U.S. patent application number 14/443369 was filed with the patent office on 2015-10-15 for anti apob antisense conjugate compounds.
This patent application is currently assigned to Roche Innovation Center Copenhagen A/S. The applicant listed for this patent is SANTARIS PHARMA A/S. Invention is credited to Nanna Albaek, Henrik Frydenlund Hansen, Susanne Kammler, Marie Lindholm, Henrik Orum, Jacob Ravn, Mark Turner.
Application Number | 20150291958 14/443369 |
Document ID | / |
Family ID | 49584733 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150291958 |
Kind Code |
A1 |
Albaek; Nanna ; et
al. |
October 15, 2015 |
ANTI APOB ANTISENSE CONJUGATE COMPOUNDS
Abstract
The present invention relates to conjugates of LNA antisense
oligonucleotides (oligomers) that target ApoB.
Inventors: |
Albaek; Nanna; (Birkerod,
DK) ; Hansen; Henrik Frydenlund; (Rodovre, DK)
; Kammler; Susanne; (Holte, DK) ; Ravn; Jacob;
(Skovlunde, DK) ; Orum; Henrik; (Vaerlose, DK)
; Turner; Mark; (Horsholm, DK) ; Lindholm;
Marie; (Gerlachsgatan, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SANTARIS PHARMA A/S |
Horsholm |
|
DK |
|
|
Assignee: |
Roche Innovation Center Copenhagen
A/S
Horsholm
DK
|
Family ID: |
49584733 |
Appl. No.: |
14/443369 |
Filed: |
November 14, 2013 |
PCT Filed: |
November 14, 2013 |
PCT NO: |
PCT/EP2013/073859 |
371 Date: |
May 15, 2015 |
Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
A61P 7/04 20180101; A61P
31/14 20180101; C12N 2310/351 20130101; A61P 35/00 20180101; C12N
15/111 20130101; C12N 2310/341 20130101; C12N 2310/314 20130101;
A61P 3/06 20180101; C12N 2310/3517 20130101; C12N 2310/3515
20130101; C12N 2320/32 20130101; A61P 7/06 20180101; A61P 3/04
20180101; A61P 3/00 20180101; C12N 15/113 20130101; A61P 9/00
20180101; C12N 2330/30 20130101; A61P 29/00 20180101; A61P 7/00
20180101; A61P 1/16 20180101; A61P 31/12 20180101; C12N 2310/11
20130101; C12N 2310/315 20130101; C12N 2310/3231 20130101; A61P
7/02 20180101; A61P 3/10 20180101; A61P 9/10 20180101; A61P 19/00
20180101; C12N 2310/341 20130101; C12N 2310/3231 20130101; C12N
2310/341 20130101; C12N 2310/3515 20130101; C12N 2310/341 20130101;
C12N 2310/351 20130101; C12N 2310/341 20130101; C12N 2310/3231
20130101; C12N 2310/351 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 15, 2012 |
EP |
12192773.5 |
Jan 30, 2013 |
EP |
13153296.2 |
Feb 28, 2013 |
EP |
13157237.2 |
Jun 27, 2013 |
EP |
13174092.0 |
Claims
1.-18. (canceled)
19. An antisense oligonucleotide conjugate comprising a first
region of a LNA oligomer (region A--such as an LNA gapmer
oligomer), targeting an ApoB nucleic acid, covalently joined to a
further region (region C) comprising a conjugate moiety selected
from the group consisting of an asialoglycoprotein receptor
targeting conjugate and a lipophilic conjugate, wherein the
lipophilic conjugate, and optionally the asialoglycoprotein
receptor targeting conjugate, is joined to the LNA oligomer via
biocleavable linker.
20. The antisense oligonucleotide conjugate according to claim 19,
wherein the conjugate moiety (C) comprises a sterol, such as
cholesterol or tocopherol, such as Conj 5 or Conj 6.
21. The antisense oligonucleotide conjugate according to claim 19,
wherein the conjugate moiety (C) comprises a GalNAc
(N-acetylgalactosamine) moiety, such as a trivalent GalNac
moiety.
22. The antisense oligonucleotide conjugate according to claim 19,
wherein the biocleavable linker comprises a peptide or polypeptide,
such as a lysine linker, or physiologically labile nucleotide
linker.
23. The antisense oligonucleotide conjugate according to claim 19,
wherein the LNA oligomer is covalently joined to the conjugate
moiety via a region of one or more phosphate linked nucleosides,
such as DNA or RNA nucleosides (region B), such as a phosphodiester
nucleotide linker.
24. The antisense oligomer conjugate according to claim 19, wherein
the LNA oligomer is covalently joined to the conjugate moiety via a
region B of 1-6 phosphate linked DNA nucleosides (region B).
25. The antisense oligomer conjugate according to claim 24, wherein
region B (phosphodiester nucleotide linkage) comprises 1, 2 or 3
contiguous DNA phosphodiester nucleotides, such as two contiguous
DNA phosphodiester nucleotides, such as a 5' CA 3'
dinucleotide.
26. The antisense oligomer according to claim 28, wherein the LNA
oligomer and region B form a contiguous nucleotide sequence,
wherein region A is complementary to a corresponding region of the
ApoB target, and region B may or may or may not be complementary to
the corresponding region of the ApoB target.
27. The antisense oligonucleotide conjugate according to claim 19,
wherein the conjugate moiety further comprises a linker (Y)
covalently linking the conjugate moiety to either the LNA oligomer
or to the region of one or more phosphate linked DNA or RNA
nucleotides (region B).
28. The antisense oligomer conjugate of claim 27 wherein the linker
region Y comprises a fatty acid, such as a C6 linker.
29. The antisense oligonucleotide conjugate according to claim 19,
wherein the conjugate moiety comprises a trivalent GalNac moiety
selected from the group consisting of Conj1, Conj2, Conj3, Conj4,
Conj1a, Conj2a, Conj3a and Conj4a.
30. The antisense oligonucleotide conjugate according to claim 19,
wherein the LNA oligomer comprises a contiguous nucleotide sequence
selected from the group consisting of SEQ ID No 1 or SEQ ID No 2:
TABLE-US-00016 (3833) SEQ ID NO 1 GCattggtatTCA (4955) SEQ ID NO 2
GTtgacactgTC
Wherein capital letters represent LNA nucleosides, such as
beta-D-oxy LNA, lower case letters represent DNA nucleosides, LNA
cytosines are optionally 5-methyl cytosine, and all internucleoside
linkages are phosphorothioate.
31. The antisense oligonucleotide conjugate according to claim 30,
which is selected from the group consisting of SEQ ID NO 7, 20, 28
or 30.
32. A pharmaceutical composition comprising the antisense
oligonucleotide conjugate according to claim 19, and a
pharmaceutically acceptable diluent, carrier, salt or adjuvant.
33. The antisense oligonucleotide conjugate or pharmaceutical
composition according to claim 19, for use as a medicament, such as
for the treatment of acute coronary syndrome, or
hypercholesterolemia or related disorder, such as a disorder
selected from the group consisting of atherosclerosis,
hyperlipidemia, hypercholesterolemia, HDL/LDL cholesterol
imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL),
acquired hyperlipidemia, statin-resistant hypercholesterolemia,
coronary artery disease (CAD), and coronary heart disease
(CHD).
34. The use of an antisense oligonucleotide conjugate or
pharmaceutical composition according to claim 19, for the
manufacture of a medicament for the treatment of acute coronary
syndrome, or hypercholesterolemia or a related disorder, such as a
disorder selected from the group consisting of atherosclerosis,
hyperlipidemia, hypercholesterolemia, HDL/LDL cholesterol
imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL),
acquired hyperlipidemia, statin-resistant hypercholesterolemia,
coronary artery disease (CAD), and coronary heart disease
(CHD).
35. A method of treating acute coronary syndrome, or
hypercholesterolemia or a related disorder, such as a disorder
selected from the group consisting atherosclerosis, hyperlipidemia,
hypercholesterolemia, HDL/LDL cholesterol imbalance, dyslipidemias,
e.g., familial hyperlipidemia (FCHL), acquired hyperlipidemia,
statin-resistant hypercholesterolemia, coronary artery disease
(CAD), and coronary heart disease (CHD), said method comprising
administering an effective amount of an antisense oligonucleotide
conjugate or pharmaceutical composition according to claim 19, to a
patient suffering from, or likely to suffer from
hypercholesterolemia or a related disorder.
36. An in vivo or in vitro method for the inhibition of ApoB in a
cell which is expressing ApoB, said method comprising administering
an oligomer or conjugate or pharmaceutical composition according to
claim 19 to said cell so as to inhibit ApoB in said cell.
Description
FIELD OF INVENTION
[0001] The present invention relates to conjugates of LNA antisense
oligonucleotides (oligomers) that target ApoB.
RELATED CASES
[0002] This application claims priority from EP12192773.5,
EP13153296.2, EP13157237.2 and EP13174092.0, which are hereby
incorporated by reference.
BACKGROUND
[0003] See the background sections of WO2007/031081, WO2008/113830,
WO2010142805, and WO2010076248 which are hereby incorporated by
reference. SPC3833 and SPC4955 (which have SEQ ID NO 1 and 2) are
two LNA compounds which have been previously identified as potent
compounds which target human ApoB mRNA.
[0004] WO2007/146511 reports on short bicyclic (LNA) gapmer
antisense oligonucleotides which apparently are more potent and
less toxic than longer compounds. The exemplified compounds appear
to be 14 nts in length.
[0005] According to van Poelgeest et al., (American Journal of
Kidney Disease, 2013 October; 62(4):796-800), the administration of
LNA antisense oligonucleotide SPC5001 in human clinical trials may
result in acute kidney injury.
[0006] According to EP 1 984 381 B1, Seth et al., Nucleic Acids
Symposium Series 2008 No. 52 553-554 and Swayze et al., Nucleic
Acid Research 2007, vol 35, pp 687-700, LNA oligonucleotides cause
significant hepatotoxicity in animals. According to WO2007/146511,
the toxicity of LNA oligonucleotides may be avoided by using LNA
gapmers as short as 12-14 nucleotides in length. EP 1 984 381 B1
recommends using 6' substituted bicyclic nucleotides to decrease
the hepatotoxicity potential of LNA oligonucleotides. According to
Hagedorn et al., Nucleic Acid Therapeutics 2013, the hepatotoxic
potential of antisense oligonucleotide may be predicted from their
sequence and modification pattern.
[0007] Oligonucleotide conjugates have been extensively evaluated
for use in siRNAs, where they are considered essential in order to
obtain sufficient in vivo potency. For example, see WO2004/044141
refers to modified oligomeric compounds that modulate gene
expression via an RNA interference pathway. The oligomeric
compounds include one or more conjugate moieties that can modify or
enhance the pharmacokinetic and pharmacodynamic properties of the
attached oligomeric compound.
[0008] WO2012/083046 reports on a galactose cluster-pharmacokinetic
modulator targeting moiety for siRNAs.
[0009] In contrast, single stranded antisense oligonucleotides are
typically administered therapeutically without conjugation or
formulation. The main target tissues for antisense oligonucleotides
are the liver and the kidney, although a wide range of other
tissues are also accessible by the antisense modality, including
lymph node, spleen, bone marrow.
[0010] WO 2005/086775 refers to targeted delivery of therapeutic
agents to specific organs using a therapeutic chemical moiety, a
cleavable linker and a labeling domain. The cleavable linker may
be, for example, a disulfide group, a peptide or a restriction
enzyme cleavable oligonucleotide domain.
[0011] WO 2011/126937 refers to targeted intracellular delivery of
oligonucleotides via conjugation with small molecule ligands.
[0012] WO2009/025669 refers to polymeric (polyethylene glycol)
linkers containing pyridyl disulphide moieties. See also Zhao et
al., Bioconjugate Chem. 2005 16 758-766.
[0013] Chaltin et al., Bioconjugate Chem. 2005 16 827-836 reports
on cholesterol modified mono- di- and tetrameric oligonucleotides
used to incorporate antisense oligonucleotides into cationic
liposomes, to produce a dendrimeric delivery system. Cholesterol is
conjugated to the oligonucleotides via a lysine linker.
[0014] Other non-cleavable cholesterol conjugates have been used to
target siRNAs and antagomirs to the liver--see for example,
Soutscheck et al., Nature 2004 vol. 432 173-178 and Krutzfeldt et
al., Nature 2005 vol 438, 685-689. For the partially
phosphorothiolated siRNAs and antagomirs, the use of cholesterol as
a liver targeting entity was found to be essential for in vivo
activity.
[0015] There is therefore a need for ApoB targeting LNA antisense
compounds have enhanced efficacy and a reduced toxicity risk.
SUMMARY OF INVENTION
[0016] The invention provides for an antisense oligonucleotide
conjugate (the compound of the invention) comprising a first region
of an oligomer (region A--such as an LNA oligomer, a gapmer
oligomer or an LNA gapmer oligomer), targeting an ApoB nucleic
acid, covalently joined to a further region (region C) comprising a
conjugate moiety selected from the group consisting of an
asialoglycoprotein receptor targeting conjugate and a lipophilic
conjugate, wherein the lipophilic conjugate, and optionally the
asialoglycoprotein receptor targeting conjugate, is joined to the
LNA oligomer via biocleavable linker.
[0017] The invention provides for a conjugate comprising an LNA
antisense oligomer (the compound of the invention) targeting to a
ApoB nucleic acid (A) and at least one non-nucleotide or
non-polynucleotide moiety (C) covalently attached to said oligomer
(A), wherein the non-polynucleotide moiety is selected from the
group consisting of an asialoglycoprotein receptor targeting
conjugate and a lipophilic conjugate, wherein the lipophilic
conjugate, and optionally the asialoglycoprotein receptor targeting
conjugate, is covalently joined to the LNA antisense oligomer via a
biocleavable linker (region B) In some embodiments, the invention
provides for an oligomeric compound (the compound of the
invention), which targets an ApoB nucleic acid target, which
comprises three regions: [0018] i) a first region (region A), which
comprises 7-26 contiguous nucleotides which are complementary to a
ApoB nucleic acid target; [0019] ii) a second region (region B)
which comprises between 1-10 nucleotides, which is covalently
linked to the 5' or 3' nucleotide of the first region, such as via
a internucleoside linkage group such as a phosphodiester linkage,
wherein either [0020] a. the internucleoside linkage between the
first and second region is a phosphodiester linkage and the
nucleoside of the second region [such as immediately] adjacent to
the first region is either DNA or RNA; and/or [0021] b. at least 1
nucleoside of the second region is a phosphodiester linked DNA or
RNA nucleoside; [0022] iii) a third region (C) which comprises a
conjugate moiety, a targeting moiety, a reactive group, an
activation group, or a blocking moiety, wherein the third region is
covalent linked to the second region.
[0023] In some embodiments, region A and region B form a single
contiguous nucleotide sequence of 8-35 nucleotides in length.
[0024] In some aspects the internucleoside linkage between the
first and second regions may be considered part of the second
region.
[0025] In some embodiments, there is a phosphorus containing
linkage group between the second and third region. The phosphorus
linkage group, may, for example, be a phosphate (phosphodiester), a
phosphorothioate, a phosphorodithioate or a boranophosphate group.
In some embodiments, this phosphorus containing linkage group is
positioned between the second region and a linker region which is
attached to the third region. In some embodiments, the phosphate
group is a phosphodiester.
[0026] Therefore, in some aspects the oligomeric compound comprises
at least two phosphodiester groups, wherein at least one is as
according to the above statement of invention, and the other is
positioned between the second and third regions, optionally between
a linker group and the second region.
[0027] In some embodiments, the third region is an activation
group, such as an activation group for use in conjugation. In this
respect, the invention also provides activated oligomers comprising
region A and B and a activation group, e.g an intermediate which is
suitable for subsequent linking to the third region, such as
suitable for conjugation.
[0028] In some embodiments, the third region is a reactive group,
such as a reactive group for use in conjugation. In this respect,
the invention also provides oligomers comprising region A and B and
a reactive group, e.g an intermediate which is suitable for
subsequent linking to the third region, such as suitable for
conjugation. The reactive group may, in some embodiments comprise
an amine of alcohol group, such as an amine group.
[0029] In some embodiments region A comprises at least one, such as
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, or 24 internucleoside linkages other than
phosphodiester, such as internucleoside linkages which are
(optionally independently] selected from the group consisting of
phosphorothioate, phosphorodithioate, and boranophosphate, and
methylphosphonate, such as phosphorothioate. In some embodiments
region A comprises at least one phosphorothioate linkage. In some
embodiments at least 50%, such as at least 75%, such as at least
90% of the internucleoside linkages, such as all the
internucleoside linkages within region A are other than
phosphodiester, for example are phosphorothioate linkages. In some
embodiments, all the internucleoside linkages in region A are other
than phosphodiester.
[0030] In some embodiments, the oligomeric compound comprises an
antisense oligonucleotide, such as an antisense oligonucleotide
conjugate. The antisense oligonucleotide may be or may comprise the
first region, and optionally the second region. In this respect, in
some embodiments, region B may form part of a contiguous nucleobase
sequence which is complementary to the (nucleic acid) target. In
other embodiments, region B may lack complementarity to the
target.
[0031] Alternatively stated, in some embodiments, the invention
provides a non-phosphodieser linked, such as a phosphorothioate
linked, oligonucleotide (e.g. an antisense oligonucleotide) which
has at least one terminal (5' and/or 3') DNA or RNA nucleoside
linked to the adjacent nucleoside of the oligonucleotide via a
phosphodiester linkage, wherein the terminal DNA or RNA nucleoside
is further covalently linked to a conjugate moiety, a targeting
moiety or a blocking moiety, optionally via a linker moiety.
[0032] The invention provides for pharmaceutical composition
comprising the compound of the invention, and a pharmaceutically
acceptable diluent, carrier, salt or adjuvant.
[0033] The invention provides for the compound or pharmaceutical
composition of the invention, for use as a medicament, such as for
the treatment of acute coronary syndrome, or hypercholesterolemia
or related disorder, such as a disorder selected from the group
consisting of atherosclerosis, hyperlipidemia,
hypercholesterolemia, HDL/LDL cholesterol imbalance, dyslipidemias,
e.g., familial hyperlipidemia (FCHL), acquired hyperlipidemia,
statin-resistant hypercholesterolemia, coronary artery disease
(CAD), and coronary heart disease (CHD).
[0034] The invention provides for the use of the compound or
pharmaceutical composition of the invention, for the manufacture of
a medicament for the treatment of acute coronary syndrome, or
hypercholesterolemia or a related disorder, such as a disorder
selected from the group consisting of atherosclerosis,
hyperlipidemia, hypercholesterolemia, HDL/LDL cholesterol
imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL),
acquired hyperlipidemia, statin-resistant hypercholesterolemia,
coronary artery disease (CAD), and coronary heart disease
(CHD).
[0035] The invention provides for a method of treating acute
coronary syndrome, or hypercholesterolemia or a related disorder,
such as a disorder selected from the group consisting
atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL/LDL
cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidemia
(FCHL), acquired hyperlipidemia, statin-resistant
hypercholesterolemia, coronary artery disease (CAD), and coronary
heart disease (CHD), said method comprising administering an
effective amount of the compound or pharmaceutical composition
according to the invention, to a patient suffering from, or likely
to suffer from hypercholesterolemia or a related disorder.
[0036] The invention provides for an in vivo or in vitro method for
the inhibition of ApoB in a cell which is expressing ApoB, said
method comprising administering the compound of the invention to
said cell so as to inhibit ApoB in said cell.
[0037] The invention provides for the compound of the invention for
use in medicine, such as for use as a medicament.
[0038] The invention also provides for an LNA oligomer, comprising
a contiguous region of 12-24 phosphorothioate linked nucleosides
which are complementary to a corresponding region of a ApoB nucleic
acid target, and further comprising between 1 and 6 DNA nucleosides
which are contiguous with the LNA oligomer, wherein the
internucleoside linkages between the DNA, and/or adjacent to the
DNA nucleoside(s), is physiologically labile, such as is/are
phosphodiester linkages. Such an LNA oligomer may be in the form of
a conjugate, as described herein, or may, for example be an
intermediate to be used in a subsequent conjugation step. When
conjugated, the conjugate may, for example be or comprise a sterol,
such as cholesterol or tocopherol, or may be or comprise a
(non-nucleotide) carbohydrate, such as a GalNac conjugate, such as
a GalNac cluster, e.g. triGalNac, or another conjugate as described
herein.
[0039] The invention provides for an LNA antisense oligomer (which
may be referred to as region A herein) comprising an antisense
oligomer comprising a contiguous region of 12-24 phosphorothioate
linked nucleosides which are complementary to a corresponding
region of a ApoB nucleic acid target, and an asialoglycoprotein
receptor targeting moiety conjugate moiety, such as a GalNAc
moiety, which may form part of a further region (referred to as
region C). The LNA antisense oligomer may be 12-24, and may be in
the form of a gapmer oligomer.
BRIEF DESCRIPTION OF FIGURES
[0040] FIG. 1: Non-limiting illustration of oligomers of the
invention attached to an activation group (i.e. a protected
reactive group--as the third region). The internucleoside linkage L
may be, for example phosphodiester, phosphorothioate,
phosphorodithioate, boranophosphate or methylphosphonate, such as
phosphodiester. PO is a phosphodiester linkage. Compound a) has a
region B with a single DNA or RNA, the linkage between the second
and the first region is PO. Compound b) has two DNA/RNA (such as
DNA) nucleosides linked by a phosphodiester linkage. Compound c)
has three DNA/RNA (such as DNA) nucleosides linked by a
phosphodiester linkages. In some embodiments, Region B may be
further extended by further phosphodiester DNA/RNA (such as DNA
nucleosides). The activation group is illustrated on the left side
of each compound, and may, optionally be linked to the terminal
nucleoside of region B via a phosphorus nucleoside linkage group,
such as phosphodiester, phosphorothioate, phosphorodithioate,
boranophosphate or methylphosphonate, or in some embodiments a
triazole linkage. Compounds d), e), & f) further comprise a
linker (Y) between region B and the activation group, and region Y
may be linked to region B via, for example, a phosphorus nucleoside
linkage group, such as phosphodiester, phosphorothioate,
phosphorodithioate, boranophosphate or methylphosphonate, or in
some embodiments a triazole linkage.
[0041] FIG. 2: Equivalent compounds as shown in FIG. 1, however a
reactive group is used in place of the activation group. The
reactive group may, in some embodiments be the result of activation
of the activation group (e.g. deprotection). The reactive group
may, in non-limiting examples, be an amine or alcohol.
[0042] FIG. 3: Non-limiting Illustration of compounds of the
invention. Same nomenclature as FIG. 1. X may in some embodiments
be a conjugate, such as a lipophilic conjugate such as cholesterol,
or another conjugate such as those described herein. In addition,
or alternatively X may be a targeting group or a blocking group. In
some aspects X may be an activation group (see FIG. 1), or a
reactive group (see FIG. 2). X may be covalently attached to region
B via a phosphorus nucleoside linkage group, such as
phosphodiester, phosphorothioate, phosphorodithioate,
boranophosphate or methylphosphonate, or may be linked via via an
alternative linkage, e.g. a triazol linkage (see L in compounds d),
e), and f)).
[0043] FIG. 4. Non-limiting Illustration of compounds of the
invention, where the compounds comprise the optional linker between
the third region (X) and the second region (region B). Same
nomenclature as FIG. 1. Suitable linkers are disclosed herein, and
include, for example alkyl linkers, for example C6 linkers. In
compounds A, B and C, the linker between X and region B is attached
to region B via a phosphorus nucleoside linkage group, such as
phosphodiester, phosphorothioate, phosphorodithioate,
boranophosphate or methylphosphonate, or may be linked via an
alternative linkage eg. a triazol linkage (Li). In these compounds
Lii represents the internucleoside linkage between the first (A)
and second regions (B).
