTL 8} WO 2003/011887
[0020] {PTL 9} WO 2007/131238
Non Patent Literature
[0021] {NPL 1} Kazutaka Nishina et al., Molecular Therapy, Vol. 16,
734-740 (2008)
[0022] {NPL 2} Jurgen Soutscheck et al., Nature, Vol. 432, 173-178
(2004)
[0023] {NPL 3} Kazutaka Nishina et al., Molecular Therapy, Vol. 16,
734-740 (2008)
[0024] {NPL 4} Dan Peer et al., Science, Vol. 319, 627-630
(2008)
[0025] {NPL 5} Ellen Marie Straarup et al., Nucleic Acids Research,
Vol. 38(20), 7100-7111 (2010)
[0026] {NPL 6} Tsuyoshi Yamamoto et al., J. Nucleic Acids, Article
ID 707323, 7 pages (2012)
[0027] {NPL 7} Jan Stenvang et al., Silence, Vol. 3:1, 17 pages
(2012)
SUMMARY OF INVENTION
[0028] A new chimeric antisense polynucleotide and the
double-stranded antisense agent are provided which retains the
efficacy and potency of shorter (.about.13 bases) polynucleotides
despite having an increased length. The new chimeric antisense
oligonucleotide comprises a short gapmer antisense oligonucleotide
to which additional nucleotides are added at the 5' end, at the 3'
end, or at both ends of the gapmer. The additional nucleotides
display low affinity for protein binding. Otherwise, the additional
nucleotides have higher DNase/RNase resistance than a natural DNA
or RNA nucleotide and are missing in a cell when the chimeric
antisense polynucleotide is delivered. The double-stranded
antisense agent comprises the chimeric antisense polynucleotide.
Thus, for example, a 13 base gapmer can be redesigned, according to
the disclosure herein, as a 20 base gapmer that adds wing regions
at one or both ends comprising nucleotides that display low binding
affinity for proteins and protein-like cellular components, have
higher DNase/RNase resistance than a natural DNA or RNA nucleotide
and/or are missing in a cell when the chimeric antisense
polynucleotide is delivered. The gapmer portion of the chimeric
antisense strand, that is, the center region, the first 5'-wing
region, and the first 3'-wing region may be any of the antisense
strands described in PCT/JP2012/083180, entitled "Chimeric
Double-Stranded Nucleic Acid," which is incorporated herein by
reference in its entirety.
[0029] The inventors have determined that the new chimeric
single-stranded polynucleotide and the double-stranded antisense
agent, when introduced into a cell, can modify the activity or
function of a transcription product. The transcription product may
be a protein-encoding transcription product or a
non-protein-encoding product such as miRNA. The application further
contemplates methods for altering the expressed level of a protein
in a cell, and for changing a protein structure by means of an
antisense effect.
[0030] The chimeric antisense polynucleotide and the
double-stranded antisense agent are also useful for treating
patients having a condition characterized by an altered gene
expression level, such that, for example, a protein is
overexpressed. By treating the patient with a pharmaceutical
composition comprising the chimeric antisense polynucleotide or the
double-stranded antisense agent, the gene expression level can be
specifically suppressed or inhibited to a degree that the protein
levels decrease, thereby ameliorating the condition.
[0031] In certain embodiments, the following are provided.
(1) A chimeric antisense polynucleotide comprising: a center
nucleotide region comprising at least 5 nucleotides; a first
5'-wing region joined to the 5' end of the center nucleotide region
comprising 1-10 nucleotides wherein at least 1 is a nucleotide
analog; a first 3'-wing region joined to the 3' end of the center
nucleotide region comprising 1-10 nucleotides wherein at least 1 is
a nucleotide analog; and a second 5'-wing region and/or a second
3'-wing region, wherein: the second 5'-wing region is joined to the
5' end of the first 5'-wing region and comprises at least 1 low
protein-affinity nucleotide; and the second 3'-wing region is
joined to the 3' end of the first 3'-wing region and comprises at
least 1 low protein-affinity nucleotide; wherein the total number
of nucleotides, nucleotide analogs and low protein-affinity
nucleotides is no more than 100 nucleotides. (2) A chimeric
antisense polynucleotide comprising: a center nucleotide region
comprising at least 5 nucleotides; a first 5'-wing region joined to
the 5' end of the center nucleotide region comprising 1-10
nucleotides wherein at least 1 is a nucleotide analog; a first
3'-wing region joined to the 3' end of the center nucleotide region
comprising 1-10 nucleotides wherein at least 1 is a nucleotide
analog; and a second 5'-wing region and/or a second 3'-wing region,
wherein: the second 5'-wing region is joined to the 5' end of the
first 5'-wing region, has higher resistance to DNase or RNase than
a natural DNA or RNA and is missing in a cell when the chimeric
polynucleotide delivered; and the second 3'-wing region is joined
to the 3' end of the first 3'-wing region, has higher resistance to
DNase or RNase than a natural DNA or RNA and is missing in a cell
when the chimeric polynucleotide delivered, wherein the total
number of nucleotides and nucleotide analogs is no more than 100
nucleotides. (3) The chimeric antisense polynucleotide of items (1
or 2), wherein the center nucleotide region comprises nucleotides
that, when hybridized to an RNA polynucleotide, the center
nucleotide region/RNA polynucleotide duplex is recognized by RNase
H. (4) The chimeric antisense polynucleotide of item (3), wherein
the nucleotides of the center nucleotide region are independently
selected from DNA and phosphorothioate DNA nucleotides. (5) The
chimeric antisense polynucleotide of item (4), wherein the center
nucleotide region comprises 5-20 DNA nucleotides. (6) The chimeric
antisense polynucleotide of item (4), wherein the center nucleotide
region comprises 5-12 DNA nucleotides. (7) The chimeric antisense
polynucleotide of items (1 or 2), wherein the center nucleotide
region comprises nucleotides independently selected from RNA
nucleotides and nucleotide analogs. (8) The chimeric antisense
polynucleotide of item (7), wherein the nucleotide analogs are
independently selected from LNA nucleotides, BNA nucleotides,
2'-O-Me RNA nucleotides, 2'-O-methoxyethyl RNA nucleotides. (9) The
chimeric antisense polynucleotide of item (8), wherein the center
nucleotide region comprises nucleotides independently selected from
2'-O-Me RNA nucleotides and 2'-O-methoxyethyl RNA nucleotides. (10)
The chimeric antisense polynucleotide of any one of items (1-9),
wherein at least one of the nucleotides in the center nucleotide
region is phosphorothioated. (11) The chimeric antisense
polynucleotide of item (10), wherein all nucleotides in the center
nucleotide region are phosphorothioated. (12) The chimeric
antisense polynucleotide of any one of items (1-11), wherein the
nucleotides in the first wing regions are bridged nucleotides. (13)
The chimeric antisense polynucleotide of any one of items (1-12),
wherein the bridged nucleotides are independently selected from
LNA, cEt BNA, amideBNA, and cMOE BNA. (14) The chimeric antisense
polynucleotides of any one of items (1-13), wherein at least one of
the nucleotide analogs in the center nucleotide region and the
first wing region(s) are phosphorothioated. (15) The chimeric
antisense polynucleotide of any one of items (1-14), wherein the
low protein-affinity nucleotides are independently selected from
2'-O-methyl RNA nucleotides, 2'-O-methoxyethyl RNA nucleotides,
LNA, cMOE BNA, 2-fluoro RNA nucleotides, boranophosphate
nucleotides, methylphosphonate nucleotides, phosphoramidite
nucleotides, 5-methylcytosine, UNA and 5-propynyluridine. (16) The
chimeric antisense polynucleotide of any one of items (1-15),
wherein the chimeric antisense polynucleotide includes the second
5'-wing region. (17) The chimeric antisense polynucleotide of any
one of items (1-15), wherein the chimeric polynucleotide includes
the second 3'-wing region. (18) The chimeric antisense
polynucleotide of any one of items (1-15), wherein the chimeric
antisense polynucleotide includes the second 5'-wing region and the
second 3'-wing region. (19) The chimeric antisense polynucleotide
of any one of items (1-18), wherein the chimeric antisense
polynucleotide further comprises a functional moiety joined to the
3'-end and/or the 5'-end of the chimeric antisense polynucleotide.
(20) The chimeric antisense polynucleotide of item (19), wherein
the functional moiety has a function selected from a labeling
function, a purification function, and a targeted delivery
function. (21) The chimeric antisense polynucleotide of item (19),
wherein the functional moiety is joined to the chimeric antisense
polynucleotide via a cleavable linker moiety. (22) The chimeric
antisense polynucleotide of item (19), wherein the functional
moiety is a molecule selected from a lipid, a peptide, and a
protein. (23) The chimeric antisense polynucleotides of item (22),
wherein the lipid is selected from cholesterol, a fatty acid, a
lipid-soluble vitamin, a glycolipid, and a glyceride. (24) The
chimeric antisense polynucleotides of item (22), wherein the lipid
is selected from cholesterol, a tocopherol, and a tocotrienol. (25)
The chimeric antisense polynucleotides of item (22), wherein the
peptide is selected from a receptor ligand fragment and an antibody
fragment. (26) The chimeric antisense polynucleotide of item (22),
wherein the protein is selected from a receptor ligand and an
antibody. (27) The chimeric antisense polynucleotide of item (1 or
2), wherein the chimeric antisense polynucleotide can hybridize to
a cellular transcription product in a 100 mM sodium chloride, 10 mM
sodium phosphate buffer, pH 7.2, at 25 degrees C. (28) The chimeric
antisense polynucleotide of item (27), wherein the center region is
fully complementary to the cellular transcription product to which
the chimeric antisense polynucleotide can hybridize. (29) The
chimeric antisense polynucleotide of item (27), wherein the second
5'-wing region and/or the second 3'-wing region comprise at least
one mismatched base when the chimeric antisense polynucleotide
hybridizes to the cellular transcription product to which the
chimeric antisense polynucleotide can hybridize. (30) The chimeric
antisense polynucleotide of item (29), wherein all bases of the
second 5'-wing region and/or the second 3'-wing region are
mismatched. (31) A double-stranded antisense agent comprising the
chimeric antisense polynucleotide of any one of items (1-18) and
(27-30) and a complementary strand annealed to the chimeric
antisense polynucleotide. (32) The double-stranded antisense agent
comprising the chimeric antisense polynucleotide of item (31),
wherein the complementary strand further comprises a functional
moiety joined to the 3'-end and/or the 5'-end. (33) The
double-stranded antisense agent of item (32), wherein the
functional moiety has a function independently selected from a
labeling function, a purification function, and a targeted delivery
function. (34) The double-stranded antisense agent of item (32),
wherein the functional moiety is independently joined to the
chimeric antisense polynucleotide and/or the complementary strand
via a cleavable linker moiety. (35) The double-stranded antisense
agent of item (32), wherein the functional moiety is a molecule
independently selected from a lipid, a peptide, and a protein. (36)
The double-stranded antisense agent of item (35), wherein the lipid
is independently selected from cholesterol, a fatty acid, a
lipid-soluble vitamin, a glycolipid, and a glyceride. (37) The
double-stranded antisense agent of item (35), wherein the lipid is
independently selected from cholesterol, a tocopherol, and a
tocotrienol. (38) The double-stranded antisense agent of item (35),
wherein the peptide is independently selected from a receptor
ligand fragment and an antibody fragment. (39) The double-stranded
antisense agent of item (35), wherein the protein is independently
selected from a receptor ligand and an antibody. (40) The
double-stranded antisense agent comprising the chimeric antisense
polynucleotide of item (31), wherein the chimeric antisense
polynucleotide can hybridize to a cellular transcription product in
a 100 mM sodium chloride, 10 mM sodium phosphate buffer, pH 7.2, at
25 degrees C. (41) A pharmaceutical composition comprising the
chimeric antisense polynucleotide of any one of items (1-30) or the
double-stranded antisense agent of any one of items (31-40), and a
pharmaceutically acceptable carrier. (42) A method of modifying the
function of a transcription product in a cell comprising the step
of administering to the cell a composition comprising the chimeric
antisense polynucleotide of any one of items (1-30) or the
double-stranded antisense agent of any one of items (31-40). (43) A
method of changing the expressed level of a protein in a cell
comprising the step of administering to the cell a composition
comprising the chimeric antisense polynucleotide of any one of
items (1-30) or the double-stranded antisense agent of any one of
items (31-40). (44) A method of changing a protein structure in a
cell comprising the step of administering to the cell a composition
comprising the chimeric antisense polynucleotide of any one of
items (1-30) or the double-stranded antisense agent of any one of
items (31-40). (45) A method for treating a patient having a
condition characterized by changing expression level, function or
editing of a target gene, comprising: administering to said patient
a therapeutically effective amount of a pharmaceutical composition
comprising (a) at least one chimeric antisense polynucleotide of
any one of items (1-30) or the double-stranded antisense agent of
any one of items (31-40); and (b) a pharmaceutically acceptable
carrier.
