U.S. patent application number 13/870951 was filed with the patent office on 2014-09-25 for novel trialkyl cationic lipids and methods of use thereof.
This patent application is currently assigned to Protiva Biotherapeutics, Inc.. The applicant listed for this patent is PROTIVA BIOTHERAPEUTICS, INC.. Invention is credited to James Heyes, Alan Martin, Mark Wood.
Application Number | 20140288146 13/870951 |
Document ID | / |
Family ID | 46381302 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140288146 |
Kind Code |
A1 |
Heyes; James ; et
al. |
September 25, 2014 |
NOVEL TRIALKYL CATIONIC LIPIDS AND METHODS OF USE THEREOF
Abstract
The present invention provides compositions and methods for the
delivery of therapeutic agents to cells. In particular, these
include novel cationic lipids and nucleic acid-lipid particles that
provide efficient encapsulation of nucleic acids and efficient
delivery of the encapsulated nucleic acid to cells in vivo. The
compositions of the present invention are highly potent, thereby
allowing effective knock-down of a specific target protein at
relatively low doses. In addition, the compositions and methods of
the present invention are less toxic and provide a greater
therapeutic index compared to compositions and methods previously
known in the art.
Inventors: |
Heyes; James; (Vancouver,
CA) ; Wood; Mark; (Port Moody, CA) ; Martin;
Alan; (Burnaby, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PROTIVA BIOTHERAPEUTICS, INC. |
Burnaby |
|
CA |
|
|
Assignee: |
Protiva Biotherapeutics,
Inc.
Burnaby
CA
|
Family ID: |
46381302 |
Appl. No.: |
13/870951 |
Filed: |
April 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13235253 |
Sep 16, 2011 |
8466122 |
|
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13870951 |
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61384191 |
Sep 17, 2010 |
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Current U.S.
Class: |
514/44A ;
514/44R; 514/785; 560/155; 560/159 |
Current CPC
Class: |
A61K 31/7088 20130101;
C07C 271/20 20130101; A61K 31/7105 20130101; C07C 229/12 20130101;
A61K 47/34 20130101; A61K 47/44 20130101; A61K 31/713 20130101;
A61K 9/1272 20130101 |
Class at
Publication: |
514/44.A ;
560/155; 560/159; 514/785; 514/44.R |
International
Class: |
A61K 47/44 20060101
A61K047/44; C07C 271/20 20060101 C07C271/20; A61K 31/7088 20060101
A61K031/7088; C07C 229/12 20060101 C07C229/12 |
Claims
1. A cationic lipid of Formula I having the following structure:
##STR00076## or salts and isomers thereof, wherein: R.sup.1 and
R.sup.2 are independently selected from the group consisting of
hydrogen (H), optionally substituted C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl;
alternatively, R.sup.1 and R.sup.2 may join to form an optionally
substituted heterocycloalkyl of 4 to 6 carbon atoms and 1 to 2
heteroatoms selected from the group consisting of nitrogen (N),
oxygen (O), and mixtures thereof; R.sup.3 is absent or is hydrogen
(H) or a C.sub.1-C.sub.6 alkyl to provide a quaternary amine;
R.sup.4 is absent or is selected from the group consisting of
--CH.sub.2--, optionally substituted C.sub.10-C.sub.30 alkyl,
C.sub.10-C.sub.30 alkenyl, C.sub.10-C.sub.30 alkynyl,
C.sub.10-C.sub.30 acyl, and NR.sup.4aR.sup.4b, wherein R.sup.4a and
R.sup.4b are independently selected from the group consisting of
hydrogen (H), an optionally substituted C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl; R.sup.5 is
selected from the group consisting of hydrogen (H), oxygen (O),
optionally substituted C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30
alkenyl, C.sub.10-C.sub.30 alkynyl, and OR.sup.5a, wherein R.sup.5a
is selected from the group consisting of hydrogen (H), an
optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, and C.sub.2-C.sub.6 alkynyl; alternatively, R.sup.4 and
R.sup.5 may join to form a C.sub.3-10 heterocycloalkyl having from
1 to 3 heteroatoms each independently selected from the group
consisting of nitrogen (N), oxygen (O), and sulfur (S), and
optionally substituted with from 1 to 3 groups each independently
selected from the group consisting of hydrogen (H) and
C.sub.1-C.sub.6 alkyl; R.sup.6 and R.sup.7 are independently
selected from the group consisting of optionally substituted
C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30 alkenyl,
C.sub.10-C.sub.30 alkynyl, and C.sub.10-C.sub.30 acyl; R.sup.8 is
selected from the group consisting of optionally substituted
C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30 alkenyl,
C.sub.10-C.sub.30 alkynyl, and C.sub.10-C.sub.30 acyl; R.sup.13 and
R.sup.14 are independently selected from the group consisting of
hydrogen (H), optionally substituted C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl; X is selected
from the group consisting of --CH--, nitrogen (N), oxygen (O),
sulfur (S), N(R.sup.9), C(O), C(O)O, OC(O), C(O)N(R.sup.9),
N(R.sup.9)C(O), OC(O)N(R.sup.9), N(R.sup.9)C(O)O, C(O)S, SC(O),
C(S)O, OC(S), S(O), S(O)(O), and C(S), wherein R.sup.9 is selected
from the group consisting of hydrogen (H), optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, and ##STR00077## wherein * represents the point of
attachment to N; Y is selected from the group consisting of
--CR.sup.11R.sup.12-- and oxygen (O), wherein R.sup.11 and R.sup.12
are independently selected from the group consisting of hydrogen
(H), optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, and C.sub.2-C.sub.6 alkynyl; Z.sup.1, Z.sup.2, and Z.sup.3
are independently selected from oxygen (O), C(O)O, or OC(O);
T.sup.1, T.sup.2, and T.sup.3 are independently selected from
hydrogen (H) or OR.sup.10, wherein R.sup.10 is hydrogen (H) or
optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, or C.sub.2-C.sub.6 alkynyl; subscripts a, b, c, d, e, f,
and g are independently selected from the group consisting of 0, 1,
2, 3, 4, 5, 6, 7 and 8; subscripts h, i, j, k, and m are
independently selected from the group consisting of 0 and 1;
provided that when R.sup.5 is an optionally substituted
C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30 alkenyl,
C.sub.10-C.sub.30 alkynyl, and C.sub.10-C.sub.30 acyl, then either
h is 0 or at least one of Z.sup.1 and Z.sup.2 is oxygen (O); and
provided that at least three of R.sup.4, R.sup.5, R.sup.6, R.sup.7,
and R.sup.8 are selected from the group consisting of optionally
substituted C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30 alkenyl, and
C.sub.10-C.sub.30 alkynyl.