[0044] FIGS. 5a and b. 5b shows a non-limiting example of a method
of synthesis of compounds of the invention. US represent a
oligonucleotide synthesis support, which may be a solid support. X
is the third region, such as a conjugate, a targeting group, a
blocking group etc. In an optional pre-step, X is added to the
oligonucleotide synthesis support. Otherwise the support with X
already attached may be obtained (i). In a first step, region B is
synthesized (ii), followed by region A (iii), and subsequently the
cleavage of the oligomeric compound of the invention from the
oligonucleotide synthesis support (iv). In an alternative method
the pre-step involves the provision of a oligonucleotide synthesis
support with a region X and a linker group (Y) attached (see FIG.
5a). In some embodiments, either X or Y (if present) is attached to
region B via a phosphorus nucleoside linkage group, such as
phosphodiester, phosphorothioate, phosphorodithioate,
boranophosphate or methylphosphonate, or an alternative linkage,
such as a triazol linkage.
[0045] FIG. 6. A non-limiting example of a method of synthesis of
compounds of the invention which comprise a linker (Y) between the
third region (X) and the second region (B). US represents a
oligonucleotide synthesis support, which may be a solid support. X
is the third region, such as a conjugate, a targeting group, a
blocking group etc. In an optional pre-step, Y is added to the
oligonucleotide synthesis support. Otherwise the support with Y
already attached may be obtained (i). In a first step, region B is
synthesized (ii), followed by region A (iii), and subsequently the
cleavage of the oligomeric compound of the invention from the
oligonucleotide synthesis support (iv). In some embodiments (as
shown), region X may be added to the linker (Y) after the cleavage
step (v). In some embodiments, Y is attached to region B via a
phosphorus nucleoside linkage group, such as phosphodiester,
phosphorothioate, phosphorodithioate, boranophosphate or
methylphosphonate, or an alternative linkage, such as a triazol
linkage.
[0046] FIG. 7. A non-limiting example of a method of synthesis of
compounds of the invention which utilize an activation group. In an
optional pre-step, the activation group is attached the
oligonucleotide synthesis support (i), or the oligonucleotide
synthesis support with activation group is otherwise obtained. In
step ii) region B is synthesized, followed by region A (iii). The
oligomer is then cleaved from the oligonucleotide synthesis support
(iv). The intermediate oligomer (comprising an activation group)
may then be activated (vl) or (viii) and a third region (X) added
(vi), optionally via a linker (Y) (ix). In some embodiments, X (or
Y when present) is attached to region B via a phosphorus nucleoside
linkage group, such as phosphodiester, phosphorothioate,
phosphorodithioate, boranophosphate or methylphosphonate, or an
alternative linkage, such as a triazol linkage.
[0047] FIG. 8. A non-limiting example of a method of synthesis of
compounds of the invention, wherein a bifunctional oligonucleotide
synthesis support is used (i). In such a method, either the
oligonucleotide is synthesized in an initial series of steps
(ii)-(iii), followed by the attachment of the third region
(optionally via a linker group Y), the oligomeric compound of the
invention may then be cleaved (v). Alternatively, as shown in steps
(vi)-(ix), the third region (optionally with a linker group (Y) is
attached to the oligonucleotide synthesis support (this may be an
optional pre-step)--or a oligonucleotide synthesis support with the
third region (optionally with Y) is otherwise provided, the
oligonucleotide is then synthesized (vii-viii). The oligomeric
compound of the invention may then be cleaved (ix). In some
embodiments, X (or Y when present) is attached to region B via a
phosphorus nucleoside linkage group, such as phosphodiester,
phosphorothioate, phosphorodithioate, boranophosphate or
methylphosphonate, or an alternative linkage, such as a triazol
linkage. The US may in some embodiment, prior to the method (such
as the pre-step) comprise a step of adding a bidirectional
(bifunctional) group which allows the independent synthesis of the
oligonucleotide and the covalent attachment of group X, Y (or X and
Y) to support (as shown)--this may for example be achieved using a
triazol or of nucleoside group. The bidirectional (bifunctional)
group, with the oligomer attached, may then be cleaved from the
support.
[0048] FIG. 9. A non-limiting example of a method of synthesis of
compounds of the invention: In an initial step, the first region
(A) is synthesized (ii), followed by region B. In some embodiments
the third region is then attached to region B (iii), optionally via
a phosphate nucleoside linkage (or e.g. a triazol linkage). The
oligomeric compound of the invention may then be cleaved (iv). When
a linker (Y) is used, in some embodiments the steps (v)-(viii) may
be followed: after synthesis of region B, the linker group (Y) is
added, and then either attached to (Y) or in a subsequent step,
region X is added (vi). The oligomeric compound of the invention
may then be cleaved (vii). In some embodiments, X (or Y when
present) is attached to region B via a phosphorus nucleoside
linkage group, such as phosphodiester, phosphorothioate,
phosphorodithioate, boranophosphate or methylphosphonate, or an
alternative linkage, such as a triazol linkage.
[0049] FIG. 10. A non-limiting example of a method of synthesis of
compounds of the invention: In this method an activation group is
used: Steps (i)-(iii) are as per FIG. 9. However after the
oligonucleotide synthesis (step iii), an activation group (or a
reactive group) is added to region B, optionally via a phosphate
nucleoside linkage. The oligonucleotide is then cleaved from the
support (v). The activation group may be subsequently activated to
produce a reactive group, and then the third region (X), such as
the conjugate, blocking group or targeting group, is added to the
reactive group (which may be the activated activation group or the
reactive group), to produce the oligomer (vi). As shown in
(vii)-(viii), after cleavage, a linker group (Y) is added (vii),
and then either attached to (Y) or in a subsequent step, region X
is added to produce the oligomer (viii). It should be recognized
that in an alternative all of the steps (ii)-(viii) may be
performed on the oligonucleotide synthesis support, and in such
instances a final step of cleaving the oligomer from the support
may be performed. In some embodiments, the reactive group or
activation group is attached to region B via a phosphorus
nucleoside linkage group, such as phosphodiester, phosphorothioate,
phosphorodithioate, boranophosphate or methylphosphonate, or an
alternative linkage, such as a triazol linkage.
[0050] FIG. 11. Silencing of ApoB mRNA with Cholesterol-conjugates
in vivo. Mice were injected with a single dose of 1 mg/kg
unconjugated LNA-antisense oligonucleotide (#3833) or equimolar
amounts of LNA antisense oligonucleotides conjugated to Cholesterol
with different linkers (Tab. 3) and sacrificed at days 1, 3, 7 and
10 after dosing. RNA was isolated from liver and kidney and
subjected to ApoB specific RT-qPCR A. Quantification of ApoB mRNA
from liver samples normalized to GAPDH and shown as percentage of
the average of equivalent saline controls B. Quantification of ApoB
mRNA from kidney samples normalized to GAPDH and shown as
percentage of the average of equivalent saline controls.
[0051] FIG. 12. Shows the cholesterol C6 conjugate which may be
used as X-Y- in compounds of the invention, as well as specific
compounds used in the examples, include specific compounds of the
invention.
[0052] FIG. 13: Examples of tri-GalNac conjugates which may be
used. Conjugates 1-4 illustrate 4 suitable GalNac conjugate
moieties, and conjugates 1a-4a refer to the same conjugates with an
additional linker moiety (Y) which is used to link the conjugate to
the oligomer (region A or to a biocleavable linker, such as region
B). The wavy line represents the covalent link to the oligomer.
[0053] FIG. 14: Examples of cholesterol and tocopherol conjugate
moieties. The wavy line represents the covalent link to the
oligomer.
[0054] FIG. 15: In vivo silencing of ApoB mRNA with different
conjugates (See example 4). Mice were treated with 1 mg/kg of ASO
with different conjugates either without biocleavable linker, with
Dithio-linker (SS) or with DNA/PO-linker (PO). RNA was isolated
from liver (A) and kidney samples (B) and analysed for ApoB mRNA
knock down. Data is shown compared to Saline (=1).
[0055] FIG. 16: Example 8--ApoB mRNA expression
[0056] FIG. 17: Example 8--Total cholesterol in serum
[0057] FIG. 18: Example 8--Oligonucleotide content in liver and
kidney
DETAILED DESCRIPTION OF INVENTION
[0058] In some embodiments, the invention relates to oligomeric
compounds which targets an ApoB nucleic acid, such as LNA antisense
oligonucleotides, which are covalently linked to a conjugate group,
a targeting group, a reactive group, an activation group, or a
blocking group, via a short region comprising (e.g. 1-10) of
phosphodiester linked DNA or RNA nucleoside(s).
The Oligomer
[0059] The term "oligomer" in the context of the present invention,
refers to a molecule formed by covalent linkage of two or more
nucleotides (i.e. an oligonucleotide). Herein, a single nucleotide
(unit) may also be referred to as a monomer or unit. In some
embodiments, the terms "nucleoside", "nucleotide", "unit" and
"monomer" are used interchangeably. It will be recognized that when
referring to a sequence of nucleotides or monomers, what is
referred to is the sequence of bases, such as A, T, G, C or U.
[0060] The oligomer of the invention may be an LNA oligomer, i.e.
comprises at least one LNA nucleoside unit, or a gapmer, such as an
LNA gapmer.
[0061] The oligomer of the invention may comprise between 10-22,
such as 12-22 nucleotides in length. The oligomer of the invention
may comprise a contiguous sequence of 10-20 nucleotides which are
complementary, such as fully complementary, to a corresponding
length of the ApoB nucleic acid target (such as NM.sub.--000384 or
genbank accession No: NG.sub.--011793, NM.sub.--000384.2
GI:105990531 and NG.sub.--011793.1 GI:226442987 are hereby
incorporated by reference). The contiguous sequence of 10-20
nucleotides may linked, for example, by phosphorothioate
linkages.
[0062] For example, the oligomer of the invention may comprise the
sequence of nucleobases shown in SEQ ID NO 1 or SEQ ID No 2.
[0063] The compound (e.g. oligomer or conjugate) of the invention
targets ApoB, and as such is capable of inhibiting ApoB, such as
human ApoB, in a cell expressing said ApoB.
[0064] In some embodiments, the internucleoside linkages of the
contiguous sequence may be phosphorothioate linkages, and may
comprise affinity enhancing nucleotide analogues.
[0065] In some embodiments, the nucleotide analogues are sugar
modified nucleotides, such as sugar modified nucleotides
independently or dependently selected from the group consisting of:
Locked Nucleic Acid (LNA or BNA) units; 2'-O-alkyl-RNA units,
2'-OMe-RNA units, 2'-amino-DNA units, and 2'-fluoro-DNA units.
[0066] In some embodiments, the nucleotide analogues comprise or
are Locked Nucleic Acid (LNA, also known as BNA) units.
[0067] In some embodiments, the oligomer of the invention comprises
or is a gapmer, such as a LNA gapmer oligonucleotide.
[0068] In some embodiments, the oligomer of the invention comprises
a contiguous sequence of 13, 14, 15 or 16 nucleotides which are
complementary to a corresponding length of the ApoB nucleic acid
target, and may optionally comprise a further 1-10, for example 1-6
nucleotides, which may form or comprise a biocleavable nucleotide
region, such as a phosphate nucleotide linker. Suitably, the
biocleavable nucleotide region is formed of a short stretch (eg. 1,
2, 3, 4, 5 or 6) of nucleotides which are physiologically labile.
This may be achieved by using phosphodiester linkages with DNA/RNA
nucleosides, or if physiological liability can be maintained, other
nucleosides may be used.
[0069] The oligomer of the invention may therefore comprise of a
contiguous nucleotide sequence of 10-20 nts in length which is
complementary to a corresponding length of the ApoB nucleic acid
target (A first region, or region A). The oligomer of the invention
may comprise a further nucleotide region. In some embodiments, the
further nucleotide region comprises a biocleavable nucleotide
region, such as a phosphate nucleotide sequence (a second region,
region B), which may covalently link region A to a non-nucleotide
moiety, such as a conjugate group, (a third region, or region C).
In some embodiments the contiguous nucleotide sequence of the
oligomer of the invention (region A) is directly covalently linked
to region C. In some embodiments region C is biocleavable.
[0070] The may oligomer consists or comprises of a contiguous
nucleotide sequence of from 10-22, such as 13, 14, 15, 16, 17, 18,
19, 20, 21, nucleotides in length, such as 13-16, or 13 or 14, or
15 or 16 nucleotides in length. The oligomer may therefore refer to
the combined length of region A and region B, e.g. (Region A 10-16
nt) and region B (1-6 nt).
[0071] In various embodiments, the compound of the invention does
not comprise RNA (units). In some embodiments, the compound
according to the invention, the first region, or the first and
second regions together (e.g. as a single contiguous sequence), is
a linear molecule or is synthesized as a linear molecule. The
oligomer may therefore be single stranded molecule. In some
embodiments, the oligomer does not comprise short regions of, for
example, at least 3, 4 or 5 contiguous nucleotides, which are
complementary to equivalent regions within the same oligomer (i.e.
duplexes). The oligomer, in some embodiments, may be not
(essentially) double stranded. In some embodiments, the oligomer is
essentially not double stranded, such as is not a siRNA.
The Target
[0072] Suitably the oligomer of the invention is capable of
down-regulating expression of the APO-B gene, such as ApoB-100 or
ApoB-48 (APOB). In this regards, the oligomer of the invention can
affect the inhibition of APOB, typically in a mammalian such as a
human cell, such as liver cells. In some embodiments, the oligomers
of the invention bind to the target nucleic acid and effect
inhibition of expression of at least 10% or 20% compared to the
normal expression level, more preferably at least a 30%, 40%, 50%,
60%, 70%, 80%, 90% or 95% inhibition compared to the normal
expression level. In some embodiments, such modulation is seen when
using between 0.04 and 25 nM, such as between 0.8 and 20 nM
concentration of the compound of the invention. In the same or a
different embodiment, the inhibition of expression is less than
100%, such as less than 98% inhibition, less than 95% inhibition,
less than 90% inhibition, less than 80% inhibition, such as less
than 70% inhibition. Modulation of expression level may be
determined by measuring protein levels, e.g. by the methods such as
SDS-PAGE followed by western blotting using suitable antibodies
raised against the target protein. Alternatively, modulation of
expression levels can be determined by measuring levels of mRNA,
e.g. by northern blotting or quantitative RT-PCR. When measuring
via mRNA levels, the level of down-regulation when using an
appropriate dosage, such as between 0.04 and 25 nM, such as between
0.8 and 20 nM concentration, is, In some embodiments, typically to
a level of between 10-20% the normal levels in the absence of the
compound of the invention.
[0073] The invention therefore provides a method of down-regulating
or inhibiting the expression of APO-B protein and/or mRNA in a cell
which is expressing APO-B protein and/or mRNA, said method
comprising administering the compound of the invention to the
invention to said cell to down-regulating or inhibiting the
expression of APO-B protein and/or mRNA in said cell. Suitably the
cell is a mammalian cell such as a human cell. The administration
may occur, in some embodiments, in vitro. The administration may
occur, in some embodiments, in vivo.
[0074] The term "target nucleic acid", as used herein refers to the
DNA or RNA encoding mammalian APO-B polypeptide, such as human
APO-B100, such as human APO-B100 mRNA. APO-B100 encoding nucleic
acids or naturally occurring variants thereof, and RNA nucleic
acids derived therefrom, preferably mRNA, such as pre-mRNA,
although preferably mature mRNA. In some embodiments, for example
when used in research or diagnostics the "target nucleic acid" may
be a cDNA or a synthetic oligonucleotide derived from the above DNA
or RNA nucleic acid targets. The oligomer according to the
invention is preferably capable of hybridising to the target
nucleic acid. It will be recognised that human APO-B mRNA is a cDNA
sequence, and as such, corresponds to the mature mRNA target
sequence, although uracil is replaced with thymidine in the cDNA
sequences.
[0075] The term "naturally occurring variant thereof" refers to
variants of the APO-B1 polypeptide of nucleic acid sequence which
exist naturally within the defined taxonomic group, such as
mammalian, such as mouse, monkey, and preferably human. Typically,
when referring to "naturally occurring variants" of a
polynucleotide the term also may encompass any allelic variant of
the APO-B encoding genomic DNA by chromosomal translocation or
duplication, and the RNA, such as mRNA derived therefrom.
"Naturally occurring variants" may also include variants derived
from alternative splicing of the APO-B100 mRNA. When referenced to
a specific polypeptide sequence, e.g., the term also includes
naturally occurring forms of the protein which may therefore be
processed, e.g. by co- or post-translational modifications, such as
signal peptide cleavage, proteolytic cleavage, glycosylation,
etc.
[0076] The oligomers (region A) may comprise or consist of a
contiguous nucleotide sequence which corresponds to the reverse
complement of a nucleotide sequence present in e.g. the human APO-B
mRNA.
[0077] The oligomer (region A) may comprise or consist of a
contiguous nucleotide sequence which is fully complementary
(perfectly complementary) to the equivalent region of a nucleic
acid which encodes a mammalian APO-B (e.g., human APO-B100 mRNA).
Thus, the oligomer (region A) can comprise or consist of an
antisense nucleotide sequence.
[0078] However, in some embodiments, the oligomer may tolerate 1 or
2 mismatches, when hybridising to the target sequence and still
sufficiently bind to the target to show the desired effect, i.e.
down-regulation of the target. Mismatches may, for example, be
compensated by increased length of the oligomer nucleotide sequence
and/or an increased number of nucleotide analogues, such as LNA,
present within the nucleotide sequence.
[0079] It is recognised that, in some embodiments the nucleotide
sequence of the oligomer may comprise additional 5' or 3'
nucleotides, such as, independently, 1, 2, 3, 4, 5 or 6 additional
nucleotides 5' and/or 3', which are non-complementary to the target
sequence--such non-complementary oligonucleotides may form region
B. In this respect the oligomer of the invention, may, in some
embodiments, comprise a contiguous nucleotide sequence which is
flanked 5' and or 3' by additional nucleotides. In some embodiments
the additional 5' or 3' nucleotides are naturally occurring
nucleotides, such as DNA or RNA. In some embodiments, the
additional 5' or 3' nucleotides may represent region D as referred
to in the context of gapmer oligomers herein. In some embodiments
the internucleoside linkages between the additional nucleotides,
and optionally between the additional nucleotides and the oligomer
are phosphodiester linkages.
[0080] In some embodiments the oligomer according to the invention
consists or comprises of a nucleotide sequence according to SEQ ID
NO:1, or a sub-sequence of at least 10 or 12 nucleobases
thereof.
[0081] In some embodiments the oligomer according to the invention
consists or comprises of a nucleotide sequence according to SEQ ID
NO:2, or a sub-sequence of at least 10 or 12 nucleobases
thereof.
[0082] The following Table provides specific combinations of
oligomer and conjugates:
TABLE-US-00001 TABLE 1 Oligomer/conjugate combinations. Conjugate
Number (See figures) SEQ ID Conj1 Conj2 Conj3 Conj4 Conj1a Conj2a
Conj3a Conj4a Conj5 Conj6 1 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 2 C11
C12 C13 C14 C15 C16 C17 C18 C19 C20 Please note that a biocleavable
linker (B) may or may not be present between the conjugate
moiety(C) and the oligomer(A). For Conj1-4 and 1a-4a the GalNac
conjugate itself is biocleavable, utilizing a peptide linker in the
GalNac cluster, and as such a further biocleavable linker (B) may
or may not be used. However, preliminary data indicates inclusion
of a biocleavable linker (B), such as the phosphate nucleotide
linkers disclosed herein may enhance activity of such GalNac
cluster oligomer conjugates. For use with Conj 5 and Conj 6, the
use of a biocleavable linker greatly enhances compound activity
inclusion of a biocleavable linker (B), such as the phosphate
nucleotide linkers disclosed herein is recommended. The conjugate
moiety (and region B or region Y or B and Y, may be positioned,
e.g. 5' or 3' to the SEQ ID, such as 5' to region A.
[0083] The terms "corresponding to" and "corresponds to" refer to
the comparison between the nucleotide sequence of the oligomer
(i.e. the nucleobase or base sequence) or contiguous nucleotide
sequence (a first region/region A) and the reverse complement of
the nucleic acid target, or sub-region thereof.
[0084] Nucleotide analogues are compared directly to their
equivalent or corresponding nucleotides. In a preferred embodiment,
the oligomers (or first region thereof) are complementary to the
target region or sub-region, such as fully complementary.
[0085] The terms "reverse complement", "reverse complementary" and
"reverse complementarity" as used herein are interchangeable with
the terms "complement", "complementary" and "complementarity".
[0086] The terms "corresponding nucleotide analogue" and
"corresponding nucleotide" are intended to indicate that the
nucleotide in the nucleotide analogue and the naturally occurring
nucleotide are identical. For example, when the 2-deoxyribose unit
of the nucleotide is linked to an adenine, the "corresponding
nucleotide analogue" contains a pentose unit (different from
2-deoxyribose) linked to an adenine.
[0087] The term "nucleobase" refers to the base moiety of a
nucleotide and covers both naturally occurring a well as
non-naturally occurring variants. Thus, "nucleobase" covers not
only the known purine and pyrimidine heterocycles but also
heterocyclic analogues and tautomeres thereof. It will be
recognised that the DNA or RNA nucleosides of region B may have a
naturally occurring and/or non-naturally occurring
nucleobase(s).
[0088] Examples of nucleobases include, but are not limited to
adenine, guanine, cytosine, thymidine, uracil, xanthine,
hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine,
5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine,
inosine, diaminopurine, and 2-chloro-6-aminopurine. In some
embodiments the nucleobases may be independently selected from the
group consisting of adenine, guanine, cytosine, thymidine, uracil,
5-methylcytosine. In some embodiments the nucleobases may be
independently selected from the group consisting of adenine,
guanine, cytosine, thymidine, and 5-methylcytosine.
[0089] In some embodiments, at least one of the nucleobases present
in the oligomer is a modified nucleobase selected from the group
consisting of 5-methylcytosine, isocytosine, pseudoisocytosine,
5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine,
inosine, diaminopurine, and 2-chloro-6-aminopurine.
Length
[0090] The oligomers may comprise or consist of a contiguous
nucleotide sequence of a total of between 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides in length.
Lengths may include region A or region A and B for example.
[0091] In some embodiments, the oligomers comprise or consist of a
contiguous nucleotide sequence of a total of between 10-22, such as
12-18, such as 13-17 or 12-16, such as 13, 14, 15, 16 contiguous
nucleotides in length.
[0092] In some embodiments, the oligomer according to the invention
consists of no more than 22 nucleotides, such as no more than 20
nucleotides, such as no more than 18 nucleotides, such as 15, 16 or
17 nucleotides. In some embodiments the oligomer of the invention
comprises less than 20 nucleotides.
Nucleotide Analogues
[0093] The term "nucleotide" as used herein, refers to a glycoside
comprising a sugar moiety, a base moiety and a covalently linked
group, such as a phosphate or phosphorothioate internucleotide
linkage group, and covers both naturally occurring nucleotides,
such as DNA or RNA, and non-naturally occurring nucleotides
comprising modified sugar and/or base moieties, which are also
referred to as "nucleotide analogues" herein. Herein, a single
nucleotide (unit) may also be referred to as a monomer or nucleic
acid unit.
[0094] In field of biochemistry, the term "nucleoside" is commonly
used to refer to a glycoside comprising a sugar moiety and a base
moiety, and may therefore be used when referring to the nucleotide
units, which are covalently linked by the internucleotide linkages
between the nucleotides of the oligomer.
[0095] As one of ordinary skill in the art would recognise, the 5'
nucleotide of an oligonucleotide does not comprise a 5'
internucleotide linkage group, although may or may not comprise a
5' terminal group.
[0096] Non-naturally occurring nucleotides include nucleotides
which have modified sugar moieties, such as bicyclic nucleotides or
2' modified nucleotides, such as 2' substituted nucleotides.