[0032] According to certain embodiments, a chimeric antisense
polynucleotide can be delivered to a target site with high
specificity and high efficiency by associating a delivery moiety
with the complex. According to certain embodiments, a
double-stranded antisense agent can be delivered to a target site
with high specificity and high efficiency by associating a
functional moiety, e.g., a delivery moiety, with the
double-stranded complex.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIG. 1 is a diagram illustrating the general mechanisms of
certain antisense methods. As illustrated in the diagram, when an
oligonucleotide (antisense oligonucleotide (ASO)) ("DNA" in the
diagram) that is complementary to a partial sequence of the mRNA of
a target gene is introduced into a cell, the expression of a
protein that is encoded by the target gene is selectively
inhibited. In the dashed box, a degradation mechanism is shown in
which RNase H cleaves mRNA at a location at which it is hybridized
to an ASO. As a result of RNase H cleavage, the mRNA generally will
not be translated to produce a functional gene expression
product.
[0034] FIG. 2 is a schematic diagram illustrating the structures of
RNA, DNA, and an LNA nucleotide.
[0035] FIGS. 3A-C are schematic diagrams illustrating examples of
suitable embodiments of chimeric polynucleotides. The core
5'-(1.sup.st 5' wing)(center region)(1.sup.st 3' wing)-3' may be
joined with both a (2.sup.nd 5' wing) and a (2.sup.nd 3' wing)
(FIG. 3A), only a (2.sup.nd 5' wing) (FIG. 3B), or only a (2.sup.nd
3' wing) (FIG. 3C).
[0036] FIGS. 4A-D are schematic diagrams illustrating examples of
suitable embodiments of chimeric polynucleotides. The chimeric
polynucleotide 5'-(2.sup.nd 5' wing)(1.sup.st 5' wing)(center
region)(1.sup.st 3' wing)(2.sup.nd 3' wing)-3' strands are
antisense nucleic acids that can hybridize to targeted RNA strands,
such as a transcription product. In the illustrations, "X"
represents a functional moiety, and may independently represent a
lipid (for example, cholesterol or tocopherol), a sugar or the
like, or a protein, a peptide (for example, an antibody) or the
like.
[0037] FIG. 5 shows the structural formula of various natural and
non-natural nucleic acid moieties.
[0038] FIGS. 6A-6D are schematic diagrams illustrating designs for
chimeric winged antisense oligonucleotides that incorporate
sequence mismatches into the 5' and 3' second wing regions as a
means for tuning the potency of the antisense effect.
[0039] FIG. 7 shows a graph illustrating the results obtained upon
administering to Hep 1-6 cells "20mer29PS," "20mer12PS," and
"13mer12PS," all of which have a sequence complementary to the base
sequence of ApoB1 gene, and analyzing the amount of expression of
ApoB1 gene in the cells by quantitative PCR, the sequence of each
antisense polynucleotide, and a schematic illustration comparing
the design of each polynucleotide.
[0040] FIG. 8A shows the sequence of two gapmer antisense
polynucleotides and three chimeric winged antisense
polynucleotides, and a schematic illustration comparing the design
of each polynucleotide.
[0041] FIG. 8B shows a graph illustrating the results obtained upon
administering to Hep 1-6 cells "20mer gapmer," "20mer chimera 5'/3'
wings," "20mer chimera 3' wing," "20mer chimera 5' wing," and
"13mer gapmer," all of which have a sequence complementary to the
base sequence of ApoB1 gene, and analyzing the amount of expression
of ApoB1 gene in the cells by quantitative PCR.
[0042] FIG. 9 is a graph illustrating the results obtained by
administering the chimeric polynucleotides and control
polynucleotides, shown in the upper portion of the figure, to mice,
and analyzing the amounts of expression of ApoB1 gene, whose
transcription product is targeted by the antisense strand, in the
mice.
[0043] FIG. 10 shows a result of Northern blot analysis. The
hybridized signals of T17 and T20(-) missing in liver to length of
13mer were detected (lower right panel) The mouse U6 (internal
control) is shown in upper panel.
[0044] FIG. 11 illustrates relative antisense effect of various
chimeric single-stranded polynucleotides.
[0045] FIG. 12 indicates ApoB mRNA expression level normalized by
Gapdh mRNA expression level compared to control 13mer.
[0046] FIG. 13 shows comparison or time course of antisense effect
of 13mer and Toc-20mer-8U PS(-) single-stranded
polynucleotides.
[0047] FIG. 14 indicates effect of various chimeric single-stranded
polynucleotides on lipid level (LDL-Cholesterol, total cholesterol
and triglyceride).
[0048] FIG. 15 presents schematic diagrams of an embodiment of a
chimeric antisense oligonucleotide and example embodiments of
suitable complementary strands.
[0049] FIG. 16A shows the sequence of gapmer antisense
polynucleotides and a chimeric antisense polynucleotide targeting
ApoB1, and complementary nucleotides used to form double-stranded
antisense agents with the antisense polynucleotides.
[0050] FIG. 16B is a graph illustrating the results obtained by
administering the double-stranded complexes shown in FIG. 16A, to
mice, and analyzing the amounts of expression of ApoB1 gene, whose
transcription product is targeted by the antisense strand, in the
mice.
DESCRIPTION OF EMBODIMENTS
[0051] Chimeric single-stranded polynucleotides comprising the
following structure are active in modifying or suppressing the
expression of a target gene or more generally the level of a
transcription product, by means of an antisense effect:
[0052] a center nucleotide region comprising at least 5
nucleotides;
[0053] a first 5'-wing region joined to the 5' end of the center
nucleotide region comprising 1-10 nucleotide analogs;
[0054] a first 3'-wing region joined to the 3' end of the center
nucleotide region comprising 1-10 nucleotide analogs; and
[0055] a second 5'-wing region and/or a second 3'-wing region,
wherein:
[0056] the second 5'-wing region is joined to the 5' end of the
first 5'-wing region and comprises at least 1 low protein-affinity
nucleotide; and
[0057] the second 3'-wing region is joined to the 3' end of the
first 3'-wing region and comprises at least 1 low protein-affinity
nucleotide;
[0058] wherein the total number of nucleotides, nucleotide analogs,
and low protein-affinity nucleotides is no more than 100
nucleotides.
[0059] Otherwise, chimeric single-stranded polynucleotides
comprising the following structure are active in modifying or
suppressing the expression of a target gene or more generally the
level of a transcription product, by means of an antisense
effect:
[0060] a center nucleotide region comprising at least 5
nucleotides;
[0061] a first 5'-wing region joined to the 5' end of the center
nucleotide region comprising 1-10 nucleotides wherein at least 1 is
a nucleotide analog;
[0062] a first 3'-wing region joined to the 3' end of the center
nucleotide region comprising 1-10 nucleotides wherein at least 1 is
a nucleotide analog; and
[0063] a second 5'-wing region and/or a second 3'-wing region,
wherein:
[0064] the second 5'-wing region is joined to the 5' end of the
first 5'-wing region, has higher resistance to DNase or RNase than
a natural DNA or RNA and is missing in a cell when the chimeric
polynucleotide delivered; and
[0065] the second 3'-wing region is joined to the 3' end of the
first 3'-wing region, has higher resistance to DNase or RNase than
a natural DNA or RNA and is missing in a cell when the chimeric
polynucleotide delivered,
[0066] wherein the total number of nucleotides and nucleotide
analogs is no more than 100 nucleotides.
[0067] Certain embodiments include a purified or isolated
double-stranded antisense agent comprising the chimeric single
stranded antisense polynucleotide above.
[0068] In some embodiments, the center nucleotide region of the
antisense strand comprises at least 4 consecutive nucleotides, or
in some embodiments at 5 least consecutive nucleotides, that are
recognized by RNase H when the antisense strand is hybridized to a
transcription product.
[0069] The complementary strand comprises nucleotides and
optionally nucleotide analogs, and optionally a low
protein-affinity nucleotide, and the complementary strand can
anneal to the antisense strand.
[0070] In some embodiments the complementary strand comprises:
[0071] (i) an RNA nucleotide and optionally a nucleotide analog,
and optionally a low protein-affinity nucleotide, and optionally a
DNA nucleotide; or
[0072] (ii) a DNA nucleotide and/or a nucleotide analog and/or a
low protein-affinity nucleotide; or
[0073] (iii) PNA nucleotides.
[0074] The "antisense effect" means suppressing the expression of a
target gene or the level of a targeted transcription product, which
occurs as a result of hybridization of the targeted transcription
product (RNA sense strand) with, for example, a DNA strand, or more
generally, a strand designed to cause the antisense effect,
complementary to a partial sequence of the transcription product or
the like. In certain instances, inhibition of translation or a
splicing function modifying effect such as exon skipping (see the
Description in the upper part outside the area surrounded by dotted
lines in FIG. 1) may be caused by covering of the transcription
product by the hybridization product, and/or decomposition of the
transcription product may occur as a result of recognition of the
hybridized portion (see the Description within the area surrounded
by dotted lines in FIG. 1).