2-6. (canceled)
7. The cationic lipid of claim 1, wherein R.sup.6, R.sup.7, and
R.sup.8 independently comprise at least one site of
unsaturation.
8. The cationic lipid of claim 7, wherein R.sup.6, R.sup.7, and
R.sup.8 are independently selected from the group consisting of a
dodecenyl moiety, a tetradecenyl moiety, a hexadecenyl moiety, an
octadecenyl moiety, and an icosadecenyl moiety.
9-11. (canceled)
12. The cationic lipid of claim 1, wherein R.sup.6, R.sup.7, and
R.sup.8 independently comprise at least two sites of
unsaturation.
13. The cationic lipid of claim 12, wherein R.sup.6, R.sup.7, and
R.sup.8 are independently selected from the group consisting of a
dodecadienyl moiety, a tetradecadienyl moiety, a hexadecadienyl
moiety, an octadecadienyl moiety, and an icosadienyl moiety.
14-16. (canceled)
17. The cationic lipid of claim 1, wherein R.sup.6, R.sup.7, and
R.sup.8 independently comprise at least three sites of
unsaturation.
18. The cationic lipid of claim 17, wherein R.sup.6, R.sup.7, and
R.sup.8 are independently selected from the group consisting of a
dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienyl
moiety, an octadecatrienyl moiety, and an icosatrienyl moiety.
19-21. (canceled)
22. The cationic lipid of claim 1, wherein R.sup.6, R.sup.7, and
R.sup.8 are independently selected from the group consisting of
oleyl moieties, linoleyl moieties, linolenyl moieties,
.gamma.-linolenyl moieties, and phytanyl moieties.
23-27. (canceled)
28. The cationic lipid of claim 1, wherein R.sup.6, R.sup.7, and
R.sup.8 independently comprise at least one optionally substituted
cyclic alkyl group.
29-33. (canceled)
34. The cationic lipid of claim 1, wherein subscripts g, k, and m
are all 0.
35. The cationic lipid of claim 34, wherein R.sup.5, R.sup.6, and
R.sup.7 are independently selected from the group consisting of an
optionally substituted C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30
alkenyl, and C.sub.10-C.sub.30 alkynyl.
36. The cationic lipid of claim 35, wherein R.sup.5, R.sup.6, and
R.sup.7 are independently selected from the group consisting of
oleyl moieties, linoleyl moieties, linolenyl moieties,
.gamma.-linolenyl moieties, and phytanyl moieties.
37-39. (canceled)
40. The cationic lipid of claim 1, wherein X is selected from the
group consisting of C(O)O and OC(O).
41. The cationic lipid of claim 1, wherein X is N(R.sup.9)C(O)O or
OC(O)N(R.sup.9).
42-79. (canceled)
80. A lipid particle comprising a cationic lipid of claim 1.
81-87. (canceled)
88. The lipid particle of claim 80, wherein the particle further
comprises a therapeutic agent.
89. The lipid particle of claim 88, wherein the therapeutic agent
is a nucleic acid.
90. The lipid particle of claim 89, wherein the nucleic acid is an
interfering RNA.
91-96. (canceled)
97. A pharmaceutical composition comprising a lipid particle of
claim 80 and a pharmaceutically acceptable carrier.
98-102. (canceled)
103. A method for treating a disease or disorder in a mammal in
need thereof, the method comprising: administering to the mammal a
therapeutically effective amount of a lipid particle of claim
88.
104-105. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 13/235,253, allowed, filed on Sep. 16, 2011,
which claims the benefit of priority of U.S. Provisional Patent
Application No. 61/384,191, filed on Sep. 17, 2010, the contents of
which are hereby incorporated in their entirety by reference.