[0097] "Nucleotide analogues" are variants of natural nucleotides,
such as DNA or RNA nucleotides, by virtue of modifications in the
sugar and/or base moieties. Analogues could in principle be merely
"silent" or "equivalent" to the natural nucleotides in the context
of the oligonucleotide, i.e. have no functional effect on the way
the oligonucleotide works to inhibit target gene expression. Such
"equivalent" analogues may nevertheless be useful if, for example,
they are easier or cheaper to manufacture, or are more stable to
storage or manufacturing conditions, or represent a tag or label.
Preferably, however, the analogues will have a functional effect on
the way in which the oligomer works to inhibit expression; for
example by producing increased binding affinity to the target
and/or increased resistance to intracellular nucleases and/or
increased ease of transport into the cell. Specific examples of
nucleoside analogues are described by e.g. Freier & Altmann;
Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in
Drug Development, 2000, 3(2), 293-213, and in Scheme 1:
##STR00001## ##STR00002##
[0098] The oligomer may thus comprise or consist of a simple
sequence of natural occurring nucleotides--preferably
2'-deoxynucleotides (referred here generally as "DNA"), but also
possibly ribonucleotides (referred here generally as "RNA"), or a
combination of such naturally occurring nucleotides and one or more
non-naturally occurring nucleotides, i.e. nucleotide analogues.
Such nucleotide analogues may suitably enhance the affinity of the
oligomer for the target sequence.
[0099] Examples of suitable and preferred nucleotide analogues are
provided by WO2007/031091 or are referenced therein. Other
nucleotide analogues which may be used in the oligomer of the
invention include tricyclic nucleic acids, for example please see
WO2013154798 and WO2013154798 which are hereby incorporated by
reference.
[0100] Incorporation of affinity-enhancing nucleotide analogues in
the oligomer, such as LNA or 2'-substituted sugars, can allow the
size of the specifically binding oligomer to be reduced, and may
also reduce the upper limit to the size of the oligomer before
non-specific or aberrant binding takes place.
[0101] In some embodiments the oligomer comprises at least 2
nucleotide analogues. In some embodiments, the oligomer comprises
from 3-8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues. In
the by far most preferred embodiments, at least one of said
nucleotide analogues is a locked nucleic acid (LNA); for example at
least 3 or at least 4, or at least 5, or at least 6, or at least 7,
or 8, of the nucleotide analogues may be LNA. In some embodiments
all the nucleotides analogues may be LNA.
[0102] It will be recognised that when referring to a preferred
nucleotide sequence motif or nucleotide sequence, which consists of
only nucleotides, the oligomers of the invention which are defined
by that sequence may comprise a corresponding nucleotide analogue
in place of one or more of the nucleotides present in said
sequence, such as LNA units or other nucleotide analogues, which
raise the duplex stability/T.sub.m of the oligomer/target duplex
(i.e. affinity enhancing nucleotide analogues).
[0103] T.sub.m Assay: The oligonucleotide: Oligonucleotide and RNA
target (PO) duplexes are diluted to 3 mM in 500 ml RNase-free water
and mixed with 500 ml 2.times. T.sub.m-buffer (200 mM NaCl, 0.2 mM
EDTA, 20 mM Naphosphate, pH 7.0). The solution is heated to
95.degree. C. for 3 min and then allowed to anneal in room
temperature for 30 min. The duplex melting temperatures (T.sub.m)
is measured on a Lambda 40 UV/VIS Spectrophotometer equipped with a
Peltier temperature programmer PTP6 using PE Templab software
(Perkin Elmer). The temperature is ramped up from 20.degree. C. to
95.degree. C. and then down to 25.degree. C., recording absorption
at 260 nm. First derivative and the local maximums of both the
melting and annealing are used to assess the duplex T.sub.m.
LNA
[0104] The term "LNA" refers to a bicyclic nucleoside analogue
which comprises a C2*-C4* biradical (a bridge), and is known as
"Locked Nucleic Acid". It may refer to an LNA monomer, or, when
used in the context of an "LNA oligonucleotide", LNA refers to an
oligonucleotide containing one or more such bicyclic nucleotide
analogues. In some aspects bicyclic nucleoside analogues are LNA
nucleotides, and these terms may therefore be used interchangeably,
and is such embodiments, both are be characterized by the presence
of a linker group (such as a bridge) between C2' and C4' of the
ribose sugar ring.
[0105] In some embodiments, at least one nucleoside analogue
present in the first region (A) is a bicyclic nucleoside analogue,
such as at least 2, at least 3, at least 4, at least 5, at least 6,
at least 7, at least 8, (except the DNA and or RNA nucleosides of
region B) are sugar modified nucleoside analogues, such as such as
bicyclic nucleoside analogues, such as LNA, e.g. beta-D-X-LNA or
alpha-L-X-LNA (wherein X is oxy, amino or thio), or other LNAs
disclosed herein including, but not limited to, (R/S) cET, cMOE or
5'-Me-LNA.
[0106] In some embodiments the LNA used in the oligonucleotide
compounds of the invention preferably has the structure of the
general formula II:
##STR00003##
wherein Y is selected from the group consisting of --O--,
--CH.sub.2O--, --S--, --NH--, N(R.sup.e) and/or --CH.sub.2--; Z and
Z* are independently selected among an internucleotide linkage,
R.sup.H, a terminal group or a protecting group; B constitutes a
natural or non-natural nucleotide base moiety (nucleobase), and
R.sup.H is selected from hydrogen and C.sub.1-4-alkyl; R.sup.a,
R.sup.b R.sup.c, R.sup.d and R.sup.e are, optionally independently,
selected from the group consisting of hydrogen, optionally
substituted C.sub.1-12-alkyl, optionally substituted
C.sub.2-12-alkenyl, optionally substituted C.sub.2-12-alkynyl,
hydroxy, C.sub.1-12-alkoxy, C.sub.2-12-alkoxyalkyl,
C.sub.2-12-alkenyloxy, carboxy, C.sub.1-12-alkoxycarbonyl,
C.sub.1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,
arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,
heteroarylcarbonyl, amino, mono- and di(C.sub.1-6-alkyl)amino,
carbamoyl, mono- and di(C.sub.1-6-alkyl)-amino-carbonyl,
amino-C.sub.1-6-alkyl-aminocarbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands, where aryl and heteroaryl may be
optionally substituted and where two geminal substituents R.sup.a
and R.sup.b together may designate optionally substituted methylene
(.dbd.CH.sub.2); and R.sup.H is selected from hydrogen and
C.sub.1-4-alkyl. In some embodiments R.sup.a, R.sup.b R.sup.c,
R.sup.d and R.sup.e are, optionally independently, selected from
the group consisting of hydrogen and C.sub.1-6 alkyl, such as
methyl. For all chiral centers, asymmetric groups may be found in
either R or S orientation, for example, two exemplary
stereochemical isomers include the beta-D and alpha-L isoforms,
which may be illustrated as follows:
##STR00004##
[0107] Specific exemplary LNA units are shown below:
##STR00005##
[0108] The term "thio-LNA" comprises a locked nucleotide in which Y
in the general formula above is selected from S or --CH.sub.2--S--.
Thio-LNA can be in both beta-D and alpha-L-configuration.
[0109] The term "amino-LNA" comprises a locked nucleotide in which
Y in the general formula above is selected from --N(H)--, N(R)--,
CH.sub.2--N(H)--, and --CH.sub.2--N(R)-- where R is selected from
hydrogen and C.sub.1-4-alkyl. Amino-LNA can be in both beta-D and
alpha-L-configuration.
[0110] The term "oxy-LNA" comprises a locked nucleotide in which Y
in the general formula above represents --O--. Oxy-LNA can be in
both beta-D and alpha-L-configuration.
[0111] The term "ENA" comprises a locked nucleotide in which Y in
the general formula above is --CH.sub.2--O-- (where the oxygen atom
of --CH.sub.2--O-- is attached to the 2'-position relative to the
base B). R.sup.e is hydrogen or methyl.
[0112] In some exemplary embodiments LNA is selected from
beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and
beta-D-thio-LNA, in particular beta-D-oxy-LNA.
[0113] As used herein, "bicyclic nucleosides" refer to modified
nucleosides comprising a bicyclic sugar moiety. Examples of
bicyclic nucleosides include, without limitation, nucleosides
comprising a bridge between the 4' and the 2' ribosyl ring atoms.
In some embodiments, compounds provided herein include one or more
bicyclic nucleosides wherein the bridge comprises a 4' to 2'
bicyclic nucleoside. Examples of such 4' to 2' bicyclic
nucleosides, include, but are not limited to, one of the formulae:
4'-(CH.sub.2)--O-2' (LNA); 4'-(CH.sub.2)--S-2';
4'-(CH.sub.2).sub.2--O-2' (ENA); 4'-CH(CH.sub.3)--O-2' and
4'-CH(CH.sub.2OCH.sub.3)--O-2', and analogs thereof (see, U.S. Pat.
No. 7,399,845, issued on Jul. 15, 2008);
4'-C(CH.sub.3)(CH.sub.3)--O-2', and analogs thereof (see, published
PCT International Application WO2009/006478, published Jan. 8,
2009); 4'-CH.sub.2--N(OCH.sub.3)-2', and analogs thereof (see,
published PCT International Application WO2008/150729, published
Dec. 11, 2008); 4'-CH.sub.2--O--N(CH.sub.3)-2' (see, published U.S.
Patent Application US2004/0171570, published Sep. 2, 2004);
4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.10 alkyl, or
a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep.
23, 2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see, Chattopadhyaya, et
al, J. Org. Chem., 2009, 74, 118-134); and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2', and analogs thereof (see,
published PCT International Application WO 2008/154401, published
on Dec. 8, 2008). Also see, for example: Singh et al., Chem.
Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,
3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000,
97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,
2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;
Srivastava et al., J. Am. Chem. Soc, 129(26) 8362-8379 (Jul. 4,
2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,
558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; Oram et al,
Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos.
6,670,461, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133,
6,525,191, 7,399,845; published PCT International applications WO
2004/106356, WO 94/14226, WO 2005/021570, and WO 2007/134181; U.S.
Patent Publication Nos. US2004/0171570, US2007/0287831, and
US2008/0039618; and U.S. patent Ser. No. 12/129,154, 60/989,574,
61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and
61/099,844; and PCT International Application Nos.
PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922. Each
of the foregoing bicyclic nucleosides can be prepared having one or
more stereochemical sugar configurations including for example
a-L-ribofuranose and beta-D-ribofuranose (see PCT international
application PCT DK98/00393, published on Mar. 25, 1999 as WO
99/14226).
[0114] In some embodiments, bicyclic sugar moieties of BNA
nucleosides include, but are not limited to, compounds having at
least one bridge between the 4' and the 2' position of the
pentofuranosyl sugar moiety wherein such bridges independently
comprises 1 or from 2 to 4 linked groups independently selected
from -[CiR.sub.aXR.sub.b)]--, --C(R.sub.a).dbd.C(R.sub.b)--,
--C(R.sub.a).dbd.N--, --C(.dbd.NR.sub.a)--, --C(.dbd.O)--,
--C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--,
and --N(Ra)--; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each
R.sub.a and R.sub.b is, independently, H, a protecting group,
hydroxyl, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12
alkyl, C.sub.2-Ci.sub.2 alkenyl, substituted C.sub.2-C.sub.12
alkenyl, C.sub.2-Ci.sub.2 alkynyl, substituted C.sub.2-C.sub.12
alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl,
heterocycle radical, substituted heterocycle radical, heteroaryl,
substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical,
substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl
(C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.6
alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12
alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12
alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20
aryl, substituted C.sub.5-C.sub.2o aryl, acyl (C(.dbd.O)--H),
substituted acyl, a heterocycle radical, a substituted heterocycle
radical, C1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12
aminoalkyl, or a protecting group.
[0115] In some embodiments, the bridge of a bicyclic sugar moiety
is, --[C(R.sub.a)(Rb)].sub.n--, --[C(R.sub.a)(R.sub.b)].sub.n--O--,
--C(R.sub.aR.sub.b)--N(R)--O-- or, --C(R.sub.aR.sub.b)--O--N(R)--.
In some embodiments, the bridge is 4'-CH.sub.2-2',
4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2', 4'-CH.sub.2--O-2',
4*-(CH.sub.2)2-O-2', 4'- CH.sub.2--O--N(R)-2', and
4'-CH.sub.2--N(R)--O-2'-, wherein each R is, independently, H, a
protecting group, or C.sub.1-C.sub.12 alkyl.
[0116] In some embodiments, bicyclic nucleosides are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-2' methylene-oxy bridge, may be in the a-L
configuration or in the beta-D configuration. Previously,
a-L-methyleneoxy (4'-CH.sub.2--O-2') BNA's have been incorporated
into antisense oligonucleotides that showed antisense activity
(Frieden et al, Nucleic Acids Research, 2003, 21, 6365-6372).
[0117] In some embodiments, bicyclic nucleosides include, but are
not limited to, (A) a-L-Methyleneoxy (4'-CH.sub.2--O-2') BNA, (B)
beta-D-Methyleneoxy (4'-CH.sub.2--O-2') BNA, (C) Ethyleneoxy
(4'-(CH.sub.2).sub.2--O-2') BNA, (D) Aminooxy
(4'-CH.sub.2--O--N(R)-2') BNA, (E) Oxyamino
(4'-CH.sub.2--N(R)--O-2') BNA, (F), Methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA, (G) methylene-thio (4'-CH.sub.2--S-2')
BNA, (H) methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl
carbocyclic (4'-CH.sub.2--CH(CH.sub.3)-2') BNA, and (J) propylene
carbocyclic (4'-(CH.sub.2).sub.3-2') BNA as depicted below.
##STR00006##
[0118] wherein Bx is the base moiety and R is, independently, H, a
protecting group or C.sub.1-C.sub.2 alkyl. odiments, bicyclic
nucleoside having Formula I:
##STR00007##
[0119] wherein:
[0120] Bx is a heterocyclic base moiety;
[0121] -Q.sub.a-Q.sub.b-Q.sub.c- is --CH.sub.2--N(Rc)-CH.sub.2--,
--C(.dbd.O)--N(R.sub.c)--CH.sub.2--, --CH.sub.2--O--N(Rc)-,
--CH.sub.2--N(Rc)-O--, or --N(Rc)-O--CH.sub.2;
[0122] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting
group; and
[0123] T.sub.a and T.sub.b are each, independently, H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety, or a covalent attachment to a support
medium.
[0124] In some embodiments, bicyclic nucleoside having Formula
II:
##STR00008##
[0125] wherein:
[0126] Bx is a heterocyclic base moiety;
[0127] T.sub.a and T.sub.b are each, independently, H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety, or a covalent attachment to a support medium;
Z.sub.a is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl, acyl, substituted acyl, substituted amide, thiol, or
substituted thio.
[0128] In some embodiments, each of the substituted groups is,
independently, mono or poly substituted with substituent groups
independently selected from halogen, oxo, hydroxyl, OJ.sub.c,
NJ.sub.d, SJ.sub.C, N.sub.3, OC(.dbd.X)J.sub.c, and
NJ.sub.eC(.dbd.X)NJ.sub.cJ.sub.d, wherein each J.sub.c, J.sub.d,
and J.sub.e is, independently, H, C.sub.1-C.sub.6 alkyl, or
substituted C.sub.1-C.sub.6 alkyl and X is O or NJ.sub.C.
[0129] In some embodiments, bicyclic nucleoside having Formula
III:
##STR00009##
[0130] wherein:
[0131] Bx is a heterocyclic base moiety;
[0132] T.sub.a and T.sub.b are each, independently, H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety, or a covalent attachment to a support
medium;
[0133] R.sub.d is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl, or substituted acyl (C(.dbd.O)--).
[0134] In some embodiments, bicyclic nucleoside having Formula
IV:
##STR00010##
[0135] wherein:
[0136] Bx is a heterocyclic base moiety;
[0137] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety, or a covalent attachment to a support
medium;
[0138] R.sub.d is C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted
C.sub.2-C.sub.6 alkynyl; each q.sub.b, q.sub.c and q.sub.d is,
independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-Ce alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted
C.sub.2-C6 alkynyl, C.sub.1-C.sub.6 alkoxyl, substituted Q-C.sub.6
alkoxyl, acyl, substituted acyl, C.sub.1-C.sub.6 aminoalkyl, or
substituted C.sub.1-C.sub.6 aminoalkyl;
[0139] In some embodiments, bicyclic nucleoside having Formula
V:
##STR00011##
[0140] wherein:
[0141] Bx is a heterocyclic base moiety;
[0142] T.sub.a and T.sub.b are each, independently, H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety, or a covalent attachment to a support medium;
q.sub.a, q.sub.b, q.sub.c and q.sub.f are each, independently,
hydrogen, halogen, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxy, substituted
C.sub.1-C.sub.12 alkoxy, OJ.sub.j, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j,
C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j,
O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k,
N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k; or
q.sub.e and q.sub.f together are .dbd.C(q.sub.g)(q.sub.h); q.sub.g
and q.sub.h are each, independently, H, halogen, C.sub.1-C.sub.12
alkyl, or substituted C.sub.1-C.sub.12 alkyl.
[0143] The synthesis and preparation of the methyleneoxy
(4'-CH.sub.2--O-2') BNA monomers adenine, cytosine, guanine,
5-methyl-cytosine, thymine, and uracil, along with their
oligomerization, and nucleic acid recognition properties have been
described (see, e.g., Koshkin et al., Tetrahedron, 1998, 54,
3607-3630). BNAs and preparation thereof are also described in WO
98/39352 and WO 99/14226.
[0144] Analogs of methyleneoxy (4'-CH.sub.2--O-2') BNA,
methyleneoxy (4'-CH.sub.2--O-2') BNA, and 2'-thio-BNAs, have also
been prepared {see, e.g., Kumar et al., Bioorg. Med. Chem. Lett.,
1998, 8, 2219-2222). Preparation of locked nucleoside analogs
comprising oligodeoxyribonucleotide duplexes as substrates for
nucleic acid polymerases has also been described (see, e.g., Wengel
et al., WO 99/14226). Furthermore, synthesis of 2'-amino-BNA, a
novel comformationally restricted high-affinity oligonucleotide
analog, has been described in the art (see, e.g., Singh et al., J.
Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and
2'-methylamino-BNA's have been prepared and the thermal stability
of their duplexes with complementary RNA and DNA strands has been
previously reported.
[0145] In some embodiments, the bicyclic nucleoside has Formula
VI:
##STR00012##
[0146] wherein:
[0147] Bx is a heterocyclic base moiety;
[0148] T.sub.a and T.sub.b are each, independently, H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety, or a covalent attachment to a support medium;
each qj, qj, q.sub.k and ql is, independently, H, halogen,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.1-C.sub.12 alkoxyl, substituted C.sub.2-C.sub.12 alkoxyl,
OJ.sub.j, SJ.sub.j, SOJ.sub.j, SO.sub.2J.sub.j, NJ.sub.jJ.sub.k,
N.sub.3, CN, C(.dbd.O)OJ.sub.j, C(.dbd.O)NJ.sub.jJ.sub.k,
C(.dbd.O)J.sub.j, O--C(.dbd.O)NJ.sub.jJ.sub.k,
N(H)C(.dbd.NH)NJ.sub.jJ.sub.k, N(H)C(.dbd.O)NJ.sub.jJ.sub.k, or
(H)C(.dbd.S)NJ.sub.jJ.sub.k; and qi and q.sub.j or ql and q.sub.k
together are .dbd.C(q.sub.g)(q.sub.h), wherein q.sub.g and q.sub.h
are each, independently, H, halogen, C.sub.1-C.sub.12 alkyl, or
substituted C.sub.1-C.sub.6 alkyl.
[0149] One carbocyclic bicyclic nucleoside having a
4'-(CH.sub.2).sub.3-2' bridge and the alkenyl analog, bridge
4'-CH.dbd.CH--CH.sub.2-2', have been described (see, e.g., Freier
et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek
et al, J. Org. Chem., 2006, 71, 7731-77 '40). The synthesis and
preparation of carbocyclic bicyclic nucleosides along with their
oligomerization and biochemical studies have also been described
(see, e.g., Srivastava et al, J. Am. Chem. Soc. 2007, 129(26),
8362-8379).
[0150] As used herein, "4'-2' bicyclic nucleoside" or "4' to 2'
bicyclic nucleoside" refers to a bicyclic nucleoside comprising a
furanose ring comprising a bridge connecting the 2' carbon atom and
the 4' carbon atom.
[0151] As used herein, "monocylic nucleosides" refer to nucleosides
comprising modified sugar moieties that are not bicyclic sugar
moieties. In some embodiments, the sugar moiety, or sugar moiety
analogue, of a nucleoside may be modified or substituted at any
position.
[0152] As used herein, "2'-modified sugar" means a furanosyl sugar
modified at the 2' position. In some embodiments, such
modifications include substituents selected from: a halide,
including, but not limited to substituted and unsubstituted alkoxy,
substituted and unsubstituted thioalkyl, substituted and
unsubstituted amino alkyl, substituted and unsubstituted alkyl,
substituted and unsubstituted allyl, and substituted and
unsubstituted alkynyl. In some embodiments, 2' modifications are
selected from substituents including, but not limited to:
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2),NH.sub.2,
O(CH.sub.2),CH.sub.3, O(CH.sub.2),ONH.sub.2,
OCH.sub.2C(.dbd.O)N(H)CH.sub.3, and
O(CH2).sub.nON[(CH.sub.2).sub.nCH.sub.3]2, where n and m are from 1
to about 10. Other 2'-substituent groups can also be selected from:
C.sub.1-C.sub.12 alkyl; substituted alkyl; alkenyl; alkynyl;
alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH.sub.3; OCN; Cl;
Br; CN; CF.sub.3; OCF.sub.3; SOCH.sub.3; S0.sub.2CH.sub.3;
ONO.sub.2; NO.sub.2; N.sub.3; NH.sub.2; heterocycloalkyl;
heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted
silyl; an R; a cleaving group; a reporter group; an intercalator; a
group for improving pharmacokinetic properties; and a group for
improving the pharmacodynamic properties of an antisense compound,
and other substituents having similar properties. In some
embodiments, modified nucleosides comprise a 2'-MOE side chain
{see, e.g., Baker et al., J. Biol. Chem., 1997, 272, 11944-12000).
Such 2'-MOE substitution have been described as having improved
binding affinity compared to unmodified nucleosides and to other
modified nucleosides, such as 2'-O-methyl, O-propyl, and
O-aminopropyl. Oligonucleotides having the 2-MOE substituent also
have been shown to be antisense inhibitors of gene expression with
promising features for in vivo use {see, e.g., Martin, P., He/v.
Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50,
168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637;
and Altmann et al., Nucleosides Nucleotides, 1997, 16,
917-926).
[0153] As used herein, a "modified tetrahydropyran nucleoside" or
"modified THP nucleoside" means a nucleoside having a six-membered
tetrahydropyran "sugar" substituted in for the pentofuranosyl
residue in normal nucleosides (a sugar surrogate). Modified ?THP
nucleosides include, but are not limited to, what is referred to in
the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),
manitol nucleic acid (MNA) {see Leumann, C J. Bioorg. and Med.
Chem. (2002) 10:841-854), fluoro HNA (F-HNA), or those compounds
having Formula X:
##STR00013##
[0154] X wherein independently for each of said at least one
tetrahydropyran nucleoside analog of Formula X:
[0155] Bx is a heterocyclic base moiety;
[0156] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.3 and
T.sub.4 is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.3 and T4 is H, a hydroxyl protecting group, a linked
conjugate group, or a 5' or 3'-terminal group; q.sub.1 q.sub.2
q.sub.3 q.sub.4 q.sub.5, q.sub.6 and q.sub.7 are each,
independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted
C.sub.2-C.sub.6 alkynyl; and one of R.sub.1 and R.sub.2 is hydrogen
and the other is selected from halogen, substituted or
unsubstituted alkoxy, NJ,J.sub.2, SJ, N.sub.3, OC(.dbd.X)J.sub.1,
OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and
CN, wherein X is O, S, or NJ.sub.1 and each J.sub.1, J.sub.2, and
J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.