[0075] The "target gene" or "targeted transcription product" whose
expression is suppressed by the antisense effect is not
particularly limited, and examples thereof include genes whose
expression is increased in various diseases. Also, the
"transcription product of the target gene" is an mRNA transcribed
from the genomic DNA that encodes the target gene, and also
includes an mRNA that has not been subjected to base modification,
a mRNA precursor that has not been spliced, and the like. More
generally, the "transcription product" may be any RNA synthesized
by a DNA-dependent RNA polymerase.
[0076] As used herein, the term "nucleic acid" may refer to a
monomeric nucleotide or nucleoside, or may mean an oligonucleotide
consisting of plural monomers. The term "polynucleotide" and
"nucleic acid strand" is also used herein to refer to an
oligonucleotide. Nucleic acid strands may be prepared in whole or
in part by chemical synthesis methods, including using a automated
synthesizer or by enzymatic processes, including but not limited to
polymerase, ligase, or restriction reactions.
[0077] The term "chimeric polynucleotide" as used herein means a
polynucleotide that comprises at least one natural nucleotide
(e.g., DNA or RNA nucleotide) and at least one non-natural
nucleotide (e.g., LNA, 2'-O-methyl RNA), or is comprised entirely
of non-natural nucleotides, and as a result is a polynucleotide
that does not occur in nature. The chimeric polynucleotide
according to certain embodiments is an antisense oligonucleotide
complementary to a transcription product, such as that of a target
gene.
[0078] The term "chimeric antisense polynucleotide" or "chimeric
ASO" means a chimeric polynucleotide that comprises a sequence
complementary to the targeted transcription product.
[0079] The term "double-stranded antisense agent" means a
double-stranded polynucleotide complex that can cause an antisense
effect. A double-stranded antisense agent can comprise two or more
polynucleotide strands. In some embodiments the strands are
annealed. In some embodiments, the agent comprises two strands, an
antisense strand and a complementary strand, the two of which can
anneal to form a double-stranded complex. The antisense strand is a
chimeric polynucleotide (described below). The complementary strand
is, in some embodiments, a chimeric polynucleotide, and in other
embodiments consists of natural nucleotides, and in other
embodiments comprises peptide nucleic acid units.
[0080] The term "complementary" as used herein means a relationship
in which so-called Watson-Crick base pairs (natural type base pair)
or non-Watson-Crick base pairs (Hoogsteen base pairs and the like)
can be formed via hydrogen bonding. It is not necessary that the
base sequence of the targeted transcription product, e.g., the
transcription product of a target gene, and the base sequence of
the chimeric antisense polynucleotide be perfectly complementary,
and it is acceptable if the base sequences have a complementarity
of at least 70% or higher, preferably 80% or higher, and more
preferably 90% or higher (for example, 95%, 96%, 97%, 98%, or 99%
or higher). The complementarity of sequences can be determined by
using a BLAST program or the like. A first strand can be "annealed"
or "hybridized" to a second strand when the sequences are
complementary. A person of ordinary skill in the art can readily
determine the conditions (temperature, salt concentration, etc.)
under which two strands can be annealed, taking into account the
degree of complementarity between the strands. Also, a person
having ordinary skill in the art can readily design an antisense
nucleic acid complementary to the targeted transcription product
based on the information of the base sequence of, e.g., the target
gene.
[0081] The strand length of the chimeric antisense polynucleotide
is not particularly limited, but the strand length is usually at
least 8 bases, at least 10 bases, at least 12 bases, or at least 13
bases. The strand length may be up to 20 bases, 25 bases, or 35
bases. The strand length may even be as long as about 100 bases.
Ranges of the length may be 10 to 35 bases, 12 to 25 bases, or 13
to 20 bases. In certain instances, the choice of length generally
depends on a balance of the strength of the antisense effect with
the specificity of the nucleic acid strand for the target, among
other factors such as cost, synthetic yield, and the like.
[0082] In some embodiments, the choice of the length of the
chimeric ASO and the degree of complementarity between it and the
target strand may also depend on the binding affinity between the
chimeric ASO and the target strand after it has been cleaved by
RNase H. The potency of an ASO depends both on the binding affinity
of the ASO with the target prior to cleavage as well as the
off-rate for the cleaved material to dissociate from the ASO so it
is freed to bind to another target strand.
[0083] The chimeric antisense polynucleotide comprises a "center
region," a "first 5'-wing region," and a "first 3'-wing region."
The chimeric polynucleotide further comprises a "second 5'-wing
region" or a "second 3'-wing region," or both. These regions are
further explained below. The assignment of a particular nucleotide
to one such region or another is not exclusive. There may be
several ways to assign the nucleotides of a given polynucleotide to
the different regions. These region names are for convenience,
although it should be apparent that by preparing a sequence wherein
the nucleotides provide such regions the polynucleotide will
achieve the functional performance described herein.
[0084] The "center region" comprises both natural and non-natural
nucleotides. The type of nucleotides selected for the center region
largely determines the type of antisense mechanism(s) of action
that are possible. For example, including DNA and/or DNA-like
nucleotides in the center region may lead to the formation of
DNA/RNA heteroduplex structures that can be recognized by RNase H.
Thus, RNase H-dependent mechanism of action is possible in this
case. On the other hand, if DNA and/or DNA-like nucleotides are
excluded from the center region, then RNase H-independent
mechanisms of action are expected to occur. Of course, even if
DNA/RNA heteroduplex structures are formed, RNase H-independent
mechanisms may occur.
[0085] In some embodiments, the center region comprises at least 5
nucleotides that "when hybridized to an RNA polynucleotide, the
center nucleotide region/RNA polynucleotide duplex is recognized by
Rnase H."
[0086] The "at least 5 nucleotides" that when hybridized to RNA are
"recognized by RNase H" is usually a region comprising 5 to 20
consecutive bases, a region comprising 5 to 16 consecutive bases, a
region comprising 5 to 12 consecutive bases, or a region comprising
5 to 8 consecutive bases. Furthermore, nucleotides that may be used
in this region are those that, like natural DNA, are recognized by
RNase H when hybridized to RNA nucleotides, wherein the RNase H
cleaves the RNA strand. Suitable nucleotides, such as modified DNA
nucleotides and other bases are known in the art. Nucleotides that
contain a 2'-hydroxy group, like an RNA nucleotide are known to not
be suitable. One of skill in the art can readily determine the
suitability of a nucleotide for use in this region of "at least 5
nucleotides" when an RNase H-dependent effect is desired. In one
embodiment, the nucleotides of the center nucleotide region are
independently selected from DNA and phosphorothioate DNA
nucleotides.
[0087] In some embodiments, the center region may comprises as few
as 4 nucleotides that when hybridized to an RNA polynucleotide, the
center nucleotide region/RNA polynucleotide duplex is recognized by
RNase H.
[0088] In other embodiments, the center region does not comprise
DNA. That is, in certain embodiments, an antisense nucleic acid has
an RNase H-independent effect, or, alternatively, a non-RNase
H-dependent antisense effect. The "non-RNase H-dependent antisense
effect" means an activity of suppressing the expression of a target
gene that occurs as a result of inhibition of translation or a
splicing function modifying effect such as exon skipping when a
transcription product of the target gene (RNA sense strand) and a
nucleic acid strand that is complementary to a partial sequence of
the transcription product are hybridized (see the Description of
the upper part outside the area surrounded by dotted lines in FIG.
1).
[0089] The "nucleic acid that does not comprise DNA" means an
antisense nucleic acid that does not comprise natural DNA and
modified DNA, and an example thereof may be RNA, modified RNA,
nucleotide analogs, a PNA, or a nucleic acid comprising morpholino
nucleic acid. Furthermore, in regard to the "nucleic acid that does
not comprise DNA," a portion or the entirety of the nucleic acid
may be composed of modified nucleotides and/or nucleotide analogs,
from the viewpoint that the resistance to nucleases is high.
Examples of such modification include those described below, and
the same nucleotide may be subjected to plural kinds of
modifications in combination. Furthermore, preferred embodiments
related to the number of modified nucleic acids and the position of
modification can be characterized by measuring the antisense effect
possessed by the chimeric polynucleotide after modification, as
described below.
[0090] It is not necessary that the base sequence of the
nucleotides in the center region and the base sequence of the
transcription product of a target gene be perfectly complementary
to each other, and the base sequences may have a complementarity of
at least 70% or higher, preferably 80% or higher, and more
preferably 90% or higher (for example, 95%, 96%, 97%, 98%, 99% or
higher).
[0091] There are no particular limitations on the length of the
"nucleic acid that does not comprise DNA," but the length is as
described above, and is usually 5 to 35 bases, 5 to 25 bases, 12 to
25 bases, or 13 to 20 bases.
[0092] There are no particular limitations on the length of the
"nucleic acid that does not comprise DNA," but the length is as
described above, and is usually 5 to 35 bases, 5 to 25 bases, 12 to
25 bases, or 13 to 20 bases.
[0093] As used herein, "DNA nucleotide" means a natural DNA
nucleotide, or a DNA nucleotide with a modified base, sugar, or
phosphate linkage subunit. Similarly, "RNA nucleotide" means a
natural RNA nucleotide, or an RNA nucleotide with a modified base,
sugar, or phosphate linkage subunit. A modified base, sugar, or
phosphate linkage subunit is one in which a single substituent has
been added or substituted in a subunit, and the subunit as a whole
has not been replaced with a different chemical group. From the
viewpoint that a portion or the entirety of the region comprising
the nucleotide has high resistance to deoxyribonuclease and the
like, the DNA may be a modified nucleotide. Examples of such
modification include 5-methylation, 5-fluorination, 5-bromination,
5-iodination, and N4-methylation of cytosine; 5-demethylation,
5-fluorination, 5-bromination, and 5-iodination of thymidine;
N6-methylation and 8-bromination of adenine; N2-methylation and
8-bromination of guanine; phosphorothioation, methylphosphonation,
methylthiophosphonation, chiral methylphosphonation,
phosphorodithioation, phosphoroamidation, 2'-O-methylation,
2'-methoxyethyl(MOE)ation, 2'-aminopropyl(AP)ation, and
2'-fluorination. However, from the viewpoint of having excellent
pharmacokinetics, phosphorothioation is preferred. Such
modification may be carried out such that the same DNA may be
subjected to plural kinds of modifications in combination. And, as
discussed below, RNA nucleotides may be modified to achieve a
similar effect.
[0094] In certain instances, the number of modified DNA's and the
position of modification may affect the antisense effect and the
like provided by the chimeric antisense polynucleotide as disclosed
herein. Since these embodiments may vary with the sequence of the
target gene and the like, it may depend on cases, but a person
having ordinary skill in the art can determine suitable embodiments
by referring to the Descriptions of documents related to antisense
methods. Furthermore, when the antisense effect possessed by a
chimeric antisense polynucleotide after modification is measured,
if the measured value thus obtained is not significantly lower than
the measured value of the chimeric antisense polynucleotide before
modification (for example, if the measured value obtained after
modification is lower by 30% or more than the measured value of the
chimeric antisense polynucleotide before modification), the
relevant modification can be evaluated. The measurement of the
antisense effect can be carried out, as indicated in the Examples
below, by introducing a nucleic acid compound under test into a
cell or the like, and measuring the amount of expression (amount of
mRNA, amount of cDNA, amount of a protein, or the like) of the
target gene in the cell in which the expression is suppressed by
the antisense effect provided by the nucleic acid compound under
test, by appropriately using known techniques such as Northern
Blotting, quantitative PCR, and Western Blotting. The candidate
agent may be the chimeric antisense polynucleotide itself, or as
part of a double-stranded antisense agent.