BACKGROUND OF THE INVENTION
[0002] Therapeutic nucleic acids include, e.g., small interfering
RNA (siRNA), microRNA (miRNA), antisense oligonucleotides,
ribozymes, plasmids, and immune-stimulating nucleic acids. These
nucleic acids act via a variety of mechanisms. In the case of
interfering RNA molecules such as siRNA and miRNA, these nucleic
acids can down-regulate intracellular levels of specific proteins
through a process termed RNA interference (RNAi). Following
introduction of interfering RNA into the cell cytoplasm, these
double-stranded RNA constructs can bind to a protein termed RISC.
The sense strand of the interfering RNA is displaced from the RISC
complex, providing a template within RISC that can recognize and
bind mRNA with a complementary sequence to that of the bound
interfering RNA. Having bound the complementary mRNA, the RISC
complex cleaves the mRNA and releases the cleaved strands. RNAi can
provide down-regulation of specific proteins by targeting specific
destruction of the corresponding mRNA that encodes for protein
synthesis.
[0003] The therapeutic applications of RNAi are extremely broad,
since interfering RNA constructs can be synthesized with any
nucleotide sequence directed against a target protein. To date,
siRNA constructs have shown the ability to specifically
down-regulate target proteins in both in vitro and in vivo models.
In addition, siRNA constructs are currently being evaluated in
clinical studies.
[0004] However, two problems currently faced by interfering RNA
constructs are, first, their susceptibility to nuclease digestion
in plasma and, second, their limited ability to gain access to the
intracellular compartment where they can bind RISC when
administered systemically as free interfering RNA molecules. These
double-stranded constructs can be stabilized by the incorporation
of chemically modified nucleotide linkers within the molecule,
e.g., phosphothioate groups. However, such chemically modified
linkers provide only limited protection from nuclease digestion and
may decrease the activity of the construct. Intracellular delivery
of interfering RNA can be facilitated by the use of carrier systems
such as polymers, cationic liposomes, or by the covalent attachment
of a cholesterol moiety to the molecule. However, improved delivery
systems are required to increase the potency of interfering RNA
molecules such as siRNA and miRNA and to reduce or eliminate the
requirement for chemically modified nucleotide linkers.
[0005] In addition, problems remain with the limited ability of
therapeutic nucleic acids such as interfering RNA to cross cellular
membranes (see, Vlassov et al., Biochim. Biophys. Acta,
1197:95-1082 (1994)) and in the problems associated with systemic
toxicity, such as complement-mediated anaphylaxis, altered
coagulatory properties, and cytopenia (Galbraith et al., Antisense
Nuci. Acid Drug Des., 4:201-206 (1994)).
[0006] To attempt to improve efficacy, investigators have also
employed lipid-based carrier systems to deliver chemically modified
or unmodified therapeutic nucleic acids. Zelphati et al. (J. Contr.
Rel., 41:99-119 (1996)) describes the use of anionic (conventional)
liposomes, pH sensitive liposomes, immunoliposomes, fusogenic
liposomes, and cationic lipid/antisense aggregates. Similarly,
siRNA has been administered systemically in cationic liposomes, and
these nucleic acid-lipid particles have been reported to provide
improved down-regulation of target proteins in mammals including
non-human primates (Zimmermann et al., Nature, 441: 111-114
(2006)).
[0007] In spite of this progress, there remains a need in the art
for improved lipid-therapeutic nucleic acid compositions that are
suitable for general therapeutic use. Preferably, these
compositions would encapsulate nucleic acids with high efficiency,
have high drug:lipid ratios, protect the encapsulated nucleic acid
from degradation and clearance in serum, be suitable for systemic
delivery, and provide intracellular delivery of the encapsulated
nucleic acid. In addition, these nucleic acid-lipid particles
should be well-tolerated and provide an adequate therapeutic index,
such that patient treatment at an effective dose of the nucleic
acid is not associated with significant toxicity and/or risk to the
patient. The present invention provides such compositions, methods
of making the compositions, and methods of using the compositions
to introduce nucleic acids into cells, including for the treatment
of diseases.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides novel cationic ammonium
lipids and lipid particles comprising these lipids, which are
advantageous for the in vivo delivery of nucleic acids, as well as
nucleic acid-lipid particle compositions suitable for in vivo
therapeutic use. The present invention also provides methods of
making these compositions, as well as methods of introducing
nucleic acids into cells using these compositions, e.g. for the
treatment of various disease conditions.
[0009] In one embodiment, the present invention provides a cationic
lipid of Formula I having the following structure:
##STR00001##
or salts and isomers thereof, wherein:
[0010] R.sup.1 and R.sup.2 are independently selected from hydrogen
(H), optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, and C.sub.2-C.sub.6 alkynyl. Alternatively, R.sup.1 and
R.sup.2 may join to form an optionally substituted heterocycloalkyl
of 4 to 6 carbon atoms and 1 to 2 heteroatoms selected from
nitrogen (N), oxygen (O), and mixtures thereof. R.sup.3 is absent
or is hydrogen (H) or a C.sub.1-C.sub.6 alkyl to provide a
quaternary amine R.sup.4 is absent or is selected from
--CH.sub.2--, optionally substituted C.sub.10-C.sub.30 alkyl,
C.sub.10-C.sub.30 alkenyl, C.sub.10-C.sub.30 alkynyl,
C.sub.10-C.sub.30 acyl, and NR.sup.4aR.sup.4b, wherein R.sup.4a and
R.sup.4b are independently selected from hydrogen (H), an
optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, and C.sub.2-C.sub.6 alkynyl. R.sup.5 is selected from
hydrogen (H), oxygen (O), optionally substituted C.sub.10-C.sub.30
alkyl, C.sub.10-C.sub.30 alkenyl, C.sub.10-C.sub.30 alkynyl, and
OR.sup.5a, wherein R.sup.5a is selected from hydrogen (H), an
optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, and C.sub.2-C.sub.6 alkynyl.