[0157] In some embodiments, the modified THP nucleosides of Formula
X are provided wherein q.sub.m, q.sub.n, q.sub.p, q.sub.r, q.sub.s,
q.sub.t, and q.sub.u are each H. In some embodiments, at least one
of q.sub.m, q.sub.n, q.sub.p, q.sub.r, q.sub.s, q.sub.t and q.sub.u
is other than H. In some embodiments, at least one of q.sub.m,
q.sub.n, q.sub.p, q.sub.r, q.sub.s, q.sub.t and q.sub.u is methyl.
In some embodiments, THP nucleosides of Formula X are provided
wherein one of R.sub.1 and R.sub.2 is F. In some embodiments,
R.sub.1 is fluoro and R.sub.2 is H, R.sub.1 is methoxy and R.sub.2
is H, and R.sub.1 is methoxyethoxy and R.sub.2 is H.
[0158] As used herein, "2'-modified" or "2'-substituted" refers to
a nucleoside comprising a sugar comprising a substituent at the 2'
position other than H or OH. 2'-modified nucleosides, include, but
are not limited to nucleosides with non-bridging 2' substituents,
such as allyl, amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10
alkyl, --OCF.sub.3, O--(CH.sub.2).sub.2--O--CH.sub.3,
2'-O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R, is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl. 2'-modified nucleosides may further
comprise other modifications, for example, at other positions of
the sugar and/or at the nucleobase.
[0159] As used herein, "2'-F" refers to a sugar comprising a fluoro
group at the 2' position.
[0160] As used herein, "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl"
each refers to a nucleoside comprising a sugar comprising an
--OCH.sub.3 group at the 2' position of the sugar ring.
[0161] As used herein, "oligonucleotide" refers to a compound
comprising a plurality of linked nucleosides.
[0162] In some embodiments, one or more of the plurality of
nucleosides is modified. In some embodiments, an oligonucleotide
comprises one or more ribonucleosides (RNA) and/or
deoxyribonucleosides (DNA).
[0163] Many other bicyclo and tricyclo sugar surrogate ring systems
are also known in the art that can be used to modify nucleosides
for incorporation into antisense compounds {see, e.g., review
article: Leumann, J. C, Bioorganic and Medicinal Chemistry, 2002,
10, 841-854). Such ring systems can undergo various additional
substitutions to enhance activity. Methods for the preparations of
modified sugars are well known to those skilled in the art. In
nucleotides having modified sugar moieties, the nucleobase moieties
(natural, modified, or a combination thereof) are maintained for
hybridization with an appropriate nucleic acid target.
[0164] In some embodiments, antisense compounds comprise one or
more nucleotides having modified sugar moieties. In some
embodiments, the modified sugar moiety is 2'-MOE. In some
embodiments, the 2'-MOE modified nucleotides are arranged in a
gapmer motif. In some embodiments, the modified sugar moiety is a
cEt. In some embodiments, the cEt modified nucleotides are arranged
throughout the wings of a gapmer motif.
[0165] In some embodiments, in the BNA (LNA), R.sup.4* and R.sup.2*
together designate the biradical --O--CH(CH.sub.2OCH.sub.3)--
(2'O-methoxyethyl bicyclic nucleic acid--Seth at al., 2010, J. Org.
Chem)--in either the R- or S-configuration.
[0166] In some embodiments, in the BNA (LNA), R.sup.4* and R.sup.2*
together designate the biradical --O--CH(CH.sub.2CH.sub.3)--
(2'O-ethyl bicyclic nucleic acid--Seth at al., 2010, J. Org.
Chem).--in either the R- or S-configuration.
[0167] In some embodiments, in the BNA (LNA), R.sup.4* and R.sup.2*
together designate the biradical --O--CH(CH.sub.3)--.--in either
the R- or S-configuration. In some embodiments, R.sup.4* and
R.sup.2* together designate the biradical
--O--CH.sub.2--O--CH.sub.2----(Seth at al., 2010, J. Org.
Chem).
[0168] In some embodiments, in the BNA (LNA), R.sup.4* and R.sup.2*
together designate the biradical --O--NR--CH.sub.3----(Seth at al.,
2010, J. Org. Chem).
[0169] In some embodiments, the LNA units have a structure selected
from the following group:
##STR00014##
[0170] Incorporation of affinity-enhancing nucleotide analogues in
the oligomer, such as BNA, (e.g.) LNA or 2'-substituted sugars, can
allow the size of the specifically binding oligomer to be reduced,
and may also reduce the upper limit to the size of the oligomer
before non-specific or aberrant binding takes place.
[0171] In some embodiments, the oligomer comprises at least 1
nucleoside analogue. In some embodiments the oligomer comprises at
least 2 nucleotide analogues. In some embodiments, the oligomer
comprises from 3-8 nucleotide analogues, e.g. 6 or 7 nucleotide
analogues. In the by far most preferred embodiments, at least one
of said nucleotide analogues is a BNA, such as locked nucleic acid
(LNA); for example at least 3 or at least 4, or at least 5, or at
least 6, or at least 7, or 8, of the nucleotide analogues may be
BNA, such as LNA. In some embodiments all the nucleotides analogues
may be BNA, such as LNA.
[0172] It will be recognized that when referring to a preferred
nucleotide sequence motif or nucleotide sequence, which consists of
only nucleotides, the oligomers of the invention which are defined
by that sequence may comprise a corresponding nucleotide analogue
in place of one or more of the nucleotides present in said
sequence, such as BNA units or other nucleotide analogues, which
raise the duplex stability/T.sub.m of the oligomer/target duplex
(i.e. affinity enhancing nucleotide analogues).
[0173] A preferred nucleotide analogue is LNA, such as oxy-LNA
(such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA
(such as beta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA
(such as beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as
beta-D-ENA and alpha-L-ENA).
[0174] In some embodiments, the oligomer of the invention, such as
region A, may comprise BNA or LNA units and other nucleotide
analogues. Further nucleotide analogues present within the oligomer
of the invention are independently selected from, for example:
2'-O-alkyl-RNA units, 2'-amino-DNA units, 2'-fluoro-DNA units, BNA
units, e.g. LNA units, arabino nucleic acid (ANA) units,
2'-fluoro-ANA units, HNA units, INA (intercalating nucleic
acid--Christensen, 2002. Nucl. Acids. Res. 2002 30: 4918-4925,
hereby incorporated by reference) units and 2'MOE units. In some
embodiments there is only one of the above types of nucleotide
analogues present in the oligomer of the invention, such as the
first region, or contiguous nucleotide sequence thereof.
[0175] In some embodiments, the oligomer according to the invention
(region A) may therefore comprises at least one BNA, e.g. Locked
Nucleic Acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8 BNA/LNA
units, such as from 3-7 or 4 to 8 BNA/LNA units, or 3, 4, 5, 6 or 7
BNA/LNA units. In some embodiments, all the nucleotide analogues
are BNA, such as LNA. In some embodiments, the oligomer may
comprise both beta-D-oxy-LNA, and one or more of the following LNA
units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the
beta-D or alpha-L configurations or combinations thereof. In some
embodiments all BNA, such as LNA, cytosine units are
5'methyl-Cytosine. In some embodiments of the invention, the
oligomer (such as the first and optionally second regions) may
comprise both BNA and LNA and DNA units. In some embodiments, the
combined total of LNA and DNA units is 10-25, such as 10-24,
preferably 10-20, such as 10-18, such as 12-16. In some embodiments
of the invention, the nucleotide sequence of the oligomer, of first
region thereof, such as the contiguous nucleotide sequence consists
of at least one BNA, e.g. LNA and the remaining nucleotide units
are DNA units. In some embodiments the oligomer, or first region
thereof, comprises only BNA, e.g. LNA, nucleotide analogues and
naturally occurring nucleotides (such as RNA or DNA, most
preferably DNA nucleotides), optionally with modified
internucleotide linkages such as phosphorothioate.
RNAse Recruitment
[0176] It is recognised that an oligomeric compound may function
via non RNase mediated degradation of target mRNA, such as by
steric hindrance of translation, or other methods. In some
embodiments, the oligomers of the invention are capable of
recruiting an endoribonuclease (RNase), such as RNase H.
[0177] It is preferable such oligomers, such as region A, or
contiguous nucleotide sequence, comprises of a region of at least
6, such as at least 7 consecutive nucleotide units, such as at
least 8 or at least 9 consecutive nucleotide units (residues),
including 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive
nucleotides, which, when formed in a duplex with the complementary
target RNA is capable of recruiting RNase (such as DNA units). The
contiguous sequence which is capable of recruiting RNAse may be
region Y' as referred to in the context of a gapmer as described
herein. In some embodiments the size of the contiguous sequence
which is capable of recruiting RNAse, such as region Y', may be
higher, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
nucleotide units.
[0178] EP 1 222 309 provides in vitro methods for determining
RNaseH activity, which may be used to determine the ability to
recruit RNaseH. A oligomer is deemed capable of recruiting RNase H
if, when provided with the complementary RNA target, it has an
initial rate, as measured in pmol/l/min, of at least 1%, such as at
least 5%, such as at least 10% or, more than 20% of the of the
initial rate determined using DNA only oligonucleotide, having the
same base sequence but containing only DNA monomers, with no 2'
substitutions, with phosphorothioate linkage groups between all
monomers in the oligonucleotide, using the methodology provided by
Example 91-95 of EP 1 222 309.
[0179] In some embodiments, an oligomer is deemed essentially
incapable of recruiting RNaseH if, when provided with the
complementary RNA target, and RNaseH, the RNaseH initial rate, as
measured in pmol/l/min, is less than 1%, such as less than 5%, such
as less than 10% or less than 20% of the initial rate determined
using the equivalent DNA only oligonucleotide, with no 2'
substitutions, with phosphorothioate linkage groups between all
nucleotides in the oligonucleotide, using the methodology provided
by Example 91-95 of EP 1 222 309.
[0180] In other embodiments, an oligomer is deemed capable of
recruiting RNaseH if, when provided with the complementary RNA
target, and RNaseH, the RNaseH initial rate, as measured in
pmol/l/min, is at least 20%, such as at least 40%, such as at least
60%, such as at least 80% of the initial rate determined using the
equivalent DNA only oligonucleotide, with no 2' substitutions, with
phosphorothioate linkage groups between all nucleotides in the
oligonucleotide, using the methodology provided by Example 91-95 of
EP 1 222 309. Typically the region of the oligomer which forms the
consecutive nucleotide units which, when formed in a duplex with
the complementary target RNA is capable of recruiting RNase
consists of nucleotide units which form a DNA/RNA like duplex with
the RNA target. The oligomer of the invention, such as the first
region, may comprise a nucleotide sequence which comprises both
nucleotides and nucleotide analogues, and may be e.g. in the form
of a gapmer.
Gapmer Design
[0181] In some embodiments, the oligomer of the invention, such as
the first region, comprises or is a gapmer. A gapmer oligomer is an
oligomer which comprises a contiguous stretch of nucleotides which
is capable of recruiting an RNAse, such as RNAseH, such as a region
of at least 6 or 7 DNA nucleotides, referred to herein in as region
Y' (Y'), wherein region Y' is flanked both 5' and 3' by regions of
affinity enhancing nucleotide analogues, such as from 1-6
nucleotide analogues 5' and 3' to the contiguous stretch of
nucleotides which is capable of recruiting RNAse--these regions are
referred to as regions X' (X') and Z' (Z') respectively. Examples
of gapmers are disclosed in WO2004/046160, WO2008/113832, and
WO2007/146511.
[0182] In some embodiments, the monomers which are capable of
recruiting RNAse are selected from the group consisting of DNA
monomers, alpha-L-LNA monomers, C4' alkylated DNA monomers (see
PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18
(2008) 2296-2300, hereby incorporated by reference), and UNA
(unlinked nucleic acid) nucleotides (see Fluiter et al., Mol.
Biosyst., 2009, 10, 1039 hereby incorporated by reference). UNA is
unlocked nucleic acid, typically where the C2-C3 C--C bond of the
ribose has been removed, forming an unlocked "sugar" residue.
Preferably the gapmer comprises a (poly)nucleotide sequence of
formula (5' to 3'), X'-Y'-Z', wherein; region X' (X') (5' region)
consists or comprises of at least one nucleotide analogue, such as
at least one BNA (e.g. LNA) unit, such as from 1-6 nucleotide
analogues, such as BNA (e.g. LNA) units, and; region Y' (Y')
consists or comprises of at least five consecutive nucleotides
which are capable of recruiting RNAse (when formed in a duplex with
a complementary RNA molecule, such as the mRNA target), such as DNA
nucleotides, and; region Z' (Z') (3' region) consists or comprises
of at least one nucleotide analogue, such as at least one BNA (e.g
LNA unit), such as from 1-6 nucleotide analogues, such as BNA (e.g.
LNA) units.
[0183] In some embodiments, region X' consists of 1, 2, 3, 4, 5 or
6 nucleotide analogues, such as BNA (e.g. LNA) units, such as from
2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4
nucleotide analogues, such as 3 or 4 LNA units; and/or region Z'
consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as BNA
(e.g. LNA) units, such as from 2-5 nucleotide analogues, such as
2-5 BNA (e.g. LNA units), such as 3 or 4 nucleotide analogues, such
as 3 or 4 BNA (e.g. LNA) units.
[0184] In some embodiments Y' consists or comprises of 5, 6, 7, 8,
9, 10, 11 or 12 consecutive nucleotides which are capable of
recruiting RNAse, or from 6-10, or from 7-9, such as 8 consecutive
nucleotides which are capable of recruiting RNAse. In some
embodiments region Y' consists or comprises at least one DNA
nucleotide unit, such as 1-12 DNA units, preferably from 4-12 DNA
units, more preferably from 6-10 DNA units, such as from 7-10 DNA
units, most preferably 8, 9 or 10 DNA units.
[0185] In some embodiments region X' consist of 3 or 4 nucleotide
analogues, such as BNA (e.g. LNA), region X' consists of 7, 8, 9 or
10 DNA units, and region Z' consists of 3 or 4 nucleotide
analogues, such as BNA (e.g. LNA). Such designs include (X'-Y'-Z')
3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3,
3-7-3, 3-7-4, 4-7-3.
[0186] Further gapmer designs are disclosed in WO2004/046160, which
is hereby incorporated by reference. WO2008/113832, which claims
priority from U.S. provisional application 60/977,409 hereby
incorporated by reference, refers to `shortmer` gapmer oligomers.
In some embodiments, oligomers presented here may be such shortmer
gapmers.
[0187] In some embodiments the oligomer, e.g. region X', is
consisting of a contiguous nucleotide sequence of a total of 10,
11, 12, 13 or 14 nucleotide units, wherein the contiguous
nucleotide sequence comprises or is of formula (5'-3'), X'-Y'-Z'
wherein; X' consists of 1, 2 or 3 nucleotide analogue units, such
as BNA (e.g. LNA) units; Y' consists of 7, 8 or 9 contiguous
nucleotide units which are capable of recruiting RNAse when formed
in a duplex with a complementary RNA molecule (such as a mRNA
target); and Z' consists of 1, 2 or 3 nucleotide analogue units,
such as BNA (e.g. LNA) units.
[0188] In some embodiments X' consists of 1 BNA (e.g. LNA) unit. In
some embodiments X' consists of 2 BNA (e.g. LNA) units. In some
embodiments X' consists of 3 BNA (e.g. LNA) units. In some
embodiments Z' consists of 1 BNA (e.g. LNA) units. In some
embodiments Z' consists of 2 BNA (e.g. LNA) units. In some
embodiments Z' consists of 3 BNA (e.g. LNA) units. In some
embodiments Y' consists of 7 nucleotide units. In some embodiments
Y' consists of 8 nucleotide units. In some embodiments Y' consists
of 9 nucleotide units. In certain embodiments, region Y' consists
of 10 nucleoside monomers. In certain embodiments, region Y'
consists or comprises 1-10 DNA monomers. In some embodiments Y'
comprises of from 1-9 DNA units, such as 2, 3, 4, 5, 6, 7, 8 or 9
DNA units. In some embodiments Y' consists of DNA units. In some
embodiments Y' comprises of at least one BNA unit which is in the
alpha-L configuration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units
in the alpha-L-configuration. In some embodiments Y' comprises of
at least one alpha-L-oxy BNA/LNA unit or wherein all the LNA units
in the alpha-L-configuration are alpha-L-oxy LNA units. In some
embodiments the number of nucleotides present in X'-Y'-Z' are
selected from the group consisting of (nucleotide analogue
units-region Y'-nucleotide analogue units): 1-8-1, 1-8-2, 2-8-1,
2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8- 4, 2-8-4, or; 1-9-1,
1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1, 4-9-1, 1-9-4, or;
1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1. In some embodiments
the number of nucleotides in X'-Y'-Z' are selected from the group
consisting of: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and
4-7-3. In certain embodiments, each of regions X' and Y' consists
of three BNA (e.g. LNA) monomers, and region Y' consists of 8 or 9
or 10 nucleoside monomers, preferably DNA monomers. In some
embodiments both X' and Z' consists of two BNA (e.g. LNA) units
each, and Y' consists of 8 or 9 nucleotide units, preferably DNA
units. In various embodiments, other gapmer designs include those
where regions X' and/or Z' consists of 3, 4, 5 or 6 nucleoside
analogues, such as monomers containing a 2'-O-methoxyethyl-ribose
sugar (2'-MOE) or monomers containing a 2'-fluoro-deoxyribose
sugar, and region Y' consists of 8, 9, 10, 11 or 12 nucleosides,
such as DNA monomers, where regions X'-Y'-Z' have 3-9-3, 3-10-3,
5-10-5 or 4-12-4 monomers. Further gapmer designs are disclosed in
WO 2007/146511A2, hereby incorporated by reference.
[0189] BNA and LNA Gapmers: A BNA gapmer is a gapmer oligomer
(region A) which comprises at least one BNA nucleotide. A LNA
gapmer is a gapmer oligomer (region A) which comprises at least one
LNA nucleotide. SEQ ID NO 2 and 3 are LNA gapmer oligomers. The
oligomers with a contiguous sequence of 10-16 nucleotides which are
complementary to a corresponding length of SEQ ID NO 33 or 34 may
also be gapmer oligomers such as BNA gapmers or LNA gapmers.
Internucleotide Linkages
[0190] The nucleoside monomers of the oligomers (e.g. first and
second regions) described herein are coupled together via
[internucleoside] linkage groups. Suitably, each monomer is linked
to the 3' adjacent monomer via a linkage group.
[0191] The person having ordinary skill in the art would understand
that, in the context of the present invention, the 5' monomer at
the end of an oligomer does not comprise a 5' linkage group,
although it may or may not comprise a 5' terminal group.
[0192] The terms "linkage group" or "internucleotide linkage" are
intended to mean a group capable of covalently coupling together
two nucleotides. Specific and preferred examples include phosphate
groups and phosphorothioate groups.
[0193] The nucleotides of the oligomer of the invention or
contiguous nucleotides sequence thereof are coupled together via
linkage groups. Suitably each nucleotide is linked to the 3'
adjacent nucleotide via a linkage group.
[0194] Suitable internucleotide linkages include those listed
within WO2007/031091, for example the internucleotide linkages
listed on the first paragraph of page 34 of WO2007/031091 (hereby
incorporated by reference).
[0195] It is, in some embodiments, other than the phosphodiester
linkage(s) of region B (where present), the preferred to modify the
internucleotide linkage from its normal phosphodiester to one that
is more resistant to nuclease attack, such as phosphorothioate or
boranophosphate--these two, being cleavable by RNase H, also allow
that route of antisense inhibition in reducing the expression of
the target gene.
[0196] Suitable sulphur (S) containing internucleotide linkages as
provided herein may be preferred, such as phosphorothioate or
phosphodithioate. Phosphorothioate internucleotide linkages are
also preferred, particularly for the first region, such as in
gapmers, mixmers, antimirs splice switching oligomers, and
totalmers.
[0197] For gapmers, the internucleotide linkages in the oligomer
may, for example be phosphorothioate or boranophosphate so as to
allow RNase H cleavage of targeted RNA. Phosphorothioate is
preferred, for improved nuclease resistance and other reasons, such
as ease of manufacture.
[0198] In one aspect, with the exception of the phosphodiester
linkage between the first and second region, and optionally within
region B, the remaining internucleoside linkages of the oligomer of
the invention, the nucleotides and/or nucleotide analogues are
linked to each other by means of phosphorothioate groups. In some
embodiments, at least 50%, such as at least 70%, such as at least
80%, such as at least 90% such as all the internucleoside linkages
between nucleosides in the first region are other than
phosphodiester (phosphate), such as are selected from the group
consisting of phosphorothioate phosphorodithioate, or
boranophosphate. In some embodiments, at least 50%, such as at
least 70%, such as at least 80%, such as at least 90% such as all
the internucleoside linkages between nucleosides in the first
region are phosphorothioate.
[0199] WO09124238 refers to oligomeric compounds having at least
one bicyclic nucleoside attached to the 3' or 5' termini by a
neutral internucleoside linkage. The oligomers of the invention may
therefore have at least one bicyclic nucleoside attached to the 3'
or 5' termini by a neutral internucleoside linkage, such as one or
more phosphotriester, methylphosphonate, MMI, amide-3, formacetal
or thioformacetal. The remaining linkages may be
phosphorothioate.
Oligomer Conjugates
[0200] Representative conjugate moieties which have been used with
oligonucleotides can include lipophilic molecules (aromatic and
non-aromatic) including steroid molecules; proteins (e.g.,
antibodies, enzymes, serum proteins); peptides; vitamins
(water-soluble or lipid-soluble); polymers (water-soluble or
lipid-soluble); small molecules including drugs, toxins, reporter
molecules, and receptor ligands; carbohydrate complexes; nucleic
acid cleaving complexes; metal chelators (e.g., porphyrins,
texaphyrins, crown ethers, etc.); intercalators including hybrid
photonuclease/intercalators; crosslinking agents (e.g.,
photoactive, redox active), and combinations and derivatives
thereof. Numerous suitable conjugate moieties, their preparation
and linkage to oligomeric compounds are provided, for example, in
WO 93/07883 and U.S. Pat. No. 6,395,492, each of which is
incorporated herein by reference in its entirety. Oligonucleotide
conjugates and their syntheses are also reported in comprehensive
reviews by Manoharan in Antisense Drug Technology, Principles,
Strategies, and Applications, S. T. Crooke, ed., Ch. 16, Marcel
Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug
Development, 2002, 12, 103, each of which is incorporated herein by
reference in its entirety.
[0201] In some embodiments the oligomer of the invention is
targeted to the liver--i.e. after systemic administration the
compound accumulates in the liver cells (such as hepatocytes).
Targeting to the liver can be greatly enhanced by the addition of a
conjugate moiety (C). However, in order to maximize the efficacy of
the oligomer it is often desirable that the conjugate (or targeting
moiety) is linked to the oligomer via a biocleavable linker (B),
such as a nucleotide phosphate linker. It is therefore desirable to
use a conjugate moiety which enhances uptake and activity in
hepatocytes. The enhancement of activity may be due to enhanced
uptake or it may be due to enhanced potency of the compound in
hepatocytes.
[0202] In some embodiments, the oligomeric compound is a BNA or LNA
oligomer, such as a gapmer, or for example an LNA antisense
oligomer, (which may be referred to as region A herein) comprising
an antisense oligomer, optionally a biocleavable linker, such as
region B, and a carbohydrate conjugate (which may be referred to as
region C). The LNA antisense oligomer may be 7-30, such as 8-26
nucleosides in length and it comprises at least one LNA unit
(nucleoside). In some embodiments the carbohydrate moiety is not a
linear carbohydrate polymer.