[0095] As used herein, "RNA nucleotide" means a naturally occurring
RNA nucleotide, or an RNA nucleotide with a modified base, sugar,
or phosphate linkage subunit. A modified base, sugar, or phosphate
linkage subunit is one in which a single substituent has been added
or substituted in a subunit, and the subunit as a whole has not
been replaced with a different chemical group.
[0096] A portion or the entirety of the nucleic acid may be a
modified nucleotide, from the viewpoint of having high resistance
to a nuclease such as a ribonuclease (RNase). Examples of such
modification include 5-methylation, 5-fluorination, 5-bromination,
5-iodination and N4-methylation of cytosine; 5-demethylation,
5-fluorination, 5-bromination, and 5-iodination of thymidine;
N6-methylation and 8-bromination of adenine; N2-methylation and
8-bromination of guanine; phosphorothioation, methylphosphonation,
methylthiophosphonation, chiral methylphosphonation,
phosphorodithioation, phosphoroamidation, 2'-O-methylation,
2'-methoxyethyl(MOE)ation, 2'-aminopropyl(AP)lation, cleavage
between 2'-carbon and 3'-carbon of deoxyribose (resulting in `UNA`)
and 2'-fluorination. Also, an RNA nucleotide with a thymidine base
substituted for a uracil base is also contemplated. However, from
the viewpoint of having excellent pharmacokinetics,
phosphorothioation is used. Furthermore, such modification may be
carried out such that the same nucleic acid may be subjected to
plural kinds of modifications in combination. For example, as used
in the Examples described below, the same RNA may be subjected to
phosphorothioation and 2'-O-methylation in order to provide
resistance to enzymatic cleavage.
[0097] As used herein, "nucleotide analog" means a non-naturally
occurring nucleotide, wherein the base, sugar, or phosphate linkage
subunit has more than one substituent added or substituted in a
subunit, or that the subunit as a whole has been replaced with a
different chemical group. An example of an analog with more than
one substitution is a bridged nucleic acid, wherein a bridging unit
has been added by virtue of two substitutions on the sugar ring,
typically linked to the 2' and 4' carbon atoms. In regard to the
first wing according to certain embodiments, from the viewpoint of
increasing the affinity to a partial sequence of the transcription
product of the target gene and/or the resistance of the target gene
to a nuclease, the first wing further comprises a nucleotide
analog. The "nucleotide analog" may be any nucleic acid in which,
owing to the modifications (bridging groups, substituents, etc.),
the affinity to a partial sequence of the transcription product of
the target gene and/or the resistance of the nucleic acid to a
nuclease is enhanced, and examples thereof include nucleic acids
that are disclosed to be suitable for use in antisense methods, in
JP 10-304889 A, WO 2005/021570, JP 10-195098 A, JP 2002-521310 W,
WO 2007/143315, WO 2008/043753, WO 2008/029619, and WO 2008/049085
(hereinafter, these documents will be referred to as "documents
related to antisense methods"). That is, examples thereof include
the nucleic acids disclosed in the documents described above: a
hexitol nucleic acid (HNA), a cyclohexane nucleic acid (CeNA), a
peptide nucleic acid (PNA), a glycol nucleic acid (GNA), a threose
nucleic acid (TNA), a morpholino nucleic acid, a tricyclo-DNA
(tcDNA), a 2'-O-methylated nucleic acid, a 2'-MOE
(2'-O-methoxyethyl)lated nucleic acid, a 2'-AP
(2'-O-aminopropyl)lated nucleic acid, a 2'-fluorinated nucleic
acid, a 2'-F-arabinonucleic acid (2'-F-ANA), and a BNA (bridged
nucleic acid).
[0098] The BNA according to certain embodiments may be any
ribonucleotide or deoxyribonucleotide in which the 2' carbon atom
and 4' carbon atom are bridged by two or more atoms. Examples of
bridged nucleic acids are known to those of skill in the art. One
subgroup of such BNA's can be described as having the carbon atom
at the 2'-position and the carbon atom at the 4'-position bridged
by
4'-(CH.sub.2).sub.p--O-2',4'-(CH.sub.2).sub.p--S-2',4'-(CH.sub.2).sub.p---
OCO-2',4'-(CH.sub.2).sub.n--N(R.sub.3)--O--(CH.sub.2).sub.m-2'
(here, p, m and n represent an integer from 1 to 4, an integer from
0 to 2, and an integer from 1 to 3, respectively; and R.sub.3
represents a hydrogen atom, an alkyl group, an alkenyl group, a
cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a
sulfonyl group, and a unit substituent (a fluorescent or
chemiluminescent labeling molecule, a functional group having
nucleic acid cleavage activity, an intracellular or intranuclear
localization signal peptide, or the like)). Furthermore, in regard
to the BNA according certain embodiments, in the OR.sub.2
substituent on the carbon atom at the 3'-position and the OR.sub.1
substituent on the carbon atom at the 5'-position, R.sub.1 and
R.sub.2 are typically hydrogen atoms, but may be identical with or
different from each other, and may also be a protective group of a
hydroxyl group for nucleic acid synthesis, an alkyl group, an
alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group,
an acyl group, a sulfonyl group, a silyl group, a phosphoric acid
group, a phosphoric acid group protected by a protective group for
nucleic acid synthesis, or --P(R.sub.4)R.sub.5 (here, R.sub.4 and
R.sub.5, which may be identical with or different from each other,
each represent a hydroxyl group, a hydroxyl group protected by a
protective group for nucleic acid synthesis, a mercapto group, a
mercapto group protected by a protective group for nucleic acid
synthesis, an amino group, an alkoxy group having 1 to 5 carbon
atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy
group having 1 to 6 carbon atoms, or an amino group substituted
with an alkyl group having 1 to 5 carbon atoms). Non-limiting
examples of such a BNA include
alpha-L-methyleneoxy(4'-CH.sub.2--O-2') BNA or
beta-D-methyleneoxy(4'-CH.sub.2--O-2') BNA, which are also known as
LNA (Locked Nucleic Acid (registered trademark), 2',4'-BNA),
ethyleneoxy(4'-CH.sub.2).sub.2--O-2') BNA which is also known as
ENA, beta-D-thio(4'-CH.sub.2--S-2') BNA,
aminooxy(4'-CH.sub.2--O--N(R.sub.3)-2') BNA,
oxyamino(4'-CH.sub.2--N(R.sub.3)--O-2') BNA which is also known as
2',4'-BNANc, 2',4'-BNAmc, 3'-amino-2',4'-BNA, 5'-methyl BNA,
(4'-CH(CH.sub.3)--O-2') BNA, which is also known as cEt BNA,
(4'-CH(CH.sub.2OCH.sub.3)--O-2') BNA, which is also known as cMOE
BNA, amideBNA (4'-C(O)--N(R)-2') BNA (R, H, Me), which is also
known as AmNA, and other BNA's known to those of skill in the
art.
[0099] The unlocked nucleic acids (UNA) according to certain
embodiments may be any ribonucleotide or deoxyribonucleotide in
which the covalent bond between 2' carbon atom and 3' carbon atom
are cleaved to give rise to increase flexibility. Examples of
unlocked nucleic acids are known to those of skill in the art.
[0100] Furthermore, in the nucleotide analog, according to certain
embodiments, a base moiety may be modified. Examples of the
modification at a base moiety include 5-methylation,
5-fluorination, 5-bromination, 5-iodination, and N4-methylation of
cytosine; 5-demethylation, 5-fluorination, 5-bromination, and
5-iodination of thymidine; N6-methylation and 8-bromination of
adenine; and N2-methylation and 8-bromination of guanine.
Furthermore, in the modified nucleic acid according to certain
embodiments, a phosphoric acid diester binding site may be
modified. Examples of the modification of the phosphoric acid
diester binding site include phosphorothioation,
methylphosphonation, methylthiophosphonation, chiral
methylphosphonation, phosphorodithioation, and phosphoroamidation.
However, from the viewpoint of having excellent pharmacokinetics,
phosphorothioation may be used, for example, in the central region
and the first wing region. Also, such modification of a base moiety
or modification of a phosphoric acid diester binding site may be
carried out such that the same nucleic acid may be subjected to
plural kinds of modifications in combination.
[0101] Generally, modified nucleotides and modified nucleotide
analogs are not limited to those exemplified herein. Numerous
modified nucleotides and modified nucleotide analogs are known in
art, such as, for example those disclosed in U.S. Pat. No.
8,299,039 to Tachas et al., particularly at col. 17-22, and may be
used in the embodiments of this application. Examples of a natural
nucleotides, modified nucleotides, and nucleotide analogs are shown
in FIG. 5.
[0102] A person having ordinary skill in the art can appropriately
select and use a nucleotide analog among such modified nucleic
acids while taking consideration of the antisense effect, affinity
to a partial sequence of the transcription product of the target
gene, resistance to a nuclease, and the like. However, the
nucleotide analog in some embodiments is a LNA represented by the
following formula (1):
##STR00001##
[0103] In formula (1), "Base" represents an aromatic heterocyclic
group or aromatic hydrocarbon ring group which may be substituted,
for example, a base moiety (purine base or pyrimidine base) of a
natural nucleoside, or a base moiety of a non-natural (modified)
nucleoside, while examples of modification of the base moiety
include those described above; and
[0104] R.sub.1 and R.sub.2, which may be identical with or
different from each other, each represent a hydrogen atom, a
protective group of a hydroxyl group for nucleic acid synthesis, an
alkyl group, an alkenyl group, a cycloalkyl group, an aryl group,
an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a
phosphoric acid group, a phosphoric acid group protected by a
protective group for nucleic acid synthesis, or --P(R.sub.4)R.sub.5
{here, R.sub.4 and R.sub.5, which may be identical or different
from each other, each represent a hydroxyl group, a hydroxyl group
protected by a protective group for nucleic acid synthesis, a
mercapto group, a mercapto group protected by a protective group
for nucleic acid synthesis, an amino group, an alkoxy group having
1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms,
a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group
substituted with an alkyl group having 1 to 5 carbon atoms.
[0105] The compounds shown by the above chemical formulas are
represented as nucleosides, but the "LNA" and more generally, the
BNA according to certain embodiments include nucleotide forms in
which a phosphoric acid derived group is bound to the relevant
nucleoside (nucleotide). In other words, BNA's, such as LNA, are
incorporated as nucleotides in the nucleic strands that comprise
the double stranded nucleic acid complex.
[0106] Furthermore, the nucleotide analog in some embodiments is a
unlocked nucleic acid (UNA) represented by the following formula
(2) and (3):
##STR00002##
[0107] The unlocked nucleic acid (UNA) oligonucleotides are
flexible RNA mimics that enable them to modulate their affinity to
other nucleotides and specificity. The unlocked nucleic acid UNA
are an acyclic-RNA analogues and are also known as 2',3'-seco-RNA
of which the C2'-C3' bond are cleaved. The bound cleavage make them
flexible and give rise to them modulation of the thermodynamic
stability of various nucleic acid.