[0011] Alternatively, R.sup.4 and R.sup.5 may join to form a
C.sub.3-10 heterocycloalkyl having from 1 to 3 heteroatoms each
independently selected from nitrogen (N), oxygen (O), and sulfur
(S), and optionally substituted with from 1 to 3 groups each
independently selected from hydrogen (H) and C.sub.1-C.sub.6 alkyl.
R.sup.6 and R.sup.7 independently selected from optionally
substituted C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30 alkenyl,
C.sub.10-C.sub.30 alkynyl, and C.sub.10-C.sub.30 acyl. R.sup.8 is
selected from optionally substituted C.sub.10-C.sub.30 alkyl,
C.sub.10-C.sub.30 alkenyl, C.sub.10-C.sub.30 alkynyl, and
C.sub.10-C.sub.30 acyl. R.sup.13 and R.sup.14 are independently
selected from hydrogen (H), optionally substituted C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl. X is
selected from --CH--, nitrogen (N), oxygen (O), sulfur (S),
N(R.sup.9), C(O), C(O)O, OC(O), C(O)N(R.sup.9), N(R.sup.9)C(O),
OC(O)N(R.sup.9), N(R.sup.9)C(O)O, C(O)S, SC(O), C(S)O, OC(S), S(O),
S(O)(O), and C(S), wherein R.sup.9 is selected from hydrogen (H),
optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, and
##STR00002##
wherein * represents the point of attachment to N. Y is selected
from --CR.sup.11R.sup.12-- and oxygen (O), wherein R.sup.11 and
R.sup.12 are independently selected from hydrogen (H), optionally
substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, and
C.sub.2-C.sub.6 alkynyl. Z.sup.1, Z.sup.2, and Z.sup.3 are
independently selected from oxygen (O), C(O)O, or OC(O). T.sup.1,
T.sup.2, and T.sup.3 are independently selected from hydrogen (H)
or OR.sup.10, wherein R.sup.10 is hydrogen (H) or optionally
substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.6 alkynyl. Subscripts a, b, c, d, e, f, and g are
independently selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8.
Subscripts h, i, j, k, and m are independently selected from 0 and
1. When R.sup.5 is an optionally substituted C.sub.10-C.sub.30
alkyl, C.sub.10-C.sub.30 alkenyl, C.sub.10-C.sub.30 alkynyl, and
C.sub.10-C.sub.30 acyl, then either h is 0 or at least one of
Z.sup.1 and Z.sup.2 is oxygen (O). At least three of R.sup.4,
R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are selected from optionally
substituted C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30 alkenyl, and
C.sub.10-C.sub.30 alkynyl.
[0012] In particular embodiments, the cationic lipid of Formula I
has one of the following structures:
##STR00003## ##STR00004##
[0013] In a further aspect, the present invention provides a lipid
particle comprising one or more of the above cationic lipids of
Formula I or salts thereof. In certain embodiments, the lipid
particle further comprises one or more non-cationic lipids such as
neutral lipids. In certain other embodiments, the lipid particle
further comprises one or more conjugated lipids capable of reducing
or inhibiting particle aggregation. In additional embodiments, the
lipid particle further comprises one or more active agents or
therapeutic agents.
[0014] In certain embodiments, the non-cationic lipid component of
the lipid particle may comprise a phospholipid, cholesterol (or
cholesterol derivative), or a mixture thereof. In one particular
embodiment, the phospholipid comprises
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC), or a mixture thereof. In some
embodiments, the conjugated lipid component of the lipid particle
comprises a polyethyleneglycol (PEG)-lipid conjugate. In certain
instances, the PEG-lipid conjugate comprises a PEG-diacylglycerol
(PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or
a mixture thereof. In other embodiments, the lipid conjugate
comprises a polyoxazoline (POZ)-lipid conjugate such as a POZ-DAA
conjugate.
[0015] In some embodiments, the active agent or therapeutic agent
comprises a nucleic acid. In certain instances, the nucleic acid
comprises an interfering RNA molecule such as, e.g., an siRNA,
aiRNA, miRNA, Dicer-substrate dsRNA, shRNA, or mixtures thereof. In
certain other instances, the nucleic acid comprises single-stranded
or double-stranded DNA, RNA, or a DNA/RNA hybrid such as, e.g., an
antisense oligonucleotide, a ribozyme, a plasmid, an
immunostimulatory oligonucleotide, or mixtures thereof.
[0016] In other embodiments, the active agent or therapeutic agent
is fully encapsulated within the lipid portion of the lipid
particle such that the active agent or therapeutic agent in the
lipid particle is resistant in aqueous solution to enzymatic
degradation, e.g., by a nuclease or protease. In further
embodiments, the lipid particle is substantially non-toxic to
mammals such as humans.