[0203] In some embodiments, the oligomeric compound is a LNA
oligomer, for example an LNA antisense oligomer, (which may be
referred to as region A herein) comprising an antisense oligomer,
region B as defined herein, and an asialoglycoprotein receptor
targeting moiety conjugate moiety, such as a GalNAc moiety (which
may be referred to as region C). The carbohydrate moiety may be
multi-valent, such as, for example 2, 3, 4 or 4 identical or
non-identical carbohydrate moieties may be covalently joined to the
oligomer, optionally via a linker or linkers (such as region
Y).
GalNac Conjugate Moieties
[0204] In some embodiments the carbohydrate moiety is not a linear
carbohydrate polymer. The carbohydrate moiety may however be
multi-valent, such as, for example 2, 3, 4 or 4 identical or
non-identical carbohydrate moieties may be covalently joined to the
oligomer, optionally via a linker or linkers. In some embodiments
the invention provides a conjugate comprising the oligomer of the
invention and a carbohydrate conjugate moiety. In some embodiments
the invention provides a conjugate comprising the oligomer of the
invention and an asialoglycoprotein receptor targeting moiety
conjugate moiety, such as a GalNAc moiety, which may form part of a
further region (referred to as region C).
[0205] The invention also provides LNA antisense oligonucleotides
which are conjugated to an asialoglycoprotein receptor targeting
moiety. In some embodiments, the conjugate moiety (such as the
third region or region C) comprises an asialoglycoprotein receptor
targeting moiety, such as galactose, galactosamine,
N-formyl-galactosamine, Nacetylgalactosamine,
N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and
N-isobutanoylgalactos-amine. In some embodiments the conjugate
comprises a galactose cluster, such as N-acetylgalactosamine
trimer. In some embodiments, the conjugate moiety comprises an
GalNAc (N-acetylgalactosamine), such as a mono-valent, di-valent ,
tri-valent of tetra-valent GalNAc. Trivalent GalNAc conjugates may
be used to target the compound to the liver. GalNAc conjugates have
been used with methylphosphonate and PNA antisense oligonucleotides
(e.g. U.S. Pat. No. 5,994517 and Hangeland et al., Bioconjug Chem.
1995 November-December; 6(6):695-701) and siRNAs (e.g.
WO2009/126933, WO2012/089352 & WO2012/083046). The GalNAc
references and the specific conjugates used therein are hereby
incorporated by reference. WO2012/083046 discloses siRNAs with
GalNAc conjugate moieties which comprise cleavable pharmacokinetic
modulators, which are suitable for use in the present invention,
the preferred pharmacokinetic modulators are C16 hydrophobic groups
such as palmitoyl, hexadec-8-enoyl, oleyl,
(9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. The
'046 cleavable pharmacokinetic modulators may also be
cholesterol.
[0206] The `targeting moieties (conjugate moieties) may be selected
from the group consisting of: galactose, galactosamine,
N-formyl-galactosamine, N-acetylgalactosamine,
Npropionyl-galactosamine, N-n-butanoyl-galactosamine,
N-iso-butanoylgalactos-amine, galactose cluster, and
N-acetylgalactosamine trimer and may have a pharmacokinetic
modulator selected from the group consisting of: hydrophobic group
having 16 or more carbon atoms, hydrophobic group having 16-20
carbon atoms, palmitoyl, hexadec-8-enoyl, oleyl,
(9E,12E)-octadeca-9,12dienoyl, dioctanoyl, and C16-C20 acyl, and
cholesterol. Certain GalNac clusters disclosed in '046 include:
(E)-hexadec-8-enoyl (C16), oleyl (C18),
(9,E,12E)-octadeca-9,12-dienoyl (C18), octanoyl (C8), dodececanoyl
(C12), C-20 acyl, C24 acyl, dioctanoyl (2.times.C8). The targeting
moiety-pharmacokinetic modulator targeting moiety may be linked to
the polynucleotide via a physiologically labile bond or, e.g. a
disulfide bond, or a PEG linker. The invention also relates to the
use of phosphodiester linkers between the oligomer and the
conjugate group (these are referred to as region B herein, and
suitably are positioned between the LNA oligomer and the
carbohydrate conjugate group).
[0207] For targeting hepatocytes in liver, a preferred targeting
ligand is a galactose cluster.
[0208] A galactose cluster comprises a molecule having e.g.
comprising two to four terminal galactose derivatives. As used
herein, the term galactose derivative includes both galactose and
derivatives of galactose having affinity for the asialoglycoprotein
receptor equal to or greater than that of galactose. A terminal
galactose derivative is attached to a molecule through its C--I
carbon. The asialoglycoprotein receptor (ASGPr) is unique to
hepatocytes and binds branched galactose-terminal glycoproteins. A
preferred galactose cluster has three terminal galactosamines or
galactosamine derivatives each having affinity for the
asialoglycoprotein receptor. A more preferred galactose cluster has
three terminal N-acetyl-galactosamines. Other terms common in the
art include tri-antennary galactose, tri-valent galactose and
galactose trimer. It is known that tri-antennary galactose
derivative clusters are bound to the ASGPr with greater affinity
than bi-antennary or mono-antennary galactose derivative structures
(Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al.,
1982, 1. Biol. Chem., 257,939-945). Multivalency is required to
achieve nM affinity. According to WO 2012/083046 the attachment of
a single galactose derivative having affinity for the
asialoglycoprotein receptor does not enable functional delivery of
the RNAi polynucleotide to hepatocytes in vivo when co-administered
with the delivery polymer.
[0209] A galactose cluster may comprise two or preferably three
galactose derivatives each linked to a central branch point. The
galactose derivatives are attached to the central branch point
through the C--I carbons of the saccharides. The galactose
derivative is preferably linked to the branch point via linkers or
spacers. A preferred spacer is a flexible hydrophilic spacer (U.S.
Pat. No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p.
1538-1546). A preferred flexible hydrophilic spacer is a PEG
spacer. A preferred PEG spacer is a PEG3 spacer. The branch point
can be any small molecule which permits attachment of the three
galactose derivatives and further permits attachment of the branch
point to the oligomer. An exemplary branch point group is a
di-lysine. A di-lysine molecule contains three amine groups through
which three galactose derivatives may be attached and a carboxyl
reactive group through which the di-lysine may be attached to the
oligomer. Attachment of the branch point to oligomer may occur
through a linker or spacer. A preferred spacer is a flexible
hydrophilic spacer. A preferred flexible hydrophilic spacer is a
PEG spacer. A preferred PEG spacer is a PEG3 spacer (three ethylene
units). The galactose cluster may be attached to the 3' or 5' end
of the oligomer using methods known in the art.
[0210] A preferred galactose derivative is an
N-acetyl-galactosamine (GalNAc). Other saccharides having affinity
for the asialoglycoprotein receptor may be selected from the list
comprising: galactosamine, N-n-butanoylgalactosamine, and
N-iso-butanoylgalactosamine. The affinities of numerous galactose
derivatives for the asialoglycoprotein receptor have been studied
(see for example: Jobst, S. T. and Drickamer, K. JB.C. 1996,
271,6686) or are readily determined using methods typical in the
art.
##STR00015##
[0211] Further Examples of the conjugate of the invention are
illustrated below:
##STR00016##
[0212] Where at the hydrophobic or lipophilic (or further
conjugate) moiety (i.e. pharmacokinetic modulator) in the above
GalNac cluster conjugates is, when using BNA or LNA oligomers, such
as LNA antisense oligonucleotides, optional.
[0213] See the figures for specific Galnac clusters used in the
present study, Conj 1, 2, 3, 4 and Conj 1a, 2a, 3a and 4a (which
are shown with an optional C6 linker which joins the GalNac cluster
to the oligomer).
[0214] Each carbohydrate moiety of a Galnac cluster (e.g. GalNAc)
may therefore be joined to the oligomer via a spacer, such as
(poly)ethylene glycol linker (PEG), such as a di, tri, tetra,
penta, hexa-ethylene glycol linker. As is shown above the PEG
moiety forms a spacer between the galactose sugar moiety and a
peptide (trilysine is shown) linker.
[0215] In some embodiments, the GalNac cluster comprises a peptide
linker, e.g. a Tyr-Asp(Asp) tripeptide or Asp(Asp) dipeptide, which
is attached to the oligomer (or to region Y or region B) via a
biradical linker, for example the GalNac cluster may comprise the
following biradical linkers:
##STR00017##
[0216] R.sup.1 is a biradical preferably selected from
--C.sub.2H.sub.4--, --C.sub.3H.sub.6--, --C.sub.4H.sub.8--,
--C.sub.5H.sub.10--, --C.sub.6H.sub.12--, 1,4- cyclohexyl
(--C.sub.6H.sub.10--), 1,4-phenyl (--C.sub.6H.sub.4--),
--C.sub.2H.sub.4OC.sub.2H.sub.4--,
--C.sub.2H.sub.4(OC.sub.2H.sub.4).sub.2-- or
--C.sub.2H.sub.4(OC.sub.2H.sub.4).sub.3--.
[0217] The carbohydrate conjugate (e.g. GalNAc), or
carbohydrate-linker moiety (e.g. carbohydrate-PEG moiety) may be
covalently joined (linked) to the oligomer via a branch point group
such as, an amino acid, or peptide, which suitably comprises two or
more amino groups (such as 3, 4, or 5), such as lysine, di-lysine
or tri-lysine or tetra-lysine. A tri-lysine molecule contains four
amine groups through which three carbohydrate conjugate groups,
such as galactose & derivatives (e.g. GalNAc) and a further
conjugate such as a hydrophobic or lipophilic moiety/group may be
attached and a carboxyl reactive group through which the tri-lysine
may be attached to the oligomer. The further conjugate, such as
lipophilic/hydrophobic moiety may be attached to the lysine residue
that is attached to the oligomer.
Pharmacokinetic Modulators
[0218] The compound of the invention may further comprise one or
more additional conjugate moieties, of which lipophilic or
hydrophobic moieties are particularly interesting, such as when the
conjugate group is a carbohydrate moiety. Such lipophilic or
hydrophobic moieties may act as pharmacokinetic modulators, and may
be covalently linked to either the carbohydrate conjugate, a linker
linking the carbohydrate conjugate to the oligomer or a linker
linking multiple carbohydrate conjugates (multi-valent) conjugates,
or to the oligomer, optionally via a linker, such as a bio
cleavable linker.
[0219] The oligomer or conjugate moiety may therefore comprise a
pharmacokinetic modulator, such as a lipophilic or hydrophobic
moieties. Such moieties are disclosed within the context of siRNA
conjugates in WO2012/082046. The hydrophobic moiety may comprise a
C8-C36 fatty acid, which may be saturated or un-saturated. In some
embodiments, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, C30,
C32 and C34 fatty acids may be used. The hydrophobic group may have
16 or more carbon atoms. Exemplary suitable hydrophobic groups may
be selected from the group comprising: sterol, cholesterol,
palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl,
dioctanoyl, and C16-C20 acyl. According to WO '346, hydrophobic
groups having fewer than 16 carbon atoms are less effective in
enhancing polynucleotide targeting, but they may be used in
multiple copies (e.g. 2.times., such as 2.times.C8 or C10, C12 or
C14) to enhance efficacy. Pharmacokinetic modulators useful as
polynucleotide targeting moieties may be selected from the group
consisting of: cholesterol, alkyl group, alkenyl group, alkynyl
group, aryl group, aralkyl group, aralkenyl group, and aralkynyl
group, each of which may be linear, branched, or cyclic.
Pharmacokinetic modulators are preferably hydrocarbons, containing
only carbon and hydrogen atoms. However, substitutions or
heteroatoms which maintain hydrophobicity, for example fluorine,
may be permitted.
[0220] In some embodiments, the conjugate is or may comprise a
carbohydrate or comprises a carbohydrate group. In some
embodiments, the carbohydrate is selected from the group consisting
of galactose, lactose, n-acetylgalactosamine, mannose, and
mannose-6-phosphate. In some embodiments, the conjugate group is or
may comprise mannose or mannose-6-phosphate. Carbohydrate
conjugates may be used to enhance delivery or activity in a range
of tissues, such as liver and/or muscle. See, for example,
EP1495769, WO99/65925, Yang et al., Bioconjug Chem (2009) 20(2):
213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10):
1401-17.
[0221] Surprisingly, the present inventors have found that GalNac
conjugates for use with LNA oligomers do not require a
pharmacokinetic modulator, and as such, in some embodiments, the
GalNac conjugate is not covalently linked to a lipophilic or
hydrophobic moiety, such as those described here in, e.g. do not
comprise a C8-C36 fatty acid or a sterol. The invention therefore
also provides for LNA oligomer GalNac conjugates which do not
comprise a lipophilic or hydrophobic pharmacokinetic modulator or
conjugate moiety/group.
Lipophilic Conjugates
[0222] In some embodiments, the conjugate group is or may comprise
a lipophilic moiety, such as a sterol (for example, cholesterol,
cholesteryl, cholestanol, stigmasterol, cholanic acid and
ergosterol). In some embodiments the conjugate is or comprises
tocopherol. In some embodiments, the conjugate is or may comprise
cholesterol.
[0223] In some embodiments, the conjugate is, or may comprise a
lipid, a phospholipid or a lipophilic alcohol, such as a cationic
lipids, a neutral lipids, sphingolipids, and fatty acids such as
stearic, oleic, elaidic, linoleic, linoleaidic, linolenic, and
myristic acids. In some embodiments the fatty acid comprises a
C4-C30 saturated or unsaturated alkyl chain. The alkyl chain may be
linear or branched.
[0224] Lipophilic conjugate moieties can be used, for example, to
counter the hydrophilic nature of an oligomeric compound and
enhance cellular penetration.
[0225] Lipophilic moieties include, for example, sterols stanols,
and steroids and related compounds such as cholesterol (U.S. Pat.
No. 4,958,013 and Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553), thiocholesterol (Oberhauser et al, Nucl Acids
Res., 1992, 20, 533), lanosterol, coprostanol, stigmasterol,
ergosterol, calciferol, cholic acid, deoxycholic acid, estrone,
estradiol, estratriol, progesterone, stilbestrol, testosterone,
androsterone, deoxycorticosterone, cortisone,
17-hydroxycorticosterone, their derivatives, and the like. In some
embodiments, the conjugate may be selected from the group
consisting of cholesterol, thiocholesterol, lanosterol,
coprostanol, stigmasterol, ergosterol, calciferol, cholic acid,
deoxycholic acid, estrone, estradiol, estratriol, progesterone,
stilbestrol, testosterone, androsterone, deoxycorticosterone,
cortisone, and 17-hydroxycorticosterone. Other lipophilic conjugate
moieties include aliphatic groups, such as, for example, straight
chain, branched, and cyclic alkyls, alkenyls, and alkynyls. The
aliphatic groups can have, for example, 5 to about 50, 6 to about
50, 8 to about 50, or 10 to about 50 carbon atoms. Example
aliphatic groups include undecyl, dodecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, terpenes, bornyl, adamantyl, derivatives
thereof and the like. In some embodiments, one or more carbon atoms
in the aliphatic group can be replaced by a heteroatom such as O,
S, or N (e.g., geranyloxyhexyl). Further suitable lipophilic
conjugate moieties include aliphatic derivatives of glycerols such
as alkylglycerols, bis(alkyl)glycerols, tris(alkyl)glycerols,
monoglycerides, diglycerides, and triglycerides. In some
embodiments, the lipophilic conjugate is di-hexyldecyl-rac-glycerol
or 1,2-di-O-hexyldecyl-rac-glycerol (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651; Shea, et al., Nuc. Acids Res., 1990, 18,
3777) or phosphonates thereof. Saturated and unsaturated fatty
functionalities, such as, for example, fatty acids, fatty alcohols,
fatty esters, and fatty amines, can also serve as lipophilic
conjugate moieties. In some embodiments, the fatty functionalities
can contain from about 6 carbons to about 30 or about 8 to about 22
carbons. Example fatty acids include, capric, caprylic, lauric,
palmitic, myristic, stearic, oleic, linoleic, linolenic,
arachidonic, eicosenoic acids and the like.
[0226] In further embodiments, lipophilic conjugate groups can be
polycyclic aromatic groups having from 6 to about 50, 10 to about
50, or 14 to about 40 carbon atoms. Example polycyclic aromatic
groups include pyrenes, purines, acridines, xanthenes, fluorenes,
phenanthrenes, anthracenes, quinolines, isoquinolines,
naphthalenes, derivatives thereof and the like. Other suitable
lipophilic conjugate moieties include menthols, trityls (e.g.,
dimethoxytrityl (DMT)), phenoxazines, lipoic acid, phospholipids,
ethers, thioethers (e.g., hexyl-S-tritylthiol), derivatives thereof
and the like. Preparation of lipophilic conjugates of oligomeric
compounds are well-described in the art, such as in, for example,
Saison-Behmoaras et al, EMBO J., 1991, 10, 1111; Kabanov et al.,
FEBS Lett., 1990, 259, 327; Svinarchuk et al, Biochimie, 1993, 75,
49; (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229, and
Manoharan et al., Tetrahedron Lett., 1995, 36, 3651.
[0227] Oligomeric compounds containing conjugate moieties with
affinity for low density lipoprotein (LDL) can help provide an
effective targeted delivery system. High expression levels of
receptors for LDL on tumor cells makes LDL an attractive carrier
for selective delivery of drugs to these cells (Rump, et al.,
Bioconjugate Chem., 1998, 9, 341; Firestone, Bioconjugate Chem.,
1994, 5, 105; Mishra, et al., Biochim. Biophys. Acta, 1995, 1264,
229). Moieties having affinity for LDL include many lipophilic
groups such as steroids (e.g., cholesterol), fatty acids,
derivatives thereof and combinations thereof. In some embodiments,
conjugate moieties having LDL affinity can be dioleyl esters of
cholic acids such as chenodeoxycholic acid and lithocholic
acid.
[0228] In some embodiments, the lipophilic conjugates may be or may
comprise biotin. In some embodiments, the lipophilic conjugate may
be or may comprise a glyceride or glyceride ester.
[0229] Lipophilic conjugates, such as sterols, stanols, and stains,
such as cholesterol or as disclosed herein, may be used to enhance
delivery of the oligonucleotide to, for example, the liver
(typically hepatocytes).
[0230] The following references also refer to the use of lipophilic
conjugates: Kobylanska et al., Acta Biochim Pol. (1999); 46(3):
679-91. Felber et al., Biomaterials (2012) 33(25): 599-65);
Grijalvo et al., J Org Chem (2010) 75(20): 6806-13. Koufaki et al.,
Curr Med Chem (2009) 16(35): 4728-42. Godeau et al J. Med. Chem.
(2008) 51(15): 4374-6.
Linkers (e.g. Region Y)
[0231] A linkage or linker is a connection between two atoms that
links one chemical group or segment of interest to another chemical
group or segment of interest via one or more covalent bonds.
Conjugate moieties (or targeting or blocking moieties) can be
attached to the oligomeric compound directly or through a linking
moiety (linker or tether)--a linker. Linkers are bifunctional
moieties that serve to covalently connect a third region, e.g. a
conjugate moiety, to an oligomeric compound (such as to region B).
In some embodiments, the linker comprises a chain structure or an
oligomer of repeating units such as ethylene glyol or amino acid
units. The linker can have at least two functionalities, one for
attaching to the oligomeric compound and the other for attaching to
the conjugate moiety. Example linker functionalities can be
electrophilic for reacting with nucleophilic groups on the oligomer
or conjugate moiety, or nucleophilic for reacting with
electrophilic groups. In some embodiments, linker functionalities
include amino, hydroxyl, carboxylic acid, thiol, phosphoramidate,
phosphorothioate, phosphate, phosphite, unsaturations (e.g., double
or triple bonds), and the like. Some example linkers include
8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl
4-(N-maleimidomethyl)cyclohexane-I-carboxylate (SMCC),
6-aminohexanoic acid (AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric
acid, 4-aminocyclohexylcarboxylic acid, succinimidyl
4-(N-maleimidomethyl)cyclohexane-I-carboxy-(6-amido-caproate)
(LCSMCC), succinimidyl m-maleimido-benzoylate (MBS), succinimidyl
N-e-maleimido-caproylate (EMCS), succinimidyl
6-(beta-maleimido-propionamido)hexanoate (SMPH), succinimidyl
N-(a-maleimido acetate) (AMAS), succinimidyl
4-(p-maleimidophenyl)butyrate (SMPB), beta-alanine (beta-ALA),
phenylglycine (PHG), 4-aminocyclohexanoic acid (ACHC),
beta-(cyclopropyl)alanine (beta-CYPR), amino dodecanoic acid (ADC),
alylene diols, polyethylene glycols, amino acids, and the like.
[0232] A wide variety of further linker groups are known in the art
that can be useful in the attachment of conjugate moieties to
oligomeric compounds. A review of many of the useful linker groups
can be found in, for example, Antisense Research and Applications,
S. T. Crooke and B. Lebleu, Eds., CRC Press, Boca Raton, Fla.,
1993, p. 303-350. A disulfide linkage has been used to link the 3'
terminus of an oligonucleotide to a peptide (Corey, et al., Science
1987, 238, 1401; Zuckermann, et al, J Am. Chem. Soc. 1988, 110,
1614; and Corey, et al., J Am. Chem. Soc. 1989, 111, 8524). Nelson,
et al., Nuc. Acids Res. 1989, 17, 7187 describe a linking reagent
for attaching biotin to the 3'-terminus of an oligonucleotide. This
reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is commercially
available from Clontech Laboratories (Palo Alto, Calif.) under the
name 3'-Amine. It is also commercially available under the name
3'-Amino-Modifier reagent from Glen Research Corporation (Sterling,
Va.). This reagent was also utilized to link a peptide to an
oligonucleotide as reported by Judy, et al., Tetrahedron Letters
1991, 32, 879. A similar commercial reagent for linking to the
5'-terminus of an oligonucleotide is 5'-Amino-Modifier C6. These
reagents are available from Glen Research Corporation (Sterling,
Va.). These compounds or similar ones were utilized by Krieg, et
al, Antisense Research and Development 1991, 1, 161 to link
fluorescein to the 5'-terminus of an oligonucleotide. Other
compounds such as acridine have been attached to the 3'-terminal
phosphate group of an oligonucleotide via a polymethylene linkage
(Asseline, et al., Proc. Natl. Acad. Sci. USA 1984, 81, 3297).
[0074] Any of the above groups can be used as a single linker or in
combination with one or more further linkers.
[0233] Linkers and their use in preparation of conjugates of
oligomeric compounds are provided throughout the art such as in WO
96/11205 and WO 98/52614 and U.S. Pat. Nos. 4,948,882; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,580,731; 5,486,603; 5,608,046;
4,587,044; 4,667,025; 5,254,469; 5,245,022; 5,112,963; 5,391,723;
5,510475; 5,512,667; 5,574,142; 5,684,142; 5,770,716; 6,096,875;
6,335,432; and 6,335,437,Wo2012/083046 each of which is
incorporated by reference in its entirety.
[0234] As used herein, a physiologically labile bond is a labile
bond that is cleavable under conditions normally encountered or
analogous to those encountered within a mammalian body (also
referred to as a cleavable linker). Physiologically labile linkage
groups are selected such that they undergo a chemical
transformation (e.g., cleavage) when present in certain
physiological conditions. Mammalian intracellular conditions
include chemical conditions such as pH, temperature, oxidative or
reductive conditions or agents, and salt concentration found in or
analogous to those encountered in mammalian cells. Mammalian
intracellular conditions also include the presence of enzymatic
activity normally present in a mammalian cell such as from
proteolytic or hydrolytic enzymes. In some embodiments, the
cleavable linker is susceptible to nuclease(s) which may for
example, be expressed in the target cell--and as such, as detailed
herein, the linker may be a short region (e.g. 1-10) phosphodiester
linked nucleosides, such as DNA nucleosides.