[0108] The "first 5'-wing region" and the "first 3'-wing region"
are, according to certain embodiments, located on the 5' side and
the 3' side, respectively, and continuously linked to the terminal
nucleotide on the respective ends of the center region.
[0109] The region comprising a nucleotide analog that is disposed
immediately to the 5'-side of the center region (hereinafter, also
called "first 5' wing region") and the region comprising a
nucleotide analog that is disposed immediately to the 3'-side of
the center region (hereinafter, also called "first 3' wing region")
may each independently comprise at least one kind of a nucleotide
analog that is discussed in the documents related to antisense
methods, and may further comprise a natural nucleic acid (DNA or
RNA) in addition to such a nucleotide analog. Furthermore, the
strand lengths of the first 5' wing region and the first 3' wing
region are independently usually 1 to 10 bases, 1 to 7 bases, or 2
to 5 bases.
[0110] Furthermore, there are suitable embodiments of the number of
kinds and position of the nucleotide analog and the natural
nucleotide in the first 5' wing region and the first 3' wing
region, since the number and the position of those nucleic acids
may affect the antisense effect and the like provided by the
double-stranded nucleic acid complex in certain embodiments. Since
these suitable embodiments may vary with the sequence and the like,
it may depend on cases, but a person having ordinary skill in the
art can determine the suitable embodiments by referring to the
Descriptions of documents related to antisense methods.
Furthermore, when the antisense effect possessed by the antisense
strand alone or by the double-stranded antisense agent after
modification is measured in the same manner as in the case of the
region comprising "at least 5 nucleotides," if the measured value
thus obtained is not significantly lower than that of the strand or
the agent before modification, the relevant modification can be
evaluated as a preferred embodiment.
[0111] As noted above, the chimeric antisense polynucleotide
comprises at least one "second wing" region. Some embodiments may
comprise just one second wing region, at either the 5' end or the
3' end of the polynucleotide, or the chimeric antisense
polynucleotide may comprise both a second 5'-wing region and a
second 3'-wing region. These structural designs are illustrated in
FIGS. 3A-3C.
[0112] The "second 5'-wing region" and "second 3'-wing region," if
present, comprise at least 1 low protein-affinity nucleotide.
[0113] A "low protein-affinity nucleotide" is a nucleotide that is
(i) more resistant to DNase or RNase than a natural DNA or RNA
nucleotide and (ii) possesses low affinity for binding to protein
or protein-like cellular components. In particular, the nucleotide
has a lower binding affinity towards proteins than a
phosphorothioated nucleotide. Accordingly, a low protein-affinity
nucleotide is a modified nucleotide or a nucleotide analog as
described above, but the nucleotide is not phosphorothioated.
[0114] Examples of low protein-affinity nucleotides include
2'-O-methyl RNA nucleotides, 2'-O-methoxyethyl RNA nucleotides,
LNA, cMOE BNA, 2-fluoro RNA nucleotides, boranophosphate
nucleotides, methylphosphonate nucleotides, phosphoramidite
nucleotides, 5-methylcytosine, unlocked nucleic acids (UNA) and
5-propynyluridine.
[0115] The second wings as disclosed herein extend the length of
the subject chimeric polynucleotides beyond the length that is
usually desired for ASO's. However, described here and in the
examples below, by including low protein-affinity nucleotides in
the second wing region(s), the performance decrease usually
observed in longer length ASO's is reduced or even eliminated. In
other words, the degree of gene expression inhibition that can be
achieved with the chimeric antisense polynucleotides described
herein approaches the performance of conventional gapmer ASO's that
do not have a second wing region(s) are thus necessarily shorter in
length. And, the degree of gene expression inhibition that can be
achieved with the double-stranded antisense agents described herein
approaches, equals, and in some embodiments exceeds the performance
of conventional single-stranded gapmer ASO's that do not have a
second wing region(s) are thus necessarily shorter in length.
[0116] The length of each second wing region is independent. There
is no particular limitation on the length of each second wing,
other than the overall limitation on the entire length of the
chimeric antisense polynucleotide being no more than 100
nucleotides.
[0117] It may be desirable to adjust the binding affinity of the
bases in the second wing region(s). At one extreme, the nucleotides
in the second wing region(s) can be selected to be fully
complementary to the targeted sequence in the transcription
product. Or, the second wing region(s) sequence can be completely
mismatched with respect to the corresponding target. And, any
combination of matched and mismatched base pairs in the second wing
region(s) is also contemplated. These options are schematically
illustrated in FIGS. 6A-6D. It is known in the art that the binding
affinity is an important consideration in the performance of
antisense inhibition. As discussed in Non-Patent Document 6, there
is an optimum range of binding affinity between an ASO and a target
for achieving maximum potency of the ASO. Accordingly, the length,
base content, and pattern of matched and mismatched base
incorporated into the second 5'-wing region and the second 3'-wing
region can be optimized for use according to the embodiments
disclosed herein.
[0118] The complementary strand according to some embodiments is a
polynucleotide complementary to the chimeric antisense
polynucleotide described above. It is not necessary that the base
sequence of the complementary strand and the base sequence of the
chimeric antisense strand be perfectly complementary to each other,
and the base sequences may have a complementarity of at least 70%
or higher, preferably 80% or higher, and more preferably 90% or
higher (for example, 95%, 96%, 97%, 98%, 99% or higher).
[0119] The complementary strand is a polynucleotide comprising at
least one kind of nucleic acid selected from RNA, DNA, PNA (peptide
nucleic acid) and BNA (e.g., LNA). More specifically, the
complementary strand comprises (i) an RNA nucleotide and optionally
a nucleotide analog, and optionally a low protein-affinity
nucleotide, and optionally a DNA nucleotide; or (ii) a DNA
nucleotide and/or a nucleotide analog and/or a low protein-affinity
nucleotide; or (iii) PNA nucleotides.
[0120] The term "RNA nucleotides and optionally nucleotide analogs,
and optionally a low protein-affinity nucleotide, and optionally a
DNA nucleotide" means that the complementary strand includes RNA
nucleotides, and optionally may further include nucleotide analogs
in the strand, and optionally may further include low
protein-affinity nucleotides in the strand, and optionally may
further include DNA nucleotides in the strand. The term "DNA
nucleotides and/or nucleotide analogs and/or a low protein-affinity
nucleotide" means that the second strand may include either DNA
nucleotides or nucleotide analogs, or low protein-affinity
nucleotides, or may include some combination of DNA nucleotides,
nucleotide analogs, or low protein-affinity nucleotides. The term
"PNA nucleotides" means that the second strand may substantially
comprise PNA nucleotides, although other nucleotides may be
included.
[0121] When the double-stranded antisense agent of certain
embodiments is recognized by RNase H in the cell and the
complementary nucleic acid strand is thus decomposed, releasing the
chimeric antisense strand, the second nucleic acid strand generally
comprises RNA nucleotides. In some embodiments, from the viewpoint
that a functional molecule such as a peptide can be easily bound to
the double-stranded complex, the second nucleic acid strand
comprises PNA.
[0122] The length of the complementary strand is not particularly
limited. The complementary strand may be shorter than, the same
size as, or longer than the chimeric antisense polynucleotide. In
some embodiments, a complementary strand that can anneal to two or
more chimeric antisense polynucleotides is used. The two or more
chimeric antisense strands may target the same sequence or
different sequences.
[0123] In some embodiments, the double-stranded antisense agent
comprises more than two polynucleotides. For example, two antisense
strands may form a double-stranded complex with one complementary
strand. Various multicomponent double-stranded complexes are
disclosed in PCT/JP2012/083180, and those may also be applied to
the double-stranded antisense agents disclosed in this
application.
[0124] In some embodiments, the complementary polynucleotide
comprises in whole or in part modified nucleotides or nucleotide
analogs that are, compared to natural nucleotides, resistant to a
nuclease such as a ribonuclease (RNase). Examples of such
modification include 5-methylation, 5-fluorination, 5-bromination,
5-iodination and N4-methylation of cytosine; 5-demethylation,
5-fluorination, 5-bromination, and 5-iodination of thymidine;
N6-methylation and 8-bromination of adenine; N2-methylation and
8-bromination of guanine; phosphorothioation, methylphosphonation,
methylthiophosphonation, chiral methylphosphonation,
phosphorodithioation, phosphoroamidation, 2'-O-methylation,
2'-methoxyethyl(MOE)ation, 2'-aminopropyl(AP)lation, and
2'-fluorination. Also, an RNA nucleotide with a thymidine base
substituted for a uracil base is also contemplated. The low
protein-affinity nucleotides may also be incorporated in the
complementary strand. However, in some embodiments,
phosphorothioation is used. Furthermore, such modification may be
carried out such that the same nucleic acid may be subjected to
plural kinds of modifications in combination. For example, as used
in the Examples described below, the same RNA may be subjected to
phosphorothioation and 2'-O-methylation in order to provide
resistance to enzymatic cleavage. However, where it is expected or
desired for an RNA nucleotide to be cleaved by RNase H, then only
either phosphorothioation or 2'-O-methylation is generally
applied.
[0125] There are suitable embodiments of the number of nucleotide
analogs and the position of modification within the complementary
strand, since the number and the position of modification may
affect the antisense effect and the like provided by the
double-stranded antisense agent in certain embodiments. Since these
suitable embodiments may vary with the type, sequence and the like
of the nucleic acid to be modified, it may depend on the
circumstances, but the type, sequence and the like can be
characterized by measuring the antisense effect possessed by the
double-stranded antisense agent after modification in the same
manner as described above.
[0126] In some embodiments, from the viewpoint that while the
decomposition by a ribonuclease such as RNase A is suppressed until
the complementary strand is delivered into the nucleus of a
particular cell, the complementary strand can easily exhibit the
antisense effect by being decomposed by RNase H in the particular
cell, the complementary strand generally comprises RNA, and
nucleotides that increase the nuclease stability and/or the T.sub.m
may be incorporated. For example, in the region of the
complementary strand that is complementary to one of the wing
regions of the chimeric antisense strand (i.e., a first or second
5' wing region and/or a first or second 3' wing region), one may
optionally incorporate one or more modified nucleic acids, one or
more nucleotide analogs, and/or one or more low protein-affinity
nucleotides, which serves to suppress decomposition of the complex
by enzymes, such as a ribonuclease.
[0127] According to certain embodiments, the modification is
2'-O-methylation and/or phosphorothioation of RNA. Furthermore, in
some embodiments, the entire region of the complementary strand
that is complementary to one of the wing regions of the antisense
strand may be modified, or a portion of the region that is
complementary to the wing regions of the antisense strand may be
modified. In addition, the region that is modified may be longer
than the region comprising a modified nucleic acid of the first
nucleic acid strand, or may be shorter, as long as the region that
is modified comprises that portion. Examples of some embodiments of
the structure of the complementary strand in relation to the
structure of the antisense strand are shown in FIG. 15. First, the
upper structure is a double wing antisense polynucleotide (DW-ASO).