[0017] In preferred embodiments, the present invention provides
serum-stable nucleic acid-lipid particles (SNALP) comprising: (a)
one or more nucleic acids such as interfering RNA molecules; (b)
one or more cationic lipids of Formula I-VI or salts thereof; (c)
one or more non-cationic lipids; and (d) one or more conjugated
lipids that inhibit aggregation of particles.
[0018] In some embodiments, the present invention provides nucleic
acid-lipid particles (e.g., SNALP) comprising: (a) one or more
nucleic acids; (b) one or more cationic lipids of Formula I or
salts thereof comprising from about 50 mol % to about 85 mol % of
the total lipid present in the particle; (c) one or more
non-cationic lipids comprising from about 13 mol % to about 49.5
mol % of the total lipid present in the particle; and (d) one or
more conjugated lipids that inhibit aggregation of particles
comprising from about 0.5 mol % to about 2 mol % of the total lipid
present in the particle.
[0019] In one aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) a nucleic acid; (b) a cationic lipid of
Formula I or a salt thereof comprising from about 52 mol % to about
62 mol % of the total lipid present in the particle; (c) a mixture
of a phospholipid and cholesterol or a derivative thereof
comprising from about 36 mol % to about 47 mol % of the total lipid
present in the particle; and (d) a PEG-lipid conjugate comprising
from about 1 mol % to about 2 mol % of the total lipid present in
the particle. This embodiment of nucleic acid-lipid particle is
generally referred to herein as the "1:57" formulation. In one
particular embodiment, the 1:57 formulation is a four-component
system comprising about 1.4 mol % PEG-lipid conjugate (e.g.,
PEG2000-C-DMA), about 57.1 mol % cationic lipid of Formula I or a
salt thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3 mol %
cholesterol (or derivative thereof).
[0020] In another aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) a nucleic acid; (b) a cationic lipid of
Formula I or a salt thereof comprising from about 56.5 mol % to
about 66.5 mol % of the total lipid present in the particle; (c)
cholesterol or a derivative thereof comprising from about 31.5 mol
% to about 42.5 mol % of the total lipid present in the particle;
and (d) a PEG-lipid conjugate comprising from about 1 mol % to
about 2 mol % of the total lipid present in the particle. This
embodiment of nucleic acid-lipid particle is generally referred to
herein as the "1:62" formulation. In one particular embodiment, the
1:62 formulation is a three-component system which is
phospholipid-free and comprises about 1.5 mol % PEG-lipid conjugate
(e.g., PEG2000-C-DMA), about 61.5 mol % cationic lipid of Formula I
or a salt thereof, and about 36.9 mol % cholesterol (or derivative
thereof).
[0021] Additional embodiments related to the 1:57 and 1:62
formulations are described in PCT Publication No. WO 09/127,060 and
U.S. application Ser. No. 12/794,701, filed Jun. 4, 2010, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes.
[0022] In other embodiments, the present invention provides nucleic
acid-lipid particles (e.g., SNALP) comprising: (a) one or more
nucleic acids; (b) one or more cationic lipids of Formula I or
salts thereof comprising from about 2 mol % to about 50 mol % of
the total lipid present in the particle; (c) one or more
non-cationic lipids comprising from about 5 mol % to about 90 mol %
of the total lipid present in the particle; and (d) one or more
conjugated lipids that inhibit aggregation of particles comprising
from about 0.5 mol % to about 20 mol % of the total lipid present
in the particle.
[0023] In one aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) a nucleic acid; (b) a cationic lipid of
Formula I or a salt thereof comprising from about 30 mol % to about
50 mol % of the total lipid present in the particle; (c) a mixture
of a phospholipid and cholesterol or a derivative thereof
comprising from about 47 mol % to about 69 mol % of the total lipid
present in the particle; and (d) a PEG-lipid conjugate comprising
from about 1 mol % to about 3 mol % of the total lipid present in
the particle. This embodiment of nucleic acid-lipid particle is
generally referred to herein as the "2:40" formulation. In one
particular embodiment, the 2:40 formulation is a four-component
system which comprises about 2 mol % PEG-lipid conjugate (e.g.,
PEG2000-C-DMA), about 40 mol % cationic lipid of Formula I or a
salt thereof, about 10 mol % DPPC (or DSPC), and about 48 mol %
cholesterol (or derivative thereof).
[0024] In further embodiments, the present invention provides
nucleic acid-lipid particles (e.g., SNALP) comprising: (a) one or
more nucleic acids; (b) one or more cationic lipids of Formula I or
salts thereof comprising from about 50 mol % to about 65 mol % of
the total lipid present in the particle; (c) one or more
non-cationic lipids comprising from about 25 mol % to about 45 mol
% of the total lipid present in the particle; and (d) one or more
conjugated lipids that inhibit aggregation of particles comprising
from about 5 mol % to about 10 mol % of the total lipid present in
the particle.