[0235] Chemical transformation (cleavage of the labile bond) may be
initiated by the addition of a pharmaceutically acceptable agent to
the cell or may occur spontaneously when a molecule containing the
labile bond reaches an appropriate intra- and/or extra-cellular
environment. For example, a pH labile bond may be cleaved when the
molecule enters an acidified endosome. Thus, a pH labile bond may
be considered to be an endosomal cleavable bond. Enzyme cleavable
bonds may be cleaved when exposed to enzymes such as those present
in an endosome or lysosome or in the cytoplasm. A disulfide bond
may be cleaved when the molecule enters the more reducing
environment of the cell cytoplasm. Thus, a disulfide may be
considered to be a cytoplasmic cleavable bond. As used herein, a
pH-labile bond is a labile bond that is selectively broken under
acidic conditions (pH<7). Such bonds may also be termed
endosomally labile bonds, since cell endosomes and lysosomes have a
pH less than 7.
Oligomer Linked Biocleavable Conjugates
[0236] The oligomeric compound may optionally, comprise a second
region (region B) which is positioned between the oligomer
(referred to as region A) and the conjugate (referred to as region
C). Region B may be a linker such as a cleavable linker (also
referred to as a physiologically labile linkage). (see Example
7)
[0237] Nuclease Susceptible Physiological Labile Linkages: In some
embodiments, the oligomer (also referred to as oligomeric compound)
of the invention (or conjugate) comprises three regions: [0238] iv)
a first region (region A), which comprises 10-18 contiguous
nucleotides; [0239] v) a second region (region B) which comprises a
biocleavable linker [0240] vi) a third region (C) which comprises a
conjugate moiety, a targeting moiety, an activation moiety, wherein
the third region is covalent linked to the second region.
[0241] In some embodiments, region B may be a phosphate nucleotide
linker. For example such linkers may be used when the conjugate is
a lipophilic conjugate, such as a lipid, a fatty acid, sterol, such
as cholesterol or tocopherol. Phosphate nucleotide linkers may also
be used for other conjugates, for example carbohydrate conjugates,
such as GalNac.
Peptide Linkers
[0242] In some embodiments, the biocleavable linker (region B) is a
peptide, such as a trilysine peptide linker which may be used in a
polyGalNac conjugate, such a triGalNac conjugate. Other linkers
known in the art which may be used, include disulfide linkers.
Phosphate Nucleotide Linkers
[0243] In some embodiments, region B comprises between 1-6
nucleotides, which is covalently linked to the 5' or 3' nucleotide
of the first region, such as via a internucleoside linkage group
such as a phosphodiester linkage, wherein either [0244] a. the
internucleoside linkage between the first and second region is a
phosphodiester linkage and the nucleoside of the second region
[such as immediately] adjacent to the first region is either DNA or
RNA; and/or [0245] b. at least 1 nucleoside of the second region is
a phosphodiester linked DNA or RNA nucleoside;
[0246] In some embodiments, region A and region B form a single
contiguous nucleotide sequence of 12-22 nucleotides in length.
[0247] In some aspects the internucleoside linkage between the
first and second regions may be considered part of the second
region.
[0248] In some embodiments, there is a phosphorus containing
linkage group between the second and third region. The phosphorus
linkage group, may, for example, be a phosphate (phosphodiester), a
phosphorothioate, a phosphorodithioate or a boranophosphate group.
In some embodiments, this phosphorus containing linkage group is
positioned between the second region and a linker region which is
attached to the third region. In some embodiments, the phosphate
group is a phosphodiester.
[0249] Therefore, in some aspects the oligomeric compound comprises
at least two phosphodiester groups, wherein at least one is as
according to the above statement of invention, and the other is
positioned between the second and third regions, optionally between
a linker group and the second region.
[0250] In some embodiments, the third region is an activation
group, such as an activation group for use in conjugation. In this
respect, the invention also provides activated oligomers comprising
region A and B and a activation group, e.g an intermediate which is
suitable for subsequent linking to the third region, such as
suitable for conjugation.
[0251] In some embodiments, the third region is a reactive group,
such as a reactive group for use in conjugation. In this respect,
the invention also provides oligomers comprising region A and B and
a reactive group, e.g an intermediate which is suitable for
subsequent linking to the third region, such as suitable for
conjugation. The reactive group may, in some embodiments comprise
an amine of alcohol group, such as an amine group.
[0252] In some embodiments region A comprises at least one, such as
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
or 21 internucleoside linkages other than phosphodiester, such as
internucleoside linkages which are (optionally independently]
selected from the group consisting of phosphorothioate,
phosphorodithioate, and boranophosphate, and methylphosphonate,
such as phosphorothioate. In some embodiments region A comprises at
least one phosphorothioate linkage. In some embodiments at least
50%, such as at least 75%, such as at least 90% of the
internucleoside linkages, such as all the internucleoside linkages
within region A are other than phosphodiester, for example are
phosphorothioate linkages. In some embodiments, all the
internucleoside linkages in region A are other than
phosphodiester.
[0253] In some embodiments, the oligomeric compound comprises an
antisense oligonucleotide, such as an antisense oligonucleotide
conjugate. The antisense oligonucleotide may be or may comprise the
first region, and optionally the second region. In this respect, in
some embodiments, region B may form part of a contiguous nucleobase
sequence which is complementary to the (nucleic acid) target. In
other embodiments, region B may lack complementarity to the
target.
[0254] Alternatively stated, in some embodiments, the invention
provides a non-phosphodiester linked, such as a phosphorothioate
linked, oligonucleotide (e.g. an antisense oligonucleotide) which
has at least one terminal (5' and/or 3') DNA or RNA nucleoside
linked to the adjacent nucleoside of the oligonucleotide via a
phosphodiester linkage, wherein the terminal DNA or RNA nucleoside
is further covalently linked to a conjugate moiety, a targeting
moiety or a blocking moiety, optionally via a linker moiety.
[0255] In some embodiments, the oligomeric compound comprises an
antisense oligonucleotide, such as an antisense oligonucleotide
conjugate. The antisense oligonucleotide may be or may comprise the
first region, and optionally the second region. In this respect, in
some embodiments, region B may form part of a contiguous nucleobase
sequence which is complementary to the (nucleic acid) target. In
other embodiments, region B may lack complementarity to the
target.
[0256] In some embodiments, at least two consecutive nucleosides of
the second region are DNA nucleosides (such as at least 3 or 4 or 5
consecutive DNA nucleotides).
[0257] In such an embodiment, the oligonucleotide of the invention
may be described according to the following formula:
5'-A-PO-B [Y)X-3' or 3'-A-PO-B [Y)X-5'
[0258] wherein A is region A, PO is a phosphodiester linkage, B is
region B, Y is an optional linkage group, and X is a conjugate, a
targeting, a blocking group or a reactive or activation group.
[0259] In some embodiments, region B comprises 3'-5' or 5'-3': i) a
phosphodiester linkage to the 5' nucleoside of region A, ii) a DNA
or RNA nucleoside, such as a DNA nucleoside, and iii) a further
phosphodiester linkage
5'-A-PO-B-PO-3' or 3'-A-PO-B-PO-5'
[0260] The further phosphodiester linkage link the region B
nucleoside with one or more further nucleoside, such as one or more
DNA or RNA nucleosides, or may link to X (is a conjugate, a
targeting or a blocking group or a reactive or activation group)
optionally via a linkage group (Y).
[0261] In some embodiments, region B comprises 3'-5' or 5'-3': i) a
phosphodiester linkage to the 5' nucleoside of region A, ii)
between 2-10 DNA or RNA phosphodiester linked nucleosides, such as
a DNA nucleoside, and optionally iii) a further phosphodiester
linkage:
5'-A-[PO-B]n-[Y]-X 3' or 3'-A-[PO-B]n-[Y]-X 5'
5'-A-[PO-B]n-PO-[Y]-X 3' or 3'-A-[PO-B]n-PO-[Y]-X 5'
[0262] Wherein A represent region A, [PO-B]n represents region B,
wherein n is 1-10, such as 1, 2, 3,4, 5, 6, 7, 8, 9 or 10, PO is an
optional phosphodiester linkage group between region B and X (or Y
if present).
[0263] In some embodiments the invention provides compounds
according to (or comprising) one of the following formula:
5' [Region A]-PO-[region B] 3'-Y-X
5' [Region A]-PO-[region B]-PO 3'-Y-X
5' [Region A]-PO-[region B] 3'-X
5' [Region A]-PO-[region B]-PO 3'-X
3' [Region A]-PO-[region B] 5'-Y-X
3' [Region A]-PO-[region B]-PO 5'-Y-X
3' [Region A]-PO-[region B] 5'-X
3' [Region A]-PO-[region B]-PO 5'-X
[0264] Region B, may for example comprise or consist of:
5' DNA3'
3' DNA 5'
5' DNA-PO-DNA-3'
3' DNA-PO-DNA-5'
5' DNA-PO-DNA-PO-DNA 3'
3' DNA-PO-DNA-PO-DNA 5'
5' DNA-PO-DNA-PO-DNA-PO-DNA 3'
3' DNA-PO-DNA-PO-DNA-PO-DNA 5'
5' DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA 3'
3' DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA 5'
[0265] It should be recognized that phosphate linked biocleavable
linkers may employ nucleosides other than DNA and RNA. Bio
cleavable nucleotide linkers may, for example, be identified using
the assays in Example 7.
[0266] In some embodiments, the compound of the invention comprises
a biocleavable linker (also referred to as the physiologically
labile linker, Nuclease Susceptible Physiological Labile Linkages,
or nuclease susceptible linker), for example the phosphate
nucleotide linker (such as region B) or a peptide linker, which
joins the oligomer (or contiguous nucleotide sequence or region A),
to a conjugate moiety (or region C).
[0267] The susceptibility to cleavage in the assays shown in
Example 7 can be used to determine whether a linker is biocleavable
or physiologically labile.
[0268] Biocleavable linkers according to the present invention
refers to linkers which are susceptible to cleavage in a target
tissue (i.e. physiologically labile), for example liver and/or
kidney. It is preferred that the cleavage rate seen in the target
tissue is greater than that found in blood serum. Suitable methods
for determining the level (%) of cleavage in tissue (e.g. liver or
kidney) and in serum are found in example 6. In some embodiments,
the biocleavable linker (also referred to as the physiologically
labile linker, or nuclease susceptible linker), such as region B,
in a compound of the invention, are at least about 20% cleaved,
such as at least about 30% cleaved, such as at least about 40%
cleaved, such as at least about 50% cleaved, such as at least about
60% cleaved, such as at least about 70% cleaved, such as at least
about 75% cleaved, in the liver or kidney homogenate assay of
Example 7. In some embodiments, the cleavage (%) in serum, as used
in the assay in Example 7, is less than about 30%, is less than
about 20%, such as less than about 10%, such as less than 5%, such
as less than about 1%.
[0269] In some embodiments, which may be the same of different, the
biocleavable linker (also referred to as the physiologically labile
linker, or nuclease susceptible linker), such as region B, in a
compound of the invention, are susceptible to S1 nuclease cleavage.
Susceptibility to S1 cleavage may be evaluated using the S1
nuclease assay shown in Example 7. In some embodiments, the
biocleavable linker (also referred to as the physiologically labile
linker, or nuclease susceptible linker), such as region B, in a
compound of the invention, are at least about 30% cleaved, such as
at least about 40% cleaved, such as at least about 50% cleaved,
such as at least about 60% cleaved, such as at least about 70%
cleaved, such as at least about 80% cleaved, such as at least about
90% cleaved, such as at least 95% cleaved after 120 min incubation
with S1 nuclease according to the assay used in Example 7.
Sequence Selection in the Second Region:
[0270] In some embodiments, region B does not form a complementary
sequence when the oligonucleotide region A and B is aligned to the
complementary target sequence.
[0271] In some embodiments, region B does form a complementary
sequence when the oligonucleotide region A and B is aligned to the
complementary target sequence. In this respect region A and B
together may form a single contiguous sequence which is
complementary to the target sequence.
[0272] In some embodiments, the sequence of bases in region B is
selected to provide an optimal endonuclease cleavage site, based
upon the predominant endonuclease cleavage enzymes present in the
target tissue or cell or sub-cellular compartment. In this respect,
by isolating cell extracts from target tissues and non-target
tissues, endonuclease cleavage sequences for use in region B may be
selected based upon a preferential cleavage activity in the desired
target cell (e.g. liver/hepatocytes) as compared to a non-target
cell (e.g. kidney). In this respect, the potency of the compound
for target down-regulation may be optimized for the desired
tissue/cell.
[0273] In some embodiments region B comprises a dinucleotide of
sequence AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT,
GC, or GG, wherein C may be 5-methylcysteine, and/or T may be
replaced with U. In some embodiments region B comprises a
trinucleotide of sequence AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG,
ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA,
TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT,
CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT, CGC,
CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG,
GGA, GGT, GGC, and GGG wherein C may be 5-methylcysteine and/or T
may be replaced with U. In some embodiments region B comprises a
trinucleotide of sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX,
ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TATX,
TACX, TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX,
TGTX, TGCX, TGGX, CAAX, GATX, CALX, CAGX, CTAX, CTGX, CTCX, CTTX,
COAX, CCTX, CCCX, CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX,
CAGX, GTAX, GTTX, GTCX, GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX,
GGCX, and GGGX, wherein X may be selected from the group consisting
of A, T, U, G, C and analogues thereof, wherein C may be
5-methylcysteine and/or T may be replaced with U. It will be
recognised that when referring to (naturally occurring) nucleobases
A, T, U, G, C, these may be substituted with nucleobase analogues
which function as the equivalent natural nucleobase (e.g. base pair
with the complementary nucleoside).
Amino Alkyl Intermediates
[0274] The invention further provides for the LNA oligomer
intermediates which comprise an antisense LNA oligomer which
comprises an (e.g. terminal, 5' or 3') amino alkyl, such as a
C2-C36 amino alkyl group, including, for example C6 and C12 amino
alkyl groups. The amino alkyl group may be added to the LNA
oligomer as part of standard oligonucleotide synthesis, for example
using a (e.g. protected) amino alkyl phosphoramidite. The linkage
group between the amino alkyl and the LNA oligomer may for example
be a phosphorothioate or a phosphodiester, or one of the other
nucleoside linkage groups referred to herein, for example. The
amino alkyl group may be covalently linked to, for example, the 5'
or 3' of the LNA oligomer, such as by the nucleoside linkage group,
such as phosphorothioate or phosphodiester linkage.
[0275] The invention also provides a method of synthesis of the LNA
oligomer comprising the sequential synthesis of the LNA oligomer,
such as solid phase oligonucleotide synthesis, comprising the step
of adding a amino alkyl group to the oligomer, such as e.g. during
the first or last round of oligonucleotide synthesis. The method of
synthesis my further comprise the step of reacting the a conjugate
to the amino alkyl-LNA oligomer (the conjugation step). The a
conjugate may comprise suitable linkers and/or branch point groups,
and optionally further conjugate groups, such as hydrophobic or
lipophilic groups, as described herein. The conjugation step may be
performed whilst the oligomer is bound to the solid support (e.g.
after oligonucleotide synthesis, but prior to elution of the
oligomer from the solid support), or subsequently (i.e. after
elution). The invention provides for the use of an amino alkyl
linker in the synthesis of the oligomer of the invention.
Method of Manufacture/Synthesis
[0276] The invention provides for a method of synthesizing (or
manufacture) of an oligomeric compound, such as the oligomeric
compound of the invention, said method comprising either: [0277] a)
a step of providing a [solid phase] oligonucleotide synthesis
support to which one of the following is attached [third region]:
[0278] i) a linker group (-Y-) [0279] ii) a group selected from the
group consisting of a conjugate, a targeting group, a blocking
group, a reactive group [e.g. an amine or an alcohol] or an
activation group (X-) [0280] iii) an -Y-X group [0281] and [0282]
b) a step of [sequential] oligonucleotide synthesis of region B
followed by region A, and/or: [0283] c) a step of [sequential]
oligonucleotide synthesis of a first region (A) and a second region
(B), wherein the synthesis step is followed by [0284] d) a step of
adding a third region [phosphoramidite comprising] [0285] i) a
linker group (-Y-) [0286] ii) a group selected from the group
consisting of a conjugate, a targeting group, a blocking group, a
reactive group [e.g. an amine or an alcohol] or an activation group
(X-) [0287] iii) an -Y-X group followed by [0288] e) the cleavage
of the oligomeric compound from the [solid phase] support wherein,
optionally said method further comprises a further step selected
from: [0289] f) wherein the third group is an activation group, the
step of activating the activation group to produce a reactive
group, followed by adding a conjugate, a blocking, or targeting
group to the reactive group, optionally via a linker group (Y);
[0290] g) wherein the third region is a reactive group, the step of
adding a conjugate, a blocking, or targeting group to the reactive
group, optionally via a linker group (Y). [0291] h) wherein the
third region is a linker group (Y), the step of adding a conjugate,
a blocking, or targeting group to the linker group (Y)
[0292] wherein steps f), g) or h) are performed either prior to or
subsequent to cleavage of the oligomeric compound from the
oligonucleotide synthesis support. In some embodiments, the method
may be performed using standard phosphoramidite chemistry, and as
such the region X and/or region X or region X and Y may be
provided, prior to incorporation into the oligomer, as a
phosphoramidite. Please see FIGS. 5-10 which illustrate
non-limiting aspects of the method of the invention.
[0293] The invention provides for a method of synthesizing (or
manufacture) of an oligomeric compound, such as the oligomeric
compound of the invention, said method comprising a step of
[sequential] oligonucleotide synthesis of a first region (A) and
optionally a second region (B), wherein the synthesis step is
followed by a step of adding a third region [phosphoramidite
comprising] region X (also referred to as region C) or Y, such as a
region comprising a group selected from the group consisting of a
conjugate, a targeting group, a blocking group, a functional group,
a reactive group [e.g. an amine or an alcohol] or an activation
group (X), or an -Y-X group followed by the cleavage of the
oligomeric compound from the [solid phase] support.
[0294] It is however recognized that the region X or X-Y may be
added after the cleavage from the solid support. Alternatively, the
method of synthesis may comprise the steps of synthesizing a first
(A), and optionally second region (B), followed by the cleavage of
the oligomer from the support, with a subsequent step of adding a
third region, such as X or X-Y group to the oligomer. The addition
of the third region may be achieved, by example, by adding an amino
phosphoramidite unit in the final step of oligomer synthesis (on
the support), which can, after cleavage from the support, be used
to join to the X or X-Y group, optionally via an activation group
on the X or Y (when present) group. In the embodiments where the
cleavable linker is not a nucleotide region, region B may be a
non-nucleotide cleavable linker for example a peptide linker, which
may form part of region X (also referred to as region C) or be
region Y (or part thereof).
[0295] In some embodiments of the method, region X (such as C) or
(X-Y), such as the conjugate (e.g. a GalNAc conjugate) comprises an
activation group, (an activated functional group) and in the method
of synthesis the activated conjugate (or region x, or X-Y) is added
to the first and second regions, such as an amino linked oligomer.
The amino group may be added to the oligomer by standard
phosphoramidite chemistry, for example as the final step of
oligomer synthesis (which typically will result in amino group at
the 5' end of the oligomer).
[0296] For example during the last step of the oligonucleotide
synthesis a protected amino-alkyl phosphoramidite is used, for
example a TFA-aminoC6 phosphoramidite
(6-(Trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphor-
amidite). Region X (or region C as referred to herein), such as the
conjugate (e.g. a GalNac conjugate) may be activated via NHS ester
method and then the aminolinked oligomer is added. For example a
N-hydroxysuccinimide (NHS) may be used as activating group for
region X (or region C, such as a conjugate, such as a GalNac
conjugate moiety. The invention provides an oligomer prepared by
the method of the invention.
[0297] In some embodiments, region X and/or region X or region X
and Y may be covalently joined (linked) to region B via a phosphate
nucleoside linkage, such as those described herein, including
phosphodiester or phosphorothioate, or via an alternative group,
such as a triazol group.
[0298] In some embodiments, the internucleoside linkage between the
first and second region is a phosphodiester linked to the first (or
only) DNA or RNA nucleoside of the second region, or region B
comprises at least one phosphodiester linked DNA or RNA
nucleoside.
[0299] The second region may, in some embodiments, comprise further
DNA or RNA nucleosides which may be phosphodiester linked. The
second region is further covalently linked to a third region which
may, for example, be a conjugate, a targeting group a reactive
group, and/or a blocking group.
[0300] In some aspects, the present invention is based upon the
provision of a labile region, the second region, linking the first
region, e.g. an antisense oligonucleotide, and a conjugate or
functional group, e.g. a targeting or blocking group. The labile
region comprises at least one phosphodiester linked nucleoside,
such as a DNA or RNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7, 8,9
or 10 phosphodiester linked nucleosides, such as DNA or RNA. In
some embodiments, the oligomeric compound comprises a cleavable
(labile) linker. In this respect the cleavable linker is preferably
present in region B (or in some embodiments, between region A and
B).
[0301] Alternatively stated, in some embodiments, the invention
provides a non-phosphodiester linked, such as a phosphorothioate
linked, oligonucleotide (e.g. an antisense oligonucleotide) which
has at least one terminal (5' and/or 3') DNA or RNA nucleoside
linked to the adjacent nucleoside of the oligonucleotide via a
phosphodiester linkage, wherein the terminal DNA or RNA nucleoside
is further covalently linked to a conjugate moiety, a targeting
moiety or a blocking moiety, optionally via a linker moiety.
Compositions
[0302] The oligomer of the invention may be used in pharmaceutical
formulations and compositions. Suitably, such compositions comprise
a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
WO2007/031091 provides suitable and preferred pharmaceutically
acceptable diluent, carrier and adjuvants--which are hereby
incorporated by reference. Suitable dosages, formulations,
administration routes, compositions, dosage forms, combinations
with other therapeutic agents, pro-drug formulations are also
provided in WO2007/031091--which are also hereby incorporated by
reference.
Applications
[0303] The oligomers of the invention may be utilized as research
reagents for, for example, diagnostics, therapeutics and
prophylaxis.
[0304] In research, such oligomers may be used to specifically
inhibit the synthesis of APOB protein (typically by degrading or
inhibiting the mRNA and thereby prevent protein formation) in cells
and experimental animals thereby facilitating functional analysis
of the target or an appraisal of its usefulness as a target for
therapeutic intervention.
[0305] In diagnostics the oligomers may be used to detect and
quantitate APOB expression in cell and tissues by northern
blotting, in-situ hybridisation or similar techniques.
[0306] For therapeutics, an animal or a human, suspected of having
a disease or disorder, which can be treated by modulating the
expression of APOB is treated by administering oligomeric compounds
in accordance with this invention. Further provided are methods of
treating a mammal, such as treating a human, suspected of having or
being prone to a disease or condition, associated with expression
of APOB by administering a therapeutically or prophylactically
effective amount of one or more of the oligomers or compositions of
the invention. The oligomer, a conjugate or a pharmaceutical
composition according to the invention is typically administered in
an effective amount.
[0307] The invention also provides for the use of the compound or
conjugate of the invention as described for the manufacture of a
medicament for the treatment of a disorder as referred to herein,
or for a method of the treatment of as a disorder as referred to
herein.
[0308] The invention also provides for a method for treating a
disorder as referred to herein said method comprising administering
a compound according to the invention as herein described, and/or a
conjugate according to the invention, and/or a pharmaceutical
composition according to the invention to a patient in need
thereof.
Medical Indications
[0309] The oligomers and other compositions according to the
invention can be used for the treatment of conditions associated
with over expression or expression of mutated version of the
ApoB.
[0310] The invention further provides use of a compound of the
invention in the manufacture of a medicament for the treatment of a
disease, disorder or condition as referred to herein.