Below that are four examples of complementary strands. For example,
where it is desired that the double-stranded agent be susceptible
to cleavage by RNase H, then the portion of the complementary
strand that is complementary to the center region preferably
comprises RNA. (RNA(o)=RNA with diphosphate linkage). The portions
of the complementary strand that are complementary to the various
wing regions of the DW-ASO may also comprise modified nucleotides
and/or nucleotide analogues. For example, the double wing structure
may also be included in the complementary strand (cRNA(DW)). Or,
the portions of the complementary strand that are complementary to
the first and second wing regions at each end may comprise modified
nucleotides and/or nucleotide analogues, though not necessarily low
protein-affinity nucleotides (cRNA(G) or cLNA(G); G referring to a
"gapmer" structure). Or, the complementary strand may comprise
peptide nucleic acids. FIG. 6 presents a simplified view of the
structure, and the various complementary regions do not necessarily
have to be in registration. One of skill in art can readily
understand that the structure can be modified without departing
from the desired functionality of the double-stranded antisense
agent, including the functionality of the constituent
polynucleotides. Finally, the complementary strands shown in FIG.
15 include a functional group "X" at the 5' terminus. Such a group
is optionally included, and is described below.
[0128] In the double-stranded antisense agent, one or more of the
constituent polynucleotides may further comprise a functional
moiety.
[0129] In some embodiments, the strand complementary to the
antisense polynucleotide may comprise a functional moiety bonded to
the polynucleotide. Referring back to FIG. 15, a functional moiety
"X" is illustrated joined to the 5'-end of the various examples of
complementary strands. The functional moiety, further described
below, could alternatively be joined at the 3'-end, or at a
position internal to the polynucleotide. In other embodiments, the
complementary strand comprises more than one functional moiety,
which may be joined at a plurality of positions, and/or joined as a
group to one position of the polynucleotide.
[0130] In some embodiments, the chimeric antisense polynucleotide
may comprise a functional moiety bonded to the polynucleotide. For
example, the functional moiety may be joined to the 5'-terminal
nucleotide, or the 3'-terminal nucleotide, or to both the 5' and 3'
terminal nucleotides of the chimeric antisense polynucleotide, as
illustrated in FIGS. 4B, 4C, and 4D. Of course, the functional
moiety could also be joined to an internal position, and more than
one moiety could be joined, as described above.
[0131] In the chimeric antisense polynucleotide of certain
embodiments, a functional moiety may be bonded to the
polynucleotide. The bonding between the polynucleotide (e.g.,
antisense polynucleotide or complementary polynucleotide) and the
functional moiety may be direct bonding, or may be indirect bonding
mediated by another material. However, in certain embodiments, it
is preferable that a functional moiety be directly bonded to the
polynucleotide such as the second wing via covalent bonding, ionic
bonding, hydrogen bonding or the like, and from the viewpoint that
more stable bonding may be obtained, covalent bonding is more
preferred. The functional moiety may also be bonded to the
polynucleotide via a cleavable linking group. For example, the
functional moiety may be linked via a disulfide bond.
[0132] There are no particular limitations on the structure of the
"functional moiety" according to certain embodiments, provided it
imparts the desired function to the chimeric polynucleotide. The
desired functions include a labeling function, a purification
function, and a delivery function. Examples of moieties that
provide a labeling function include compounds such as fluorescent
proteins, luciferase, and the like. Examples of moieties that
provide a purification function include compounds such as biotin,
avidin, a His tag peptide, a GST tag peptide, a FLAG tag peptide,
and the like.
[0133] Furthermore, from the viewpoint of delivering the chimeric
polynucleotide to a target site with high specificity and high
efficiency, and thereby suppressing very effectively the expression
of a target gene by the relevant nucleic acid, it is preferable
that a molecule having an activity of delivering the chimeric
antisense polynucleotide of some embodiments to a "target site"
within the body, be bonded as a functional moiety to the chimeric
antisense polynucleotide and/or another polynucleotide constituent
of the double-stranded antisense agent. In some embodiments, it is
preferred that the functional moiety is joined to the complementary
strand, that is, the polynucleotide complementary to and annealed
to the antisense polynucleotide.
[0134] The moiety having a "targeted delivery function" may be, for
example, a lipid, from the viewpoint of being capable of delivering
the chimeric polynucleotide of certain embodiments to the liver or
the like with high specificity and high efficiency. Examples of
such a lipid include lipids such as cholesterol and fatty acids
(for example, vitamin E (tocopherols, tocotrienols), vitamin A, and
vitamin D); lipid-soluble vitamins such as vitamin K (for example,
acylcarnitine); intermediate metabolites such as acyl-CoA;
glycolipids, glycerides, and derivatives thereof. However, among
these, from the viewpoint of having higher safety, in certain
embodiments, cholesterol and vitamin E (tocopherols and
tocotrienols) are used. Furthermore, from the viewpoint of being
capable of delivering the chimeric antisense polynucleotide of
certain embodiments to the brain with high specificity and high
efficiency, examples of the "functional moiety" according to the
certain embodiments include sugars (for example, glucose and
sucrose). Also, from the viewpoint of being capable of delivering
the chimeric antisense polynucleotide of certain embodiments to
various organs with high specificity and high efficiency by binding
to the various proteins present on the cell surface of the various
organs, examples of the "functional moiety" according to certain
embodiments include peptides or proteins such as receptor ligands
and antibodies and/or fragments thereof.
[0135] In regard to the chimeric antisense polynucleotide of
certain embodiments, the functional moiety may be joined to the
5'-terminal nucleotide, or the 3'-terminal nucleotide, or to both
the 5' and 3' terminal nucleotides, as illustrated in FIGS. 4B, 4C,
and 4D. Techniques for coupling labels to a nucleic acid strand
vary with the nature of the labeling moiety and the point of
attachment in the nucleic acid, and these are well-known in the
art. Although not illustrated, functional moieties may be joined to
the polynucleotide at internal sites and not at the strand terminal
sites. Again, such techniques are well-known in the art.
[0136] Thus, some suitable exemplary embodiments of the chimeric
polynucleotide of some embodiments have been described, but the
chimeric polynucleotide is not intended to be limited to the
exemplary embodiments described above. Furthermore, any person
having ordinary skill in the art can produce the chimeric
polynucleotide according to the various embodiments by
appropriately selecting a known method. For example, the nucleic
acids according to some embodiments can be produced by designing
the respective base sequences of the nucleic acids on the basis of
the information of the base sequence of the targeted transcription
product (or, in some cases, the base sequence of a targeted gene),
synthesizing the nucleic acids by using a commercially available
automated nucleic acid synthesizer (products of Applied Biosystems,
Inc.; products of Beckman Coulter, Inc.; and the like), and
subsequently purifying the resulting oligonucleotides by using a
reverse phase column or the like. Nucleic acids produced in this
manner are mixed in an appropriate buffer solution and denatured at
about 90 degrees C. to 98 degrees C. for several minutes (for
example, for 5 minutes), subsequently the nucleic acids are
annealed at about 30 degrees C. to 70 degrees C. for about 1 to 8
hours, and thus the double-stranded nucleic acid complex of some
embodiments can be produced. Furthermore, a polynucleotide bearing
a functional moiety can be produced by joining a functional moiety
to the oligonucleotide strand during or after the oligonucleotide
synthesis. Numerous methods for joining functional moieties to
nucleic acids are well-known in the art.
[0137] Thus, suitable exemplary embodiments of the chimeric
single-stranded antisense polynucleotide the complementary
polynucleotide have been described. Additional embodiments are also
disclosed in the following Examples. However, the chimeric
polynucleotide and the double-stranded antisense agent as
contemplated by the inventors is not limited to the exemplary
embodiments described above, or in the Examples below.
[0138] Compositions for modifying the expression of target gene or
level of targeted transcription product by means of antisense
effect are also contemplated.
[0139] The chimeric single-stranded polynucleotide and the double
stranded antisense agent of some embodiments can be delivered to a
target site with high specificity and high efficiency and can very
effectively suppress the expression of a target gene or the level
of a transcription product, as will be disclosed in the Examples
described below. Therefore, some embodiments provide a composition
that contains the chimeric antisense polynucleotide or the double
stranded antisense agent of some embodiments as an active
ingredient and is intended to modify or suppress, e.g., the
expression of a target gene by means of an antisense effect.
Particularly, the chimeric antisense polynucleotides or the double
stranded antisense agent of some embodiments can give high efficacy
even when administered at a low concentration, and the chimeric
design also displays reduced toxicity. Further, by directing the
antisense polynucleotide or the double stranded antisense agent to
particular organs, adverse side effects can be reduced. Therefore,
some embodiments can also provide a pharmaceutical composition
intended to treat and prevent diseases that are associated with,
e.g., increased expression of a target gene, such as metabolic
diseases, tumors, and infections.
[0140] The composition containing the chimeric antisense
polynucleotides or the double stranded antisense agent of some
embodiments can be formulated by known pharmaceutical methods. For
example, the composition can be used enterally (perorally or the
like) in the form of capsules, tablets, pills, liquids, powders,
granules, fine granules, film-coating agents, pellets, troches,
sublingual agents, peptizers, buccal preparations, pastes, syrups,
suspensions, elixirs, emulsions, coating agents, ointments,
plasters, cataplasms, transdermal preparations, lotions, inhalers,
aerosols, injections and suppositories, or non-enterally.
[0141] In regard to the formulation of these preparations,
pharmacologically acceptable carriers or carriers acceptable as
food and drink, specifically sterilized water, physiological
saline, vegetable oils, solvents, bases, emulsifiers, suspending
agents, surfactants, pH adjusting agents, stabilizers, flavors,
fragrances, excipients, vehicles, antiseptics, binders, diluents,
isotonizing agents, soothing agents, extending agents,
disintegrants, buffering agents, coating agents, lubricating
agents, colorants, sweetening agents, thickening agents,
corrigents, dissolution aids, and other additives can be
appropriately incorporated.
[0142] On the occasion of formulation, as disclosed in Non-Patent
Document 1, the chimeric antisense polynucleotides or the
double-stranded antisense agent of some embodiments to which a
lipid is bound as a functional moiety may be caused to form a
complex with a lipoprotein, such as chylomicron or chylomicron
remnant. Furthermore, from the viewpoint of increasing the
efficiency of enteral administration, complexes (mixed micelles and
emulsions) with substances having a colonic mucosal epithelial
permeability enhancing action (for example, medium-chain fatty
acids, long-chain unsaturated fatty acids, or derivatives thereof
(salts, ester forms or ether forms)) and surfactants (nonionic
surfactants and anionic surfactants) may also be used, in addition
to the lipoproteins.
[0143] There are no particular limitations on the preferred form of
administration of the composition of some embodiments, and examples
thereof include enteral (peroral or the like) or non-enteral
administration, more specifically, intravenous administration,
intraarterial administration, intraperitoneal administration,
subcutaneous administration, intracutaneous administration,
tracheobronchial administration, rectal administration, and
intramuscular administration, and administration by
transfusion.