[0025] In one aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) a nucleic acid; (b) a cationic lipid of
Formula I or a salt thereof comprising from about 50 mol % to about
60 mol % of the total lipid present in the particle; (c) a mixture
of a phospholipid and cholesterol or a derivative thereof
comprising from about 35 mol % to about 45 mol % of the total lipid
present in the particle; and (d) a PEG-lipid conjugate comprising
from about 5 mol % to about 10 mol % of the total lipid present in
the particle. This embodiment of nucleic acid-lipid particle is
generally referred to herein as the "7:54" formulation. In certain
instances, the non-cationic lipid mixture in the 7:54 formulation
comprises: (i) a phospholipid of from about 5 mol % to about 10 mol
% of the total lipid present in the particle; and (ii) cholesterol
or a derivative thereof of from about 25 mol % to about 35 mol % of
the total lipid present in the particle. In one particular
embodiment, the 7:54 formulation is a four-component system which
comprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA),
about 54 mol % cationic lipid of Formula I or a salt thereof, about
7 mol % DPPC (or DSPC), and about 32 mol % cholesterol (or
derivative thereof).
[0026] In another aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) a nucleic acid; (b) a cationic lipid of
Formula I or a salt thereof comprising from about 55 mol % to about
65 mol % of the total lipid present in the particle; (c)
cholesterol or a derivative thereof comprising from about 30 mol %
to about 40 mol % of the total lipid present in the particle; and
(d) a PEG-lipid conjugate comprising from about 5 mol % to about 10
mol % of the total lipid present in the particle. This embodiment
of nucleic acid-lipid particle is generally referred to herein as
the "7:58" formulation. In one particular embodiment, the 7:58
formulation is a three-component system which is phospholipid-free
and comprises about 7 mol % PEG-lipid conjugate (e.g.,
PEG750-C-DMA), about 58 mol % cationic lipid of Formula I or a salt
thereof, and about 35 mol % cholesterol (or derivative
thereof).
[0027] Additional embodiments related to the 7:54 and 7:58
formulations are described in U.S. application Ser. No. 12/828,189,
filed Jun. 30, 2010, the disclosure of which is herein incorporated
by reference in its entirety for all purposes.
[0028] The present invention also provides pharmaceutical
compositions comprising a lipid particle such as a nucleic
acid-lipid particle (e.g., SNALP) and a pharmaceutically acceptable
carrier.
[0029] In another aspect, the present invention provides methods
for introducing one or more therapeutic agents such as nucleic
acids into a cell, the method comprising contacting the cell with a
lipid particle described herein (e.g., SNALP). In one embodiment,
the cell is in a mammal and the mammal is a human.
[0030] In yet another aspect, the present invention provides
methods for the in vivo delivery of one or more therapeutic agents
such as nucleic acids, the method comprising administering to a
mammal a lipid particle described herein (e.g., SNALP). In certain
embodiments, the lipid particles (e.g., SNALP) are administered by
one of the following routes of administration: oral, intranasal,
intravenous, intraperitoneal, intramuscular, intraarticular,
intralesional, intratracheal, subcutaneous, and intradermal. In
particular embodiments, the lipid particles (e.g., SNALP) are
administered systemically, e.g., via enteral or parenteral routes
of administration. In preferred embodiments, the mammal is a
human.
[0031] In a further aspect, the present invention provides methods
for treating a disease or disorder in a mammal in need thereof, the
method comprising administering to the mammal a therapeutically
effective amount of a lipid particle (e.g., SNALP) comprising one
or more therapeutic agents such as nucleic acids. Non-limiting
examples of diseases or disorders include a viral infection, a
liver disease or disorder, and cancer. Preferably, the mammal is a
human.
[0032] In certain embodiments, the present invention provides
methods for treating a liver disease or disorder by administering a
nucleic acid such as an interfering RNA (e.g., siRNA) in nucleic
acid-lipid particles (e.g., SNALP), alone or in combination with a
lipid-lowering agent. Examples of lipid diseases and disorders
include, but are not limited to, dyslipidemia (e.g.,
hyperlipidemias such as elevated triglyceride levels
(hypertriglyceridemia) and/or elevated cholesterol levels
(hypercholesterolemia)), atherosclerosis, coronary heart disease,
coronary artery disease, atherosclerotic cardiovascular disease
(CVD), fatty liver disease (hepatic steatosis), abnormal lipid
metabolism, abnormal cholesterol metabolism, diabetes (including
Type 2 diabetes), obesity, cardiovascular disease, and other
disorders relating to abnormal metabolism. Non-limiting examples of
lipid-lowering agents include statins, fibrates, ezetimibe,
thiazolidinediones, niacin, beta-blockers, nitroglycerin, calcium
antagonists, and fish oil.
[0033] In one particular embodiment, the present invention provides
a method for lowering or reducing cholesterol levels in a mammal
(e.g., human) in need thereof (e.g., a mammal with elevated blood
cholesterol levels), the method comprising administering to the
mammal a therapeutically effective amount of a nucleic acid-lipid
particle (e.g., a SNALP formulation) described herein comprising
one or more interfering RNAs (e.g., siRNAs) that target one or more
genes associated with metabolic diseases and disorders. In another
particular embodiment, the present invention provides a method for
lowering or reducing triglyceride levels in a mammal (e.g., human)
in need thereof (e.g., a mammal with elevated blood triglyceride
levels), the method comprising administering to the mammal a
therapeutically effective amount of a nucleic acid-lipid particle
(e.g., a SNALP formulation) described herein comprising one or more
interfering RNAs (e.g., siRNAs) that target one or more genes
associated with metabolic diseases and disorders. These methods can
be carried out in vitro using standard tissue culture techniques or
in vivo by administering the interfering RNA (e.g., siRNA) using
any means known in the art. In preferred embodiments, the
interfering RNA (e.g., siRNA) is delivered to a liver cell (e.g.,
hepatocyte) in a mammal such as a human.