[0311] Generally stated, one aspect of the invention is directed to
a method of treating a mammal suffering from or susceptible to
conditions associated with abnormal levels and/or activity of APOB,
comprising administering to the mammal and therapeutically
effective amount of an oligomer targeted to APOB that comprises one
or more LNA units. The oligomer, a conjugate or a pharmaceutical
composition according to the invention is typically administered in
an effective amount.
[0312] The disease or disorder, as referred to herein, may, in some
embodiments be associated with a mutation in the APOB gene or a
gene whose protein product is associated with or interacts with
APOB. Therefore, in some embodiments, the target mRNA is a mutated
form of the APOB sequence.
[0313] An interesting aspect of the invention is directed to the
use of an oligomer (compound) as defined herein or a conjugate as
defined herein for the preparation of a medicament for the
treatment of a disease, disorder or condition as referred to
herein.
[0314] The methods of the invention are preferably employed for
treatment or prophylaxis against diseases caused by abnormal levels
and/or activity of APOB.
[0315] Alternatively stated, In some embodiments, the invention is
furthermore directed to a method for treating abnormal levels
and/or activity of APOB, said method comprising administering a
oligomer of the invention, or a conjugate of the invention or a
pharmaceutical composition of the invention to a patient in need
thereof.
[0316] The invention also relates to an oligomer, a composition or
a conjugate as defined herein for use as a medicament.
[0317] The invention further relates to use of a compound,
composition, or a conjugate as defined herein for the manufacture
of a medicament for the treatment of abnormal levels and/or
activity of APOB or expression of mutant forms of APOB (such as
allelic variants, such as those associated with one of the diseases
referred to herein).
[0318] Moreover, the invention relates to a method of treating a
subject suffering from a disease or condition such as those
referred to herein.
[0319] A patient who is in need of treatment is a patient suffering
from or likely to suffer from the disease or disorder.
[0320] In some embodiments, the term `treatment` as used herein
refers to both treatment of an existing disease (e.g. a disease or
disorder as herein referred to), or prevention of a disease, i.e.
prophylaxis. It will therefore be recognised that treatment as
referred to herein may, In some embodiments, be prophylactic.
[0321] In one embodiment, the invention relates to compounds or
compositions comprising compounds for treatment of
hypercholesterolemia and related disorders, or methods of treatment
using such compounds or compositions for treating
hypercholesterolemia and related disorders, wherein the term
"related disorders" when referring to hypercholesterolemia refers
to one or more of the conditions selected from the group consisting
of: atherosclerosis, hyperlipidemia, hypercholesterolemia, familiar
hypercholesterolemia e.g. gain of function mutations in APOB,
HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial
hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant
hypercholesterolemia, coronary artery disease (CAD), and coronary
heart disease (CHD.
EXAMPLES
[0322] Oligonucleotides
[0323] ApoB Targeting Compounds
[0324] Oligonucleotide Sequence Motifs
TABLE-US-00002 (SEQ ID NO 1) GCATTGGTATTCA (SEQ ID NO 2)
GTTGACACTGTC
TABLE-US-00003 SEQ Cleavable ID Seq (5'-3') Linker Region C- # NO
(Region A) (Region B) Conjugate #1 3 GCattggtatTCA no no #2 4
GCattggtatTCA no Cholesterol #3 5 GCattggtatTCA SS Cholesterol #4 6
GCattggtatTCA 3PO-DNA Cholesterol (5'tca3') #5 7 GCattggtatTCA
2PO-DNA Cholesterol (5'ca3') #6 8 GCattggtatTCA 1PO-DNA Cholesterol
(5'a3')
[0325] ApoB Targeting Compounds with FAM Label Conjugates
TABLE-US-00004 SEQ Seq Cleavable Conjugate ID (5'-3') linker (B)
(C) 9 GCattggtatTCA 3PO-DNA FAM (5'tca3') 10 GCattggtatTCA 2PO-DNA
FAM (5'ca3') 11 GCattggtatTCA 1PO-DNA FAM (5'a3') 12 GCattggtatTCA
3PO-DNA FAM (5'gac3') 13 GCattggtatTCA no FAM
TABLE-US-00005 SEQ ID Seq Cleavable NO (5'-3') Linker (B) Conjugate
14 GCattggtatTCA no Folic acid 15 GCattggtatTCA SS Folic acid 16
GCattggtatTCA 2PO-DNA Folic acid (5'ca3') 17 GCattggtatTCA no
monoGalNAc 18 GCattggtatTCA SS monoGalNAc 19 GCattggtatTCA 2PO-DNA
monoGalNAc (5'ca3') 20 GCattggtatTCA GalNAc cluster Conj2a 21
GCattggtatTCA no FAM 22 GCattggtatTCA SS FAM 23 GCattggtatTCA
2PO-DNA FAM (5'ca3') 24 GCattggtatTCA no Tocopherol 25
GCattggtatTCA SS Tocopherol 26 GCattggtatTCA 2PO-DNA Tocopherol
(5'ca3') 30 GCattggtatTCA GalNAc cluster Conj1a
TABLE-US-00006 Seq Cleavable SEQ ID NO (5'-3') Linker (B) 7
GCattggtatTCA 2PO-DNA Cholesterol (5'ca3') 20 GCattggtatTCA GalNAc
cluster Conj2a 28 GTtgacactgTC 2PO-DNA Cholesterol (5'ca3') 29
GTtgacactgTC GalNAc cluster Conj2a 31 GTtgacactgTC GalNAc cluster
Conj1a
[0326] Mouse Experiments: Unless otherwise specified, the mouse
experiments may be performed as follows:
[0327] Dose Administration and Sampling:
[0328] 7-10 week old C57Bl6-N mice were used, animals were age and
sex matched (females for study 1, 2 and 4, males in study 3).
Compounds were injected i.v. into the tail vein. For intermediate
serum sampling, 2-3 drops of blood were collected by puncture of
the vena facialis, final bleeds were taken from the vena cava
inferior. Serum was collected in gel-containing serum-separation
tubes (Greiner) and kept frozen until analysis.
[0329] C57BL6 mice were dosed i.v. with a single dose of 1 mg/kg
ASO (or amount shown) formulated in saline or saline alone
according to the information shown. Animals were sacrificed at e.g.
day 4 or 7 (or time shown) after dosing and liver and kidney were
sampled. RNA isolation and mRNA analysis: mRNA analysis from tissue
was performed using the Qantigene mRNA quantification kit
("bDNA-assay", Panomics/Affimetrix), following the manufacturers
protocol. For tissue lysates, 50-80 mg of tissue was lysed by
sonication in 1 ml lysis-buffer containing Proteinase K. Lysates
were used directly for bDNA-assay without RNA extraction. Probesets
for the target and GAPDH were obtained custom designed from
Panomics. For analysis, luminescence units obtained for target
genes were normalized to the housekeeper GAPDH.
[0330] Serum analysis for ALT, AST and cholesterol was performed on
the "Cobas INTEGRA 400 plus" clinical chemistry platform (Roche
Diagnostics), using 10 .mu.l of serum.
[0331] For quantification of Factor VII serum levels, the BIOPHEN
FVII enzyme activity kit (#221304, Hyphen BioMed) was used
according to the manufacturer's protocol.
[0332] For oligonucleotide quantification, a fluorescently-labeled
PNA probe is hybridized to the oligo of interest in the tissue
lysate. The same lysates are used as for bDNA-assays, just with
exactly weighted amounts of tissue. The heteroduplex is quantified
using AEX-HPLC and fluorescent detection.
Example 1
Synthesis of Compounds
[0333] Oligonucleotides were synthesized on uridine universal
supports using the phosphoramidite approach on an Expedite
8900/MOSS synthesizer (Multiple Oligonucleotide Synthesis System)
at 4 .mu.mol scale. At the end of the synthesis, the
oligonucleotides were cleaved from the solid support using aqueous
ammonia for 1-2 hours at room temperature, and further deprotected
for 16 hours at 65.degree. C. The oligonucleotides were purified by
reverse phase HPLC (RP-HPLC) and characterized by UPLC, and the
molecular mass was further confirmed by ESI-MS. See below for more
details.
[0334] Elongation of the Oligonucleotide
[0335] The coupling of .beta.-cyanoethyl-phosphoramidites
(DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz),
LNA-A(Bz), LNA-G(dmf), LNA-T or C6-S--S linker) is performed by
using a solution of 0.1 M of the 5'-O-DMT-protected amidite in
acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25
M) as activator. For the final cycle a commercially available
C6-linked cholesterol phosphoramidite was used at 0.1 M in DCM.
Thiolation for introduction of phosphorthioate linkages is carried
out by using xanthane hydride (0.01 M in acetonitrile/pyridine
9:1). Phosphordiester linkages are introduced using 0.02 M iodine
in THF/Pyridine/water 7:2:1. The rest of the reagents are the ones
typically used for oligonucleotide synthesis. For post solid phase
synthesis conjugation a commercially available C6 aminolinker
phorphoramidite was used in the last cycle of the solid phase
synthesis and after deprotection and cleavage from the solid
support the aminolinked deprotected oligonucleotide was isolated.
The conjugates was introduced via activation of the functional
group using standard synthesis methods.
[0336] Purification by RP-HPLC:
[0337] The crude compounds were purified by preparative RP-HPLC on
a Phenomenex Jupiter C18 10.mu. 150.times.10 mm column. 0.1 M
ammonium acetate pH 8 and acetonitrile was used as buffers at a
flowrate of 5 mL/min. The collected fractions were lyophilized to
give the purified compound typically as a white solid.
[0338] Abbreviations:
[0339] DCI: 4,5-Dicyanoimidazole
[0340] DCM: Dichloromethane
[0341] DMF: Dimethylformamide
[0342] DMT: 4,4'-Dimethoxytrityl
[0343] THF: Tetrahydrofurane
[0344] Bz: Benzoyl
[0345] Ibu: Isobutyryl
[0346] RP-HPLC: Reverse phase high performance liquid
chromatography
Example 2
Design of LNA Antisense Oligonucleotides
[0347] Oligomers used in the examples and figures. The SEQ# is an
identifier used throughout the examples and figures
TABLE-US-00007 Comp'ID (SEQ ID) Compound Sequence Comment
GCATTGGTATTCA Nucleobase (SEQ ID NO 1) sequence GTTGACACTGTC
Nucleobase (SEQ ID NO 2) sequence #1 (3) 5'- G.sub.s.sup.o
.sup.mC.sub.s.sup.o a.sub.s t.sub.s t.sub.s g.sub.s g.sub.s No
t.sub.s a.sub.s t.sub.s T.sub.s.sup.o .sup.mC.sub.s.sup.o A.sup.o
-3' conjugate #2 (4) 5'- CHOL G.sub.s.sup.o .sup.mC.sub.s.sup.o
a.sub.s t.sub.s t.sub.s g.sub.s Chol- g.sub.s t.sub.s a.sub.s
t.sub.s T.sub.s.sup.o .sup.mC.sub.s.sup.o A.sup.o -3' Compound #3
(5) 5'- Chol_C6 C6SSC6 G.sub.s.sup.o .sup.mC.sub.s.sup.o Chol-
a.sub.s t.sub.s t.sub.s g.sub.s g.sub.s t.sub.s a.sub.s t.sub.s
T.sub.s.sup.o SS-#1 .sup.mC.sub.s.sup.o A.sup.o -3' #4 (6) 5'-
Chol_C6 t c a G.sub.s.sup.o .sup.mC.sub.s.sup.o Chol- a.sub.s
t.sub.s t.sub.s g.sub.s g.sub.s t.sub.s a.sub.s t.sub.s
T.sub.s.sup.o 3PO-#1 .sup.mC.sub.s.sup.o A.sup.o -3' #5 (7) 5'-
Chol_C6 c a G.sub.s.sup.o .sup.mC.sub.s.sup.o Chol- a.sub.s t.sub.s
t.sub.s g.sub.s g.sub.s t.sub.s a.sub.s t.sub.s T.sub.s.sup.o
2PO-#1 .sup.mC.sub.s.sup.o A.sup.o -3'
Example 3
Knock Down of ApoB mRNA with Cholesterol-Conjugates in Vivo
[0348] C57BL6/J mice were injected with a single dose saline or 1
mg/kg unconjugated LNA-antisense oligonucleotide (#3) or equimolar
amounts of LNA antisense oligonucleotides conjugated to Cholesterol
with different linkers and sacrificed at days 1-10 according to the
table below. RNA was isolated from liver and kidney and subjected
to qPCR with ApoB specific primers and probe to analyse for ApoB
mRNA knockdown.
[0349] Conclusions: Cholesterol conjugated to an ApoB LNA antisense
oligonucleotide with a linker composed of 2 or 3 DNA with
Phosphodiester-backbone (Seq #4 and 5) showed a preference for
liver specific knock down of ApoB (FIG. 11). This means increases
efficiency and duration of ApoB mRNA knock down in liver tissue
compared to the unconjugated compound (Seq #3), as well as compared
to Cholesterol conjugates with stable linker (Seq #4) and with
disulphide linker (Seq. #5) and concomitant less knock down
activity of Seq #6 and #7 in kidney tissue.
[0350] Materials and Methods:
[0351] Experimental Design:
TABLE-US-00008 Compound Conc. at Animal No. of Animal strain/ Dose
level dose vol. Body Gr. no. ID no. animals gender/feed per day 10
ml/kg weight Sacrifice A 1 1-4 4 C57BL/6J- NaCl 0.9% -- Day -1, 7
Day 10 - Chow and 10 2 5-8 4 C57BL/6J- SEQ ID 3 0.1 mg/ml Day -1, 7
Day 10 - Chow 1 mg/kg and 10 3 9-12 4 C57BL/6J- SEQ ID 4 0.12 mg/ml
Day -1, 7 Day 10 - Chow 1.2 mg/kg and 10 4 13-16 4 C57BL/6J- SEQ ID
5 0.12 mg/ml Day -1, 7 Day 10 - Chow 1.2 mg/kg and 10 5 17-20 4
C57BL/6J- SEQ ID 6 0.13 mg/ml Day -1, 7 Day 10 - Chow 1.3 mg/kg and
10 6 21-24 4 C57BL/6J- SEQ ID 7 0.13 mg/ml Day -1, 7 Day 10 - Chow
1.3 mg/kg and 10 B 7 25-28 4 C57BL/6J- NaCl 0.9% -- Day -1, 7 Day 7
- Chow 8 29-32 4 C57BL/6J- SEQ ID 3 0.1 mg/ml Day -1, 7 Day 7 -
Chow 1 mg/kg 9 33-36 4 C57BL/6J- SEQ ID 4 0.12 mg/ml Day -1, 7 Day
7 - Chow 1.2 mg/kg 10 37-40 4 C57BL/6J- SEQ ID 5 0.12 mg/ml Day -1,
7 Day 7 - Chow 1.2 mg/kg 11 41-44 4 C57BL/6J- SEQ ID 6 0.13 mg/ml
Day -1, 7 Day 7 - Chow 1.3 mg/kg 12 45-48 4 C57BL/6J- SEQ ID 7 0.13
mg/ml Day -1, 7 Day 7 - Chow 1.3 mg/kg C 13 49-52 4 C57BL/6J- NaCl
0.9% -- Day 0, 3 Day 3 - Chow 14 53-56 4 C57BL/6J- SEQ ID 3 0.1
mg/ml Day 0, 3 Day 3 - Chow 1 mg/kg 15 57-60 4 C57BL/6J- SEQ ID 4
0.12 mg/ml Day 0, 3 Day 3 - Chow 1.2 mg/kg 16 61-64 4 C57BL/6J- SEQ
ID 5 0.12 mg/ml Day 0, 3 Day 3 - Chow 1.2 mg/kg 17 65-68 4
C57BL/6J- SEQ ID 6 0.13 mg/ml Day 0, 3 Day 3 - Chow 1.3 mg/kg 18
69-72 4 C57BL/6J- SEQ ID 7 0.13 mg/ml Day 0, 3 Day 3 - Chow 1.3
mg/kg D 19 73-76 4 C57BL/6J- NaCl 0.9% -- Day -1, 1 Day 1 - Chow 20
77-80 4 C57BL/6J- SEQ ID 3 0.1 mg/ml Day -1, 1 Day 1 - Chow 1 mg/kg
21 81-84 4 C57BL/6J- SEQ ID 4 0.12 mg/ml Day -1, 1 Day 1 - Chow 1.2
mg/kg 22 85-88 4 C57BL/6J- SEQ ID 5 0.12 mg/ml Day -1, 1 Day 1 -
Chow 1.2 mg/kg 23 89-92 4 C57BL/6J- SEQ ID 6 0.13 mg/ml Day -1, 1
Day 1 - Chow 1.3 mg/kg 24 93-96 4 C57BL/6J- SEQ ID 7 0.13 mg/ml Day
-1, 1 Day 1 - Chow 1.3 mg/kg
[0352] Dose administration. C57BL/6JBom female animals, app. 20 g
at arrival, were dosed with 10 ml per kg BW (according to day 0
bodyweight) i.v. of the compound formulated in saline or saline
alone according to the above table.
[0353] Sampling of liver and kidney tissue. The animals were
anaesthetised with 70% CO.sub.2-30% O.sub.2 and sacrificed by
cervical dislocation according to the table above. One half of the
large liver lobe and one kidney were minced and submerged in
RNAlater.
[0354] Total RNA Isolation and First strand synthesis. Total RNA
was extracted from maximum 30 mg of tissue homogenized by
bead-milling in the presence of RLT-Lysis buffer using the Qiagen
RNeasy kit (Qiagen cat. no. 74106) according to the manufacturer's
instructions. First strand synthesis was performed using Reverse
Transcriptase reagents from Ambion according to the manufacturer's
instructions.
[0355] For each sample 0.5 .mu.g total RNA was adjusted to (10.8
.mu.l) with RNase free H.sub.2O and mixed with 2 .mu.l random
decamers (50 .mu.M) and 4 .mu.l dNTP mix (2.5 mM each dNTP) and
heated to 70.degree. C. for 3 min after which the samples were
rapidly cooled on ice. 2 .mu.l 10.times. Buffer RT, 1 .mu.l MMLV
Reverse Transcriptase (100 U/.mu.l) and 0.25 .mu.l RNase inhibitor
(10 U/.mu.l) were added to each sample, followed by incubation at
42.degree. C. for 60 min, heat inactivation of the enzyme at
95.degree. C. for 10 min and then the sample was cooled to
4.degree. C. cDNA samples were diluted 1:5 and subjected to RT-QPCR
using Taqman Fast Universal PCR Master Mix 2.times. (Applied
Biosystems Cat #4364103) and Taqman gene expression assay (mApoB,
Mn01545150_m1 and mGAPDH #4352339E) following the manufacturers
protocol and processed in an Applied Biosystems RT-qPCR instrument
(7500/7900 or ViiA7) in fast mode.
Example 4
In Vivo Silencing of ApoB mRNA with Different Conjugates
[0356] To explore the impact of different conjugation moieties and
linkers on the activity of an ApoB compound, Seq ID #3 was
conjugated to either monoGalNAc, Folic acid, FAM or Tocopherol
using a non-cleavable linker or biocleavable linker (Dithio (SS) or
2 DNA nucleotides with Phosphodiester backbone (PO)). Additionally
the monoGalNAc was compared to a GalNAc cluster (Conjugate 2a).
C57BL6In mice were treated i.v. with saline control or with a
single dose of 1 or 0.25 mg/kg of ASO conjugates. After 7 days the
animals were sacrificed and RNA was isolated from liver and kidney
samples and analysed for ApoB mRNA expression (FIG. 15).
[0357] Conclusions: Tocopherol conjugated to the ApoB compound with
a DNA/PO-linker (#26) increased ApoB knock down in the liver
compared to the unconjugated ApoB compound (#3) while decreasing
activity in kidney (compare FIGS. 15A and B). This points towards
an ability of the Tocopherol to redirect the ApoB compound from
kidney to liver. The non-cleavable (#24) and SS-linker (#25) were
inactive in both tissues. Mono-GalNAc conjugates with a
non-cleavable (#17) and with bio-cleavable DNA/PO linker (#19) show
a tendency to preserve the activity of the unconjugated compound
(#3) in kidney while improving activity in the Liver. Introduction
of a SS-linker decreased activity in both tissues (compare FIGS.
15A and B). Conjugation of different GalNAc conjugates e.g. mono
GalNAcPO (#19) and a GalNAc cluster (#20) also allows fine tuning
of the compound activity with focus on either liver or kidney (FIG.
15C). Folic acid and FAM conjugates with the cleavable
DNA/PO-linker (SEQ ID Nos16 and 23) behave comparable to the
unconjugated compound (3). Here as well the introduction of a
non-cleavable (14 and 21) or SS-linker (15 and 22) decreases
compound activity in both tissues (compare FIGS. 15a and 15b).
[0358] Materials and Methods:
[0359] Experimental Design:
TABLE-US-00009 Animals Animal strain/ Compound Dose Adm. Dosing
Sacrifice Gr. no. per group gender/feed Seq ID # mg/kg Route Day
Day 1 5 C57BL6 3 1 i.v. 0 7 - Chow 2 5 C57BL6 14 1 i.v. 0 7 - Chow
3 5 C57BL6 15 1 i.v. 0 7 - Chow 4 5 C57BL6 16 1 i.v.. 0 7 - Chow 5
5 C57BL6 17 1 i.v. 0 7 - Chow 6 5 C57BL6 18 1 i.v. 0 7 - Chow 7 5
C57BL6 19 1 i.v. 0 7 - Chow 8 5 C57BL6 19 0.25 i.v. 0 7 - Chow 9 5
C57BL6 20 0.25 i.v. 0 7 - Chow 10 5 C57BL6 NaCl 0.9% i.v. 0 7 -
Chow 1 5 C57BL6 1 1 i.v. 0 7 - Chow 2 5 C57BL6 31 1 i.v. 0 7 - Chow
3 5 C57BL6 32 1 i.v. 0 7 - Chow 4 5 C57BL6 33 1 i.v.. 0 7 - Chow 5
5 C57BL6 34 1 i.v. 0 7 - Chow 6 5 C57BL6 35 1 i.v. 0 7 - Chow 7 5
C57BL6 36 1 i.v. 0 7 - Chow 8 5 C57BL6 NaCl 0.9% 1 i.v. 0 7 -
Chow
[0360] Dose administration and sampling. C57BL6 mice were dosed
i.v. with a single dose of 1 mg/kg or 0.25 mg/kg ASO formulated in
saline or saline alone according to the above table. Animals were
sacrificed at day 7 after dosing and liver and kidney were sampled.
RNA isolation and mRNA analysis. Total RNA was extracted from liver
and kidney samples and ApoB mRNA levels were analysed using a
branched DNA assay
Example 5
Non-Human Primate Study
[0361] The primary objective for this study is to investigate
selected lipid markers over 7 weeks after a single slow bolus
injection of anti-ApoB LNA conjugated compounds to cynomolgus
monkeys and assess the potential toxicity of compounds in monkey.
The compounds used in this study are SEQ ID NOs 7, 20, 28 & 29,
prepared in sterile saline (0.9%) at an initial concentration of
0.625 and 2.5 mg/ml).
[0362] Female monkeys of at least 24 months old are used, and given
free access to tap water and 180 g of OWM(E) SQC SHORT expanded
diet (Dietex France, SDS, Saint Gratien, France) will be
distributed daily per animal. The total quantity of food
distributed in each cage will be calculated according to the number
of animals in the cage on that day. In addition, fruit or
vegetables will be given daily to each animal. The animals will be
acclimated to the study conditions for a period of at least 14 days
before the beginning of the treatment period. During this period,
pre-treatment investigations will be performed. The animals are
dosed i.v. at a dose if, for example, 0.25 mg/kg or 1 mg/kg. The
dose volume will be 0.4 mL/kg. 2 animals are used per group. After
three weeks, the data will be analyzed and a second group of
animals using a higher or lower dosing regimen may be
initiated--preliminary dose setting is 0.5 mg/kg and 1 mg/kg, or
lower than that based on the first data set.