[0144] The composition of some embodiments can be used for animals
including human beings as subjects. However, there are no
particular limitations on the animals excluding human beings, and
various domestic animals, domestic fowls, pets, experimental
animals and the like can be the subjects of some embodiments.
[0145] When the composition of some embodiments is administered or
ingested, the amount of administration or the amount of ingestion
may be appropriately selected in accordance with the age, body
weight, symptoms and health condition of the subject, type of the
composition (pharmaceutical product, food and drink, or the like),
and the like. However, the effective amount of ingestion of the
composition according to the certain embodiments is 0.001 mg/kg/day
to 50 mg/kg/day of the chimeric polynucleotide.
[0146] The chimeric polynucleotides or the double stranded
antisense agent of some embodiments can be delivered to a target
site with high specificity and high efficiency, and can modify or
suppress the expression of a target gene or the level of a
transcription product very effectively, as will be disclosed in the
Examples that follow. Therefore, some embodiments can provide a
method of administering the chimeric polynucleotides or the double
stranded antisense agent of some embodiments to a subject, and
suppressing the expression of a target gene or transcription
product level by means of an antisense effect. Furthermore, a
method of treating or preventing various diseases that are
associated with, e.g., increased expression of target genes, by
administering the composition of some embodiments to a subject can
also be provided.
EXAMPLE
[0147] Hereinafter, some embodiments will be described more
specifically by way of Examples, but the embodiments not intended
to be limited to the following Examples.
Example 1
[0148] An experiment comparing the inhibition potency of a chimeric
polynucleotide according to one embodiment of the invention with
two conventional gapmer antisense oligonucleotide was conducted.
The polynucleotide structures and the results of the experiment are
shown in FIG. 7. The control gapmers are 13 bases (ApoB1 ASO 13mer;
SEQ ID NO:6) and 20 bases (ApoB1 ASO 20mer-4; SEQ ID NO:2) in
length. The 13mer control has a central region of 8
phosphorothioated DNA nucleotides, a first 5'-wing region of 2 LNA
bases and a first 3'-wing region of 3 LNA bases, but does not have
a second wing on either side. The 20mer control has the same
structure as the 13mer, and adds 4 phosphorothioated 2'-O-Me RNA
bases at the 5' end and 3 phosphorothioated 2'-O-Me RNA bases at
the 3' end. According to the disclosure, these bases are not low
protein-affinity nucleotides (because they are phosphorothioated)
and thus they do not constitute a second 5' or 3'-wing region. They
would actually be classified as part of the first wing region (and
thus the 20mer control is a gapmer with a 6 base 5'-wing region and
a 6 base 3'-wing region).
[0149] The chimeric polynucleotide ApoB1 ASO 20mer-5; SEQ ID NO:7)
contains an 8 base central region, a first 5'-wing region of 2 LNA
bases and a first 3'-wing region of 3 LNA bases, and adds to that a
second 5'-wing region of 4 2'-O-Me RNA bases and a second 3'-wing
region of 3 2'-O-Me RNA bases. The 2'-O-Me RNA bases are low
protein-affinity nucleotides and thus constitute second wing
regions.
[0150] The three ASO's were tested in vivo using Hep 1-6 liver
cells according to the following procedure.
[0151] The ASOs were each transfected using Lipofectamine 2000
(manufactured by Invitrogen, Inc.) according to the usage protocol
provided with the reagent. The concentration of each ASO in the
medium at the time of the transfection was 2 nM, 10 nM, and 20 nM.
Furthermore, controls having no ASO added to the cells were also
prepared. Subsequently, 24 hours after the transfection, the cells
were collected by using Isogen, and mRNA's were collected according
to the manufacturer's usage protocol and the amount of mRNA was
determined.
[0152] cDNA's were synthesized from certain amounts of the mRNA's
by using SuperScript III according to the manufacturer's protocol.
Subsequently, the cDNA obtained was used as templates, and
quantitative RT-PCR was carried out by using a TaqMan system. The
primers used in the quantitative RT-PCR were those designed and
produced by Life Technologies Corp. based on the various gene
numbers. The PCR conditions for temperature and time were as
follows: 15 seconds at 95 degrees C., 30 seconds at 60 degrees C.,
and 1 second at 72 degrees C. were designated as one cycle, and 40
cycles thereof were carried out. Based on the results of the
quantitative RT-PCR thus obtained, the amount of expression of
rApoB/amount of expression of rGAPDH (internal standard gene) were
respectively calculated, and the calculation results for the
control group and the calculation results for the nucleic
acid-administered groups were compared and evaluated by a t-test.
The results thus obtained are presented in FIG. 7.
[0153] As shown by the results in FIG. 7, all three ASOs show an
antisense effect, with greater inhibition obtained at higher
concentration of the ASO. However, comparing the inhibition potency
of the 20mer chimeric polynucleotide with the 20mer control, the
chimeric polynucleotide provides a greater degree of inhibition.
Although the potency of the 13mer control is still greater, the
presence of the second wing provides improved performance compared
to the convention 20mer control ASO, and is expected to have a
reduced toxicity compared to the 13mer control. The result reveals
that including a second wing region provides improved antisense
performance.
Example 2
[0154] An experiment comparing the inhibition potency of a chimeric
polynucleotide according to embodiments of the invention having
either a second 5'-wing region, a second 3'-wing region, or both
wing regions was conducted. The polynucleotide structures and the
results of the experiment are shown in FIG. 8A. Two conventional
gapmer controls were compared with the inhibition potency of 3
different chimeric polynucleotides.
[0155] The ASO's were tested in vivo using Hep 1-6 liver cells
according to the procedure described in Example 1, and the results
are shown in FIG. 8B. The ASOs were transfected at a concentration
of 20 nM.
[0156] As shown by the results in FIG. 8B, all five ASOs show an
antisense effect relative to the negative control (no ASO).
However, comparing the inhibition potency of the 20mer chimeric
polynucleotides with the 20mer control, the chimeric polynucleotide
provides a greater degree of inhibition in all three cases.
Significantly, the chimeric polynucleotide with a second 5'-wing
demonstrated a potency that is statistically the same as the
inhibition achieved wih the 13mer control. Thus, the presence of
the second wing yields a performance with a 20mer ASO that is the
same as that obtained with a 13mer gapmer. Moreover, the 20mer
chimeric polynucleotide is expected to have a reduced toxicity
compared to the 13mer control.
Example 3
[0157] Next, the chimeric polynucleotides were tested in vivo in
mice. The sequences and design of the ASO probes and controls are
shown in FIG. 9. The ASO were intravenously injected to a mouse in
an amount of 3.0 mg/kg each through the tail vein. The mice were
4-week old female ICR mice with body weights of 20 to 25 g. The
experiments using mice were all carried out with n=3. Also, as a
negative control group, mice to which only PBS was injected instead
of the single-stranded ASO or double-stranded nucleic acid complex
were also prepared. Seventy-two hours after the injection, the mice
were perfused with PBS, and then the mice were dissected to extract
the liver. Subsequently, extraction of mRNA, synthesis of cDNA, and
quantitative RT-PCR were carried out by the same methods as the
methods described in Example 1, the amount of expression of
mApoB/amount of expression of mGAPDH (internal standard gene) was
calculated, and comparisons were made between the group
administered with PBS (PBS only) and the groups administered with a
nucleic acid. The results thus obtained are presented in FIG.
9.
[0158] As illustrated in FIG. 9, two ASOs show an antisense effect
(13mer control and the 20mer-5 chimeric polynucleotide), but the
two 20mer controls (20mer-1 and 20mer-4), do not show an antisense
effect. Although the potency of the 13mer control is still greater
than the chimeric polynucleotide, the presence of the second wing
provides improved performance compared to the convention 20mer
control ASOs, and is expected to have a reduced toxicity compared
to the 13mer control. The result reveals that including a second
wing region provides improved antisense performance in vivo.
Example 4
[0159] Design and synthesis of Toc(tocopherol)-HDO. A series of
DNA-LNA gapmers or chimeramers of different lengths (13- to
23-mers) were designed to target mouse ApoB mRNA (NM_009693), and
were synthesized by Gene Design (Osaka, Japan) and Hokkaido System
Science (Sapporo, Japan).
[0160] The sequences of the chimeramers targeting ApoB mRNA were as
follows:
TABLE-US-00001 1) ApoB1 13mer, (SEQ ID NO: 1) 5'-G(L){circumflex
over ( )}C(L){circumflex over ( )}a{circumflex over (
)}t{circumflex over ( )}t{circumflex over ( )}g{circumflex over (
)}g{circumflex over ( )}t{circumflex over ( )}a{circumflex over (
)}t{circumflex over ( )}T(L){circumflex over ( )}C(L){circumflex
over ( )}A(L)-3'; 2) ApoB1 Toc13mer PS(-), (SEQ ID NO: 1)
5'-Toc_G(L){circumflex over ( )}C(L){circumflex over (
)}a{circumflex over ( )}t{circumflex over ( )}t{circumflex over (
)}g{circumflex over ( )}g{circumflex over ( )}t{circumflex over (
)}a{circumflex over ( )}t{circumflex over ( )}T(L){circumflex over
( )}C(L){circumflex over ( )}A(L)-3'; 3) ApoB1 Toc17merPS(-), (SEQ
ID NO: 2) 5'-Toc-U(X)-C(X)-C(X)-A(X)-G(L){circumflex over (
)}C(L){circumflex over ( )}a{circumflex over ( )}t{circumflex over
( )}t{circumflex over ( )}g{circumflex over ( )} g{circumflex over
( )}t{circumflex over ( )}a{circumflex over ( )}t{circumflex over (
)}T(L){circumflex over ( )}C(L){circumflex over ( )}A(L)-3'; 4)
ApoB1 Toc20merPS(-), (SEQ ID NO: 3)
5'-Toc-A(X)-A(X)-G(X)-U(X)-C(X)-C(X)-A(X)-G(L){circumflex over ( )}
C(L){circumflex over ( )}a{circumflex over ( )}t{circumflex over (
)}t{circumflex over ( )}g{circumflex over ( )}g{circumflex over (
)}t{circumflex over ( )}a{circumflex over ( )}t{circumflex over (
)}T(L){circumflex over ( )}C(L){circumflex over ( )}A(L)-3'; 5)
ApoB1 Toc20merPS(+) (SEQ ID NO: 4) 5'-Toc{circumflex over (
)}A(X){circumflex over ( )}A(X){circumflex over ( )}G(X){circumflex
over ( )}U(X){circumflex over ( )}C(X){circumflex over (
)}C(X){circumflex over ( )}A(X){circumflex over ( )}G(L){circumflex
over ( )} C(L){circumflex over ( )}a{circumflex over (
)}t{circumflex over ( )}t{circumflex over ( )}g{circumflex over (
)}g{circumflex over ( )}t{circumflex over ( )}a{circumflex over (
)}t{circumflex over ( )}T(L){circumflex over ( )}C(L){circumflex
over ( )}A(L)-3'; and 6) ApoB1 TocPEG13mer (SEQ ID NO: 1)
5'-Toc{circumflex over ( )}spacer18{circumflex over (
)}G(L){circumflex over ( )}C(L){circumflex over ( )}a{circumflex
over ( )}t{circumflex over ( )}t{circumflex over ( )}g{circumflex
over ( )}g{circumflex over ( )}t{circumflex over ( )}a{circumflex
over ( )}t{circumflex over ( )}T(L){circumflex over ( )}
C(L){circumflex over ( )}A(L)-3';
[0161] wherein lower case represent DNA, upper case with L be noted
in brackets represent LNA (capital C denotes LNA methylcytosine),
upper case with X be noted in brackets represent UNA or 2'-O-methyl
sugar modification, chevron mark represent phosphorothioate
linkages.