[0034] Additional embodiments related to treating a liver disease
or disorder using a lipid particle are described in, e.g., PCT
Application No. PCT/CA2010/000120, filed Jan. 26, 2010, and U.S.
Patent Publication No. 20060134189, the disclosures of which are
herein incorporated by reference in their entirety for all
purposes.
[0035] In other embodiments, the present invention provides methods
for treating a cell proliferative disorder such as cancer by
administering a nucleic acid such as an interfering RNA (e.g.,
siRNA) in nucleic acid-lipid particles (e.g., SNALP), alone or in
combination with a chemotherapy drug. The methods can be carried
out in vitro using standard tissue culture techniques or in vivo by
administering the interfering RNA (e.g., siRNA) using any means
known in the art. In preferred embodiments, the interfering RNA
(e.g., siRNA) is delivered to a cancer cell in a mammal such as a
human, alone or in combination with a chemotherapy drug. The
nucleic acid-lipid particles and/or chemotherapy drugs may also be
co-administered with conventional hormonal, immunotherapeutic,
and/or radiotherapeutic agents.
[0036] Additional embodiments related to treating a cell
proliferative disorder using a lipid particle are described in,
e.g., PCT Publication No. WO 09/082,817, U.S. Patent Publication
No. 20090149403, PCT Publication No. WO 09/129,319, and U.S.
Provisional Application No. 61/245,143, filed Sep. 23, 2009, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes.
[0037] In further embodiments, the present invention provides
methods for preventing or treating a viral infection such as an
arenavirus (e.g., Lassa virus) or Filovirus (e.g., Ebola virus,
Marburg virus, etc.) infection which causes hemorrhagic fever or a
hepatitis (e.g., Hepatitis C virus) infection which causes acute or
chronic hepatitis by administering a nucleic acid such as an
interfering RNA (e.g., siRNA) in nucleic acid-lipid particles
(e.g., SNALP), alone or in combination with the administration of
conventional agents used to treat or ameliorate the viral condition
or any of the symptoms associated therewith. The methods can be
carried out in vitro using standard tissue culture techniques or in
vivo by administering the interfering RNA using any means known in
the art. In certain preferred embodiments, the interfering RNA
(e.g., siRNA) is delivered to cells, tissues, or organs of a mammal
such as a human that are infected and/or susceptible of being
infected with the hemorrhagic fever virus, such as, e.g., cells of
the reticuloendothelial system (e.g., monocytes, macrophages,
etc.). In certain other preferred embodiments, the interfering RNA
(e.g., siRNA) is delivered to cells, tissues, or organs of a mammal
such as a human that are infected and/or susceptible of being
infected with the hepatitis virus, such as, e.g., cells of the
liver (e.g., hepatocytes).
[0038] Additional embodiments related to preventing or treating a
viral infection using a lipid particle are described in, e.g., U.S.
Patent Publication No. 20070218122, U.S. Patent Publication No.
20070135370, U.S. application Ser. No. 12/840,225, filed Jul. 20,
2010, PCT Application No. PCT/CA2010/000444, entitled "Compositions
and Methods for Silencing Hepatitis C Virus Expression," filed Mar.
19, 2010, bearing Attorney Docket No. 020801-008910PC, and U.S.
Provisional Application No. 61/319,855, filed Mar. 31, 2010, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes.
[0039] The lipid particles of the invention (e.g., SNALP)
comprising one or more cationic lipids of Formula I or salts
thereof are particularly advantageous and suitable for use in the
administration of nucleic acids such as interfering RNA to a
subject (e.g., a mammal such as a human) because they are stable in
circulation, of a size required for pharmacodynamic behavior
resulting in access to extravascular sites, and are capable of
reaching target cell populations.
[0040] Other objects, features, and advantages of the present
invention will be apparent to one of skill in the art from the
following detailed description and figures.
BRIEF DESCRIPTION OF THE DRAWING
[0041] The FIGURE shows a comparison of the liver ApoB mRNA
knockdown activity of exemplary SNALP formulations containing
cationic lipids of Formula I.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0042] The present invention is based, in part, upon the discovery
of novel cationic ammonium lipids that provide advantages when used
in lipid particles for the in vivo delivery of an active or
therapeutic agent such as a nucleic acid into a cell of a mammal.
In particular, the present invention provides nucleic acid-lipid
particle compositions comprising one or more of the novel cationic
lipids described herein that provide increased activity of the
nucleic acid (e.g., interfering RNA) and improved tolerability of
the compositions in vivo, resulting in a significant increase in
the therapeutic index as compared to nucleic acid-lipid particle
compositions previously described.