[0363] The dose formulations will be administered once on Day 1.
Animals will be observed for a period of 7 weeks following
treatment, and will be released from the study on Day 51. Day 1
corresponds to the first day of the treatment period. Clinical
observations and body weight and food intake (per group) will be
recorded prior to and during the study.
[0364] Blood is sampled and analysis at the following time
points:
TABLE-US-00010 Study Day Parameters -8 RCP, L, Apo-B, OA -1 L,
Apo-B, PK, OA 1 Dosing 4 LSB, L, Apo-B, OA 8 LSB, L, Apo-B, PK, OA
15 RCP, L, Apo-B, PK, OA 22 LSB, L, Apo-B, PK, OA 29 L, Apo-B, PK,
OA 36 LSB, L, Apo-B, PK, OA 43 L, PK, Apo-B, PK, OA 50 RCP, L,
Apo-B, PK, OA RCP 0 routine clinical pathology, LSB = liver safety
biochemistry, PK = pharmacokinetics, OA = other analysis, L =
Lipids.
[0365] Blood Biochemistry
[0366] The following parameters will be determined for all
surviving animals at the occasions indicated below: [0367] full
biochemistry panel (complete list below)--on Days-8, 15 and 50,
[0368] liver Safety (ASAT, ALP, ALAT, TBIL and GGT only)--on Days
4, 8, 22 and 36, [0369] lipid profile (Total cholesterol, HDL-C,
LDL-C and Triglycerides) and Apo-B only--on Days-1, 4, 8, 22, 29,
36, and 43.
[0370] Blood (approximately 1.0 mL) is taken into lithium heparin
tubes (using the ADVIA 1650 blood biochemistry analyzer): Apo-B,
sodium, potassium, chloride, calcium, inorganic phosphorus,
glucose, HDL-C, LDL-C, urea, creatinine, total bilirubin (TBIL),
total cholesterol, triglycerides, alkaline phosphatase (ALP),
alanine aminotransferase (ALAT), aspartate aminotransferase
(ASAT),creatine kinase, gamma-glutamyl transferase (GGT), lactate
dehydrogenase, total protein, albumin, albumin/globulin ratio.
[0371] Analysis of blood: Blood samples for ApoB analysis will be
collected from Group 1-16 animals only (i.e. animals treated with
anti-PCSK9 compounds) on Days-8, -1, 4, 8, 15, 22, 29, 36, 43 and
50. Venous blood (approximately 2 mL) will be collected from an
appropriate vein in each animal into a Serum Separating Tube (SST)
and allowed to clot for at least 60.+-.30 minutes at room
temperature. Blood will be centrifuged at 1000 g for 10 minutes
under refrigerated conditions (set to maintain +4.degree. C.). The
serum will be transferred into 3 individual tubes and stored at
-80.degree. C. until analyzed at CitoxLAB France using an ELISA
method (Circulex Human PCSK9 ELISA kit, CY-8079, validated for
samples from cynomolgus monkey).
[0372] Other Analysis: WO2010142805 provides the methods for the
following analysis: qPCR, ApoB mRNA analysis. Other analysis
includes ApoB protein ELISA, serum Lp(a) analysis with ELISA
(Mercodia No. 10-1106-01), tissue and plasma oligonucleotide
analysis (drug content), Extraction of samples, standard- and
QC-samples, Oligonucleotide content determination by ELISA.
Example 6
Liver and Kidney Toxicity Assessment in Rat
[0373] Compounds of the invention can be evaluated for their
toxicity profile in rodents, such as in mice or rats. By way of
example the following protocol may be used: Wistar Han Crl:Wl(Han)
are used at an age of approximately 8 weeks old. At this age, the
males should weigh approximately 250 g. All animals have free
access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF
Spezialdiaten GmbH, Soest, Germany) and to tap water (filtered with
a 0.22 .mu.m filter) contained in bottles. The dose level of 10 and
40 mg/kg/dose is used (sub-cutaneous administration) and dosed on
days 1 and 8. The animals are euthanized on Day 15. Urine and blood
samples are collected on day 7 and 14. A clinical pathology
assessment is made on day 14. Body weight is determined prior to
the study, on the first day of administration, and 1 week prior to
necropsy. Food consumption per group will be assessed daily. Blood
samples are taken via the tail vein after 6 hours of fasting. The
following blood serum analysis is performed: erythrocyte count mean
cell volume packed cell volume hemoglobin mean cell hemoglobin
concentration mean cell hemoglobin thrombocyte count leucocyte
count differential white cell count with cell morphology
reticulocyte count, sodium potassium chloride calcium inorganic
phosphorus glucose urea creatinine total bilirubin total
cholesterol triglycerides alkaline phosphatase alanine
aminotransferase aspartate aminotransferase total protein albumin
albumin/globulin ratio. Urinalysis are performed .alpha.-GST,
.beta.-2 Microglobulin, Calbindin, Clusterin, Cystatin C, KIM-1,
Osteopontin, TIMP-1, VEGF, and NGAL. Seven analytes (Calbindin,
Clusterin, GST-.alpha., KIM-1, Osteopontin, TIMP-1, VEGF) will be
quantified under Panel 1 (MILLIPLEX.RTM. MAP Rat Kidney Toxicity
Magnetic Bead Panel 1, RKTX1MAG-37K). Three analytes (.beta.-2
Microglobulin, Cystatin C, Lipocalin-2/NGAL) will be quantified
under Panel 2 (MILLIPLEX.RTM. MAP Rat Kidney Toxicity Magnetic Bead
Panel 2, RKTX2MAG-37K). The assay for the determination of these
biomarkers' concentration in rat urines is based on the Luminex
xMAP.RTM. technology. Microspheres coated with
anti-.alpha.-GST/.beta.-2
microglobulin/calbindin/clusterin/cystacin
C/KIM-1/osteopontin/TIMP-1/VEGF/NGAL antibodies are color-coded
with two different fluorescent dyes. The following parameters are
determined (Urine using the ADVIA 1650): Urine protein, urine
creatinine. Quantitative parameters: volume, pH (using 10-Multistix
SG test strips/Clinitek 500 urine analyzer), specific gravity
(using a refractometer). Semi-quantitative parameters (using
10-Multistix SG test strips/Clinitek 500 urine analyzer): proteins,
glucose, ketones, bilirubin, nitrites, blood, urobilinogen,
cytology of sediment (by microscopic examination). Qualitative
parameters: Appearance, color. After sacrifice, the body weight and
kidney, liver and spleen weight are determined and organ to body
weight ratio calculated. Kidney and liver samples will be taken and
either frozen or stored in formalin. Microscopic analysis is
performed.
Example 7
ApoB Targeting Compounds with FAM Label Conjugates
TABLE-US-00011 [0374] Cleavable Conjugate # Seq (5'-3') linker (B)
(C) 32 GCattggtatTCA 3PO-DNA FAM (5'tca3') 33 GCattggtatTCA 2PO-DNA
FAM (5'ca3') 34 GCattggtatTCA 1PO-DNA FAM (5'a3') 35 GCattggtatTCA
3PO-DNA FAM (5'gac3') 36 GCattggtatTCA no FAM
[0375] Capital letters are LNA nucleosides (such as beta-D-oxy
LNA), lower case letters are DNA nucleosides. Subscript s
represents a phosphorothioate internucleoside linkages. LNA
cytosines are optionally 5-methyl cytosine.
[0376] FAM-labelled ASOs with different DNA/PO-linkers were
subjected to in vitro cleavage either in S1 nuclease extract, Liver
or kidney homogenates or Serum.
[0377] FAM-labeled ASOs 100 .mu.M with different DNA/PO-linkers
were subjected to in vitro cleavage by S1 nuclease in nuclease
buffer (60 U pr. 100 .mu.L) for 20 and 120 minutes (see table
below). The enzymatic activity was stopped by adding EDTA to the
buffer solution. The solutions were then subjected to AIE HPLC
analyses on a Dionex Ultimate 3000 using an Dionex DNApac p-100
column and a gradient ranging from 10 mM-1 M sodium perchlorate at
pH 7.5. The content of cleaved and non cleaved oligonucleotide were
determined against a standard using both a fluoresense detector at
615 nm and a uv detector at 260 nm.
TABLE-US-00012 SEQ ID Linker % % cleaved after 120 min NO sequence
cleaved after 20 min S1 S1 36 -- 2 5 34 a 29.1 100 33 ca 40.8 100
32 tea 74.2 100 35 gac 22.9 n.d
[0378] Conclusion: The PO linkers (or region B as referred to
herein) results in the conjugate (or group C) being cleaved off,
and both the length and/or the sequence composition of the linker
can be used to modulate susceptibility to nucleolytic cleavage of
region B. The Sequence of DNA/PO-linkers can modulate the cleavage
rate as seen after 20 min in Nuclease S1 extract Sequence selection
for region B (e.g. for the DNA/PO-linker) can therefore also be
used to modulate the level of cleavage in serum and in cells of
target tissues.
[0379] Liver, kidney and Serum were spiked with oligonucleotide SEQ
ID NO 32 to concentrations of 200 .mu.g/g tissue (see table below).
Liver and kidney samples collected from NMRI mice were homogenized
in a homogenisation buffer (0.5% Igepal CA-630, 25 mM Tris pH 8.0,
100 mM NaCl, pH 8.0 (adjusted with 1 N NaOH). The homogenates were
incubated for 24 hours at 37.degree. and thereafter the homogenates
were extracted with phenol-chloroform. The content of cleaved and
non-cleaved oligonucleotide in the extract from liver and kidney
and from the serum were determined against a standard using the
above HPLC method.
TABLE-US-00013 % cleaved after % cleaved after % cleaved after
Linker 24 hrs liver 24 hrs kidney 24 hours in Seq ID Sequence
homogenate homogenate serum 32 tca 83 95 0
[0380] Conclusion: The PO linkers (or region B as referred to
herein) results in cleavage of the conjugate (or group C) from the
oligonucleotide in liver or kidney homogenate, but not in serum.
Note: cleavage in the above assays refers to the cleavage of the
cleavable linker, the oligomer or region A should remain
functionally intact.
[0381] The susceptibility to cleavage in the assays shown in
Example 7 may be used to determine whether a linker is biocleavable
or physiologically labile.
Example 8
Knock Down of ApoB mRNA, Tissue Content, and Total Cholesterol with
GalNAc-Conjugates in Vivo
[0382] Compounds
TABLE-US-00014 SEQ ID Seq Cleavable Conjugate NO (5'-3') (A) Linker
(B) (C) 3 G.sub.sC.sub.sa.sub.st.sub.st.sub.sg.sub.sg.sub.s no no
t.sub.sa.sub.st.sub.sT.sub.sC.sub.sA 30
G.sub.sC.sub.sa.sub.st.sub.st.sub.sg.sub.sg.sub.s GalNAc cluster
t.sub.sa.sub.st.sub.sT.sub.sC.sub.sA Conj1a 20
G.sub.sC.sub.sa.sub.st.sub.st.sub.sg.sub.sg.sub.s GalNAc cluster
t.sub.sa.sub.st.sub.sT.sub.sC.sub.sA Conj2a 7
G.sub.sC.sub.sa.sub.st.sub.st.sub.sg.sub.sg.sub.s 2PO-DNA
cholesterol t.sub.sa.sub.st.sub.sT.sub.sC.sub.sA (5'ca3')
[0383] Capital letters are LNA nucleosides (such as beta-D-oxy
LNA), lower case letters are a DNA nucleoside. Subscript s
represents a phosphorothioate internucleoside linkage (region A).
LNA cytosines are optionally 5-methyl cytosine. The 2PO linker
(region B) is 5' to the sequence region A, and comprises of two DNA
nucleosides linked by phosphodiester linkage, with the
internucleoside linkage between the 3' DNA nucleoside of region A
and the 5' LNA nucleoside of region A also being phosphodiester. A
linkage group (Y) may be used to link the conjugate group, when
present, to region B, or A (SEQ ID NO 7, 20 and 30). C57BL6/J mice
were injected either iv or sc with a single dose saline or 0.25
mg/kg unconjugated LNA-antisense oligonucleotide (SEQ ID NO3) or
equimolar amounts of LNA antisense oligonucleotides conjugated to
GalNAc1, GalNAc2, or cholesterol (2PO) and sacrificed at days 1-7
according to the table below (experimental design).
[0384] RNA was isolated from liver and kidney and subjected to qPCR
with ApoB specific primers and probe to analyse for ApoB mRNA
knockdown. The oligonucleotide content was measured using ELISA
method and total cholesterol in serum was measured.
[0385] Conclusions: GalNAc1 and GalNAc2 conjugated to an ApoB LNA
antisense oligonucleotide (SEQ ID NO 30 and 20) showed knock down
of ApoB mRNA better than the unconjugated ApoB LNA (FIG. 16). For
GalNAc 1 conjugate (SEQ ID NO 30) is seems that iv dosing is better
than sc dosing which is surprising since the opposite has been
reported for another GalNAc clusters (Alnylam, 8th Annual Meeting
of the Oligonucleotide Therapeutics Society). The total cholesterol
data show how the GalNAc cluster conjugates (SEQ ID NO 30 and 20)
gives better effect than the unconjugated and the cholesterol
conjugated compounds (SEQ ID NO 7) both at iv and sc administration
(FIG. 17, a and b). The tissue content of the oligonucleotides
(FIG. 18, a-f) shows how the conjugates enhances the uptake in
liver while giving less uptake in kidney compared to the parent
compound. This holds for both iv and sc administration. When dosing
iv the GalNAc 1 (SEQ ID NO 30) gives very much uptake in liver when
compared to GalNAc 2 (SEQ ID NO 20) but since activity is good for
both compounds the GalNAc 2 conjugate appears to induce a higher
specific activity than GalNAc 1 conjugate indicating that GalNAc
conjugates without the pharmacokinetic modulator may be
particularly useful with LNA antisense oligonucleotides.
[0386] Materials and Methods:
[0387] Experimental Design:
TABLE-US-00015 Compound Conc. at Group Animal No. of Animal strain/
Dose level dose vol. Adm. Dosing Sacrifice no. id no. Animals
gender/feed per day 10 ml/kg Route day day 1 1-3 3 C57BL/6J/ /Chow
Saline -- i.v 0 1 2 4-6 3 C57BL/6J/ /Chow SEQ ID NO 3 0.025 mg/ml
i.v 0 1 0.25 mg/kg 3 7-9 3 C57BL/6J/ /Chow SEQ ID NO 3 0.025 mg/ml
s.c 0 1 0.25 mg/kg 4 10-12 3 C57BL/6J/ /Chow SEQ ID NO 30 0.036
mg/ml i.v 0 1 0.36 mg/kg 5 13-15 3 C57BL/6J/ /Chow SEQ ID NO 30
0.036 mg/ml s.c 0 1 0.36 mg/kg 6 16-18 3 C57BL/6J/ /Chow SEQ ID NO
7 0.032 mg/ml i.v 0 1 0.32 mg/kg 7 19-21 3 C57BL/6J/ /Chow SEQ ID
NO 7 0.032 mg/ml s.c 0 1 0.32 mg/kg 8 22-24 3 C57BL/6J/ /Chow SEQ
ID NO 20 0.034 mg/ml i.v 0 1 0.34 mg/kg 9 25-27 3 C57BL/6J/ /Chow
SEQ ID NO 20 0.034 mg/ml s.c 0 1 0.34 mg/kg 10 28-30 3 C57BL/6J/
/Chow Saline -- i.v 0 3 11 31-33 3 C57BL/6J/ /Chow SEQ ID NO 3
0.025 mg/ml i.v 0 3 0.25 mg/kg 12 34-36 3 C57BL/6J/ /Chow SEQ ID NO
3 0.025 mg/ml s.c 0 3 0.25 mg/kg 13 37-39 3 C57BL/6J/ /Chow SEQ ID
NO 30 0.036 mg/ml i.v 0 3 0.36 mg/kg 14 40-42 3 C57BL/6J/ /Chow SEQ
ID NO 30 0.036 mg/ml s.c 0 3 0.36 mg/kg 15 43-45 3 C57BL/6J/ /Chow
SEQ ID NO 7 0.032 mg/ml i.v 0 3 0.32 mg/kg 16 46-48 3 C57BL/6J/
/Chow SEQ ID NO 7 0.032 mg/ml s.c 0 3 0.32 mg/kg 17 49-51 3
C57BL/6J/ /Chow SEQ ID NO 20 0.034 mg/ml i.v 0 3 0.34 mg/kg 18
52-54 3 C57BL/6J/ /Chow SEQ ID NO 20 0.034 mg/ml s.c 0 3 0.34 mg/kg
19 55-57 3 C57BL/6J/ /Chow Saline -- i.v 0 7 20 58-60 3 C57BL/6J/
/Chow SEQ ID NO 3 0.025 mg/ml i.v 0 7 0.25 mg/kg 21 61-63 3
C57BL/6J/ /Chow SEQ ID NO 3 0.025 mg/ml s.c 0 7 0.25 mg/kg 22 64-66
3 C57BL/6J/ /Chow SEQ ID NO 30 0.036 mg/ml i.v 0 7 0.36 mg/kg 23
67-69 3 C57BL/6J/ /Chow SEQ ID NO 30 0.036 mg/ml s.c 0 7 0.36 mg/kg
24 70-72 3 C57BL/6J/ /Chow SEQ ID NO 7 0.032 mg/ml i.v 0 7 0.32
mg/kg 25 73-75 3 C57BL/6J/ /Chow SEQ ID NO 7 0.032 mg/ml s.c 0 7
0.32 mg/kg 26 76-78 3 C57BL/6J/ /Chow SEQ ID NO 10 0.034 mg/ml i.v
0 7 0.34 mg/kg 27 79-81 3 C57BL/6J/ /Chow SEQ ID NO 20 0.034 mg/ml
s.c 0 7 0.34 mg/kg
[0388] Dose administration. C57BL/6JBom female animals, app. 20 g
at arrival, were dosed with 10 ml per kg BW (according to day 0
bodyweight) i.v. or s.c. of the compound formulated in saline or
saline alone according to the table above.
[0389] Sampling of liver and kidney tissue. The animals were
anaesthetised with 70% CO.sub.2-30% O.sub.2 and sacrificed by
cervical dislocation according to the above table. One half of the
large liver lobe and one kidney were minced and submerged in
RNAlater. The other half of liver and the other kidney was frozen
and used for tissue analysis.
[0390] Total RNA Isolation and First strand synthesis. Total RNA
was extracted from maximum 30 mg of tissue homogenized by
bead-milling in the presence of RLT-Lysis buffer using the Qiagen
RNeasy kit (Qiagen cat. no. 74106) according to the manufacturer's
instructions. First strand synthesis was performed using Reverse
Transcriptase reagents from Ambion according to the manufacturer's
instructions.
[0391] For each sample 0.5 .mu.g total RNA was adjusted to (10.8
.mu.l) with RNase free H.sub.2O and mixed with 2 .mu.l random
decamers (50 .mu.M) and 4 .mu.l dNTP mix (2.5 mM each dNTP) and
heated to 70.degree. C. for 3 min after which the samples were
rapidly cooled on ice. 2 .mu.l 10.times. Buffer RT, 1 .mu.l MMLV
Reverse Transcriptase (100 U/.mu.l) and 0.25 .mu.l RNase inhibitor
(10 U/.mu.l) were added to each sample, followed by incubation at
42.degree. C. for 60 min, heat inactivation of the enzyme at
95.degree. C. for 10 min and then the sample was cooled to
4.degree. C. cDNA samples were diluted 1:5 and subjected to RT-QPCR
using Taqman Fast Universal PCR Master Mix 2.times. (Applied
Biosystems Cat #4364103) and Taqman gene expression assay (mApoB,
Mn01545150_m1 and mGAPDH #4352339E) following the manufacturers
protocol and processed in an Applied Biosystems RT-qPCR instrument
(7500/7900 or ViiA7) in fast mode. Oligonucleotide content in liver
and kidney was measured by sandwich ELISA method.
[0392] Serum cholesterol analysis: Immediately before sacrifice
retro-orbital sinus blood was collected using S-monovette Serum-Gel
vials (Sarstedt, Numbrecht, Germany) for serum preparation. Serum
was analyzed for total cholesterol using ABX Pentra Cholesterol CP
(Triolab, Brondby, Denmark) according to the manufacturer's
instructions.
Sequence CWU 1
1
36113DNAArtificial SequenceOligonucleotide sequence motif
1gcattggtat tca 13212DNAartificialOligonucleotide sequence motif
2gttgacactg tc 12313DNAartificialLNA antisense gapmer
oligonucleotide 3gcattggtat tca 13413DNAartificialLNA antisense
gapmer oligonucleotide conjugate 4gcattggtat tca
13513DNAartificialLNA antisense gapmer oligonucleotide conjugate
5gcattggtat tca 13616DNAartificialLNA antisense gapmer
oligonucleotide conjugate 6tcagcattgg tattca 16715DNAartificialLNA
antisense gapmer oligonucleotide conjugate 7cagcattggt attca
15814DNAartificialLNA antisense gapmer oligonucleotide conjugate
8agcattggta ttca 14916DNAartificialLNA antisense gapmer
oligonucleotide conjugate 9tcagcattgg tattca 161015DNAartificialLNA
antisense gapmer oligonucleotide conjugate 10cagcattggt attca
151114DNAartificialLNA antisense gapmer oligonucleotide conjugate
11agcattggta ttca 141216DNAartificialLNA antisense gapmer
oligonucleotide conjugate 12gacgcattgg tattca
161313DNAartificialLNA antisense gapmer oligonucleotide conjugate
13gcattggtat tca 131413DNAartificialLNA antisense gapmer
oligonucleotide conjugate 14gcattggtat tca 131513DNAartificialLNA
antisense gapmer oligonucleotide conjugate 15gcattggtat tca
131615DNAartificialLNA antisense gapmer oligonucleotide conjugate
16cagcattggt attca 151713DNAartificialLNA antisense gapmer
oligonucleotide conjugate 17gcattggtat tca 131813DNAartificialLNA
antisense gapmer oligonucleotide conjugate 18gcattggtat tca
131915DNAartificialLNA antisense gapmer oligonucleotide conjugate
19cagcattggt attca 152013DNAartificialLNA antisense gapmer
oligonucleotide conjugate 20gcattggtat tca 132113DNAartificialLNA
antisense gapmer oligonucleotide conjugate 21gcattggtat tca
132213DNAartificialLNA antisense gapmer oligonucleotide conjugate
22gcattggtat tca 132315DNAartificialLNA antisense gapmer
oligonucleotide conjugate 23cagcattggt attca 152413DNAartificialLNA
antisense gapmer oligonucleotide conjugate 24gcattggtat tca
132513DNAartificialLNA antisense gapmer oligonucleotide conjugate
25gcattggtat tca 132615DNAartificialLNA antisense gapmer
oligonucleotide conjugate 26cagcattggt attca 152712DNAartificialLNA
antisense gapmer oligonucleotide conjugate 27gttgacactg tc
122814DNAartificialLNA antisense gapmer oligonucleotide conjugate
28cagttgacac tgtc 142912DNAartificialLNA antisense gapmer
oligonucleotide conjugate 29gttgacactg tc 123013DNAartificialLNA
antisense gapmer oligonucleotide conjugate 30gcattggtat tca
133112DNAartificialLNA antisense gapmer oligonucleotide conjugate
31gttgacactg tc 123216DNAartificialLNA antisense gapmer
oligonucleotide conjugate 32tcagcattgg tattca
163315DNAartificialLNA antisense gapmer oligonucleotide conjugate
33cagcattggt attca 153414DNAartificialLNA antisense gapmer
oligonucleotide conjugate 34agcattggta ttca 143516DNAartificialLNA
antisense gapmer oligonucleotide conjugate 35gacgcattgg tattca
163613DNAartificialLNA antisense gapmer oligonucleotide conjugate
36gcattggtat tca 13
* * * * *