[0162] Mouse Studies.
[0163] Wild type Crlj:CD1 (ICR) mice aged 4-5 weeks (Oriental
Yeast, Tokyo, Japan) were kept on a 12-h light/dark cycle in a
pathogen-free animal facility with free access to food and water.
ASO gapmer or chimeramer was administered to the mice by tail vein
injection based upon body weight. All oligonucleotides were
formulated in PBS, which was also used as the control. The
oligonucleotides were administered to wild-type mice by a single
injection of 0.75-6 mg/kg. For postmortem analyses, mice were
deeply anesthetized first with intraperitoneally administered 60
mg/kg pentobarbital, and then sacrificed by transcardiac perfusion
with PBS after confirming the absence of blink reflex. All animal
experiments were performed in accordance with the ethical and
safety guidelines for animal experiments of Tokyo Medical and
Dental University (#0140144A).
[0164] Quantitative real-time polymerase chain reaction assay.
Total RNA was extracted from mouse liver by using Isogen (Nippon
Gene, Tokyo, Japan). To detect mRNA, DNase-treated RNA (2 micro g)
was reverse transcribed with SuperScript III and Random Hexamers
(Life Technologies, Carlsbad, Calif.). To detect RNAs, quantitative
real-time polymerase chain reaction (RT-PCR) analysis was performed
by using the Light Cycler 480 Real-Time PCR Instrument (Roche
Diagnostics, Mannheim, Germany). The primers and probes for mouse
ApoB, glyceraldehyde-3-phosphate dehydrogenase (Gapdh; NM_008084)
genes were designed by Applied Biosystems. The results thus
obtained are presented in FIG.
[0165] Northern blot analysis. Total RNA was extracted from mouse
liver by using Isogen II (Nippon Gene). Total RNA (45 micro g) was
separated by electrophoresis in an 18% polyacrylamide-urea gel and
transferred to a Hybond-N+ membrane (Amersham Biosciences,
Piscataway, N.J.). The blot was hybridized with a probe
corresponding to the cRNA sequence, or with the mouse U6 micro RNA
sequence (internal control), which had been labeled with
digoxigenin-ddUTP by using a DIG Oligonucleotide 3'-End Labelling
Kit, 2nd Generation (Roche Diagnostics). The sequence of the DNA
probe for detecting gapmer or chimeramer was 5'-TGAATACCAATGCTG-3'.
The signals were visualized by Gene Images CDP-star Detection Kit
(Amersham Biosciences). The results thus obtained are presented in
FIG. 10
[0166] As illustrated in FIG. 11 and FIG. 12, three ASOs (the 13mer
control the Toc-17-mer-8U PS- and the Toc-20-mer-8U PS- chimeric
polynucleotide) show an antisense effect. On the other hand, the
two 20mer controls (20mer-9 and 20-mer-8U PS+), do not show an
antisense effect. The potency of the Toc-20-mer-8U PS- is still
greater than those of the other two chimeric polynucleotide (the
13mer control and Toc-20-mer-8U PS- chimeric polynucleotide).
Moreover, the efficacy of the Toc-20-mer-8U PS- prolonged up to 14
days after injection (FIG. 13). Furthermore, the potency of the
Toc-20-mer-8U PS- was dose dependent manner (FIG. 14).
[0167] These results suggest that the presence of the second wing
having no PS provides improved performance compared to the
convention 20mer control ASOs with PS and the set forth above
20-mer-8U PS-ASO is expected to have a reduced toxicity compared to
control.
[0168] A "high-exonuclease resistant nucleotide" is a nucleotide
that is (i) more resistant to DNase or RNase than a natural DNA or
RNA nucleotide, (ii) more resistant to exonuclease than a natural
DNA or RNA nucleotide and (III) relatively same or lower resistant
to endonuclease than a natural DNA or RNA nucleotide.
[0169] Examples of high-exonuclease resistant nucleotides include
2'-O-methyl RNA nucleotides, 2'-O-methoxyethyl RNA nucleotides,
LNA, cMOE BNA, 2-fluoro RNA nucleotides, boranophosphate
nucleotides, methylphosphonate nucleotides, phosphoramidite
nucleotides, 5-methylcytosine, 5-propynyluridine, and unlocked
nucleic acid (UNA).
Example 5
[0170] Next, a double-stranded antisense agent was tested in vivo
in mice. The sequences and design of the various antisense probes
and the complementary strands are shown in FIG. 16A. In the
complementary strands, tocopherol (Toc) was bound to the 5'
terminus. Thus, a functional moiety capable of directing the
delivery of the double-stranded agent, and presumably the chimeric
antisense polynucleotide, to the liver was incorporated in the
complementary strand.
[0171] The binding of the tocopherol moiety to the cRNA was carried
out according to a known technique, by preparing tocopherol amidite
in which the hydroxyl group at the 6-position of the chromane ring
of tocopherol was joined to the phosphoramidite, and then the
tocopherol amidite was coupled to the 5'-terminus of the RNA by
standard coupling methods.
[0172] The sequence, composition, and strand length of the
antisense polynucleotides and the complementary strands (cRNA) were
as follows:
[0173] Antisense Strands
TABLE-US-00002 1. 20mer-1 ApoB1 ASO: (SEQ ID NO: 5)
5'-T.sub.sC.sub.sC.sub.sA.sub.sG.sub.sC.sub.sa.sub.st.sub.st.sub.sg.sub.s-
g.sub.st.sub.sa.sub.st.sub.sT.sub.sC.sub.sA.sub.sG.sub.sT.sub.sG-3'
2. 20mer-4 ApoB1 ASO: (SEQ ID NO: 6)
5'-u.sub.sc.sub.sc.sub.sa.sub.sG.sub.sC.sub.sa.sub.st.sub.st.sub.sg.sub.s-
g.sub.st.sub.sa.sub.st.sub.sT.sub.sC.sub.sA.sub.sg.sub.su.sub.sg-3'
3. 20mer-5 ApoB1 ASO: (SEQ ID NO: 7)
5'-uccaG.sub.sC.sub.sa.sub.st.sub.st.sub.sg.sub.sg.sub.st.sub.sa.sub.st.s-
ub.sT.sub.sC.sub.sAgug-3' 4. 13mer ApoB1 ASO: (SEQ ID NO: 8)
5'-G.sub.sC.sub.sa.sub.st.sub.st.sub.sg.sub.sg.sub.st.sub.sa.sub.st.sub.s-
T.sub.sC.sub.sA-3'
[0174] (Upper case: LNA; lower case: DNA; underlined: 2'-O-Me RNA;
s: phosphorothioate between the bases)
[0175] Complementary Strands
TABLE-US-00003 1. 20-mer Toc-ApoB1 cRNA: (SEQ ID NO: 9)
5'-Toc-c.sub.sa.sub.sc.sub.su.sub.sg.sub.sAAUACCAAUG.sub.sc.sub.su.sub.sg-
.sub.sg.sub.sa-3' 2. 13-mer Toc-ApoB1 cRNA: (SEQ ID NO: 10)
5'-Toc-u.sub.sg.sub.sa.sub.sAUACCAAU.sub.sg.sub.sc-3'
[0176] (Upper case: RNA, underlined: 2'-OMe-RNA, s:
phosphorothioate between the bases) The antisense polynucleotides
and the respective cRNA having the same length were mixed in
equimolar amounts, and the mixtures were heated at 95 degrees C.
for 5 minutes and then were kept warm at 37 degrees C. for one hour
to thereby anneal these nucleic acid strands and form
double-stranded nucleic acid complexes. The annealed nucleic acids
were stored at 4 degrees C. or on ice.
[0177] The double-stranded complexes were intravenously injected to
a mouse in an amount of 0.75 mg/kg each through the tail vein. The
mice were 4-week old female ICR mice with body weights of 20 to 25
g. The experiments using mice were all carried out with n=3. Also,
as a negative control group, mice to which only PBS was injected
instead of the double-stranded complexes were also prepared.
Seventy-two hours after the injection, the mice were perfused with
PBS, and then the mice were dissected to extract the liver.
Subsequently, extraction of mRNA, synthesis of cDNA, and
quantitative RT-PCR were carried out by the same methods as the
methods described in Example 1, the amount of expression of
mApoB/amount of expression of mGAPDH (internal standard gene) was
calculated, and comparisons were made between the group
administered with PBS (PBS only) and the groups administered with a
nucleic acid. The results thus obtained are presented in FIG.
16B.
[0178] As illustrated in FIG. 16B, three of the double-stranded
complexes, those containing 20mer-4, 20mer-5, and 13mer ASOs showed
a statistically significant level of suppression compared to the
PBS control. Furthermore, the double-stranded agent comprising
20mer-5 ASO shows an antisense effect that is similar to that of
the double-stranded 13mer gapmer control. Thus, the double wing
structure in the 20mer-5 ASO provides improved performance compared
to the conventional 20mer control ASOs, and is expected to have a
reduced toxicity compared to the 13mer control. The result
demonstrates that double-stranded antisense agents that comprise a
chimeric antisense polynucleotide (i.e., an antisense
polynucleotide having a double wing structure, in this instance,
and second wing on both the 3' and 5' ends of the polynucleotide)
provides improved antisense performance in vivo.
Sequence CWU 1
1
18113DNAArtificialSynthetic 1gcattggtat tca
13217DNAArtificialSynthetic 2uccagcattg gtattca
17320DNAArtificialSynthetic 3aaguccagca ttggtattca
20420DNAArtificialSynthetic 4aaguccagca ttggtattca
20520DNAArtificialSynthetic 5tccagcattg gtattcagtg
20620DNAArtificialSynthetic 6uccagcattg gtattcagug
20720DNAArtificialSynthetic 7uccagcattg gtattcagug
20813DNAArtificialSynthetic 8gcattggtat tca
13920RNAArtificialSynthetic 9cacugaauac caaugcugga
201013RNAArtificialSynthetic 10ugaauaccaa ugc
131120DNAArtificialSynthetic 11tccagcattg gtattcagtg
201220DNAArtificialSynthetic 12tccagcattg gtattcagtg
201320DNAArtificialSynthetic 13gcattggtat tcagtgtgat
201420DNAArtificialSynthetic 14aagtccagca ttggtattca
201513DNAArtificialSynthetic 15gcattggtat tca
131620DNAArtificialSynthetic 16tccagcattg gtattcagtg
201720RNAArtificialSynthetic 17cacugaauac caaugcugga
201813RNAArtificialSynthetic 18ugaauaccaa ugc 13
* * * * *