[0043] In particular embodiments, the present invention provides
novel cationic lipids that enable the formulation of improved
compositions for the in vitro and in vivo delivery of interfering
RNA such as siRNA. It is shown herein that these improved lipid
particle compositions are effective in down-regulating (e.g.,
silencing) the protein levels and/or mRNA levels of target genes.
Furthermore, it is shown herein that the activity of these improved
lipid particle compositions is dependent on the presence of the
novel cationic lipids of the invention.
[0044] The lipid particles and compositions of the present
invention may be used for a variety of purposes, including the
delivery of encapsulated or associated (e.g., complexed)
therapeutic agents such as nucleic acids to cells, both in vitro
and in vivo. Accordingly, the present invention further provides
methods of treating diseases or disorders in a subject in need
thereof by contacting the subject with a lipid particle that
encapsulates or is associated with a suitable therapeutic agent,
wherein the lipid particle comprises one or more of the novel
cationic lipids described herein.
[0045] As described herein, the lipid particles of the present
invention are particularly useful for the delivery of nucleic
acids, including, e.g., interfering RNA molecules such as siRNA.
Therefore, the lipid particles and compositions of the present
invention may be used to decrease the expression of target genes
and proteins both in vitro and in vivo by contacting cells with a
lipid particle comprising one or more novel cationic lipids
described herein, wherein the lipid particle encapsulates or is
associated with a nucleic acid that reduces target gene expression
(e.g., an siRNA). Alternatively, the lipid particles and
compositions of the present invention may be used to increase the
expression of a desired protein both in vitro and in vivo by
contacting cells with a lipid particle comprising one or more novel
cationic lipids described herein, wherein the lipid particle
encapsulates or is associated with a nucleic acid that enhances
expression of the desired protein (e.g., a plasmid encoding the
desired protein).
[0046] Various exemplary embodiments of the cationic lipids of the
present invention, lipid particles and compositions comprising the
same, and their use to deliver active or therapeutic agents such as
nucleic acids to modulate gene and protein expression, are
described in further detail below.
II. Definitions
[0047] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0048] As used herein, the terms "interfering RNA" or "RNAi" or
"interfering RNA sequence" as used herein includes single-stranded
RNA (e.g., mature miRNA, ssRNAi oligonucleotides, ssDNAi
oligonucleotides) or double-stranded RNA (i.e., duplex RNA such as
siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, or pre-miRNA) that is
capable of reducing or inhibiting the expression of a target gene
or sequence (e.g., by mediating the degradation or inhibiting the
translation of mRNAs which are complementary to the interfering RNA
sequence) when the interfering RNA is in the same cell as the
target gene or sequence. Interfering RNA thus refers to the
single-stranded RNA that is complementary to a target mRNA sequence
or to the double-stranded RNA formed by two complementary strands
or by a single, self-complementary strand. Interfering RNA may have
substantial or complete identity to the target gene or sequence, or
may comprise a region of mismatch (i.e., a mismatch motif). The
sequence of the interfering RNA can correspond to the full-length
target gene, or a subsequence thereof. Preferably, the interfering
RNA molecules are chemically synthesized.
[0049] Interfering RNA includes "small-interfering RNA" or "siRNA,"
e.g., interfering RNA of about 15-60, 15-50, or 15-40 (duplex)
nucleotides in length, more typically about 15-30, 15-25, or 19-25
(duplex) nucleotides in length, and is preferably about 20-24,
21-22, or 21-23 (duplex) nucleotides in length (e.g., each
complementary sequence of the double-stranded siRNA is 15-60,
15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length,
preferably about 20-24, 21-22, or 21-23 nucleotides in length, and
the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30,
15-25, or 19-25 base pairs in length, preferably about 18-22,
19-20, or 19-21 base pairs in length). siRNA duplexes may comprise
3' overhangs of about 1 to about 4 nucleotides or about 2 to about
3 nucleotides and 5' phosphate termini. Examples of siRNA include,
without limitation, a double-stranded polynucleotide molecule
assembled from two separate stranded molecules, wherein one strand
is the sense strand and the other is the complementary antisense
strand; a double-stranded polynucleotide molecule assembled from a
single stranded molecule, where the sense and antisense regions are
linked by a nucleic acid-based or non-nucleic acid-based linker; a
double-stranded polynucleotide molecule with a hairpin secondary
structure having self-complementary sense and antisense regions;
and a circular single-stranded polynucleotide molecule with two or
more loop structures and a stem having self-complementary sense and
antisense regions, where the circular polynucleotide can be
processed in vivo or in vitro to generate an active double-stranded
siRNA molecule.
[0050] Preferably, siRNA are chemically synthesized. siRNA can also
be generated by cleavage of longer dsRNA (e.g., dsRNA greater than
about 25 nucleotides in length) with the E. coli RNase III or
Dicer. These enzymes process the dsRNA into biologically active
siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA,
99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA,
99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003);
Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et
al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol.
Chem., 243:82 (1968)). Preferably, dsRNA are at least 50
nucleotides to about 100, 200, 300, 400, or 500 nucleotides in
length. A dsRNA may be as long as 1000, 1500, 2000, 5000
nucleotides in length, or longer. The dsRNA can encode for an
entire gene transcript or a partial gene transcript. In certain
instances, siRNA may be encoded by a plasmid (e.g., transcribed as
sequences that automatically fold into duplexes with hairpin
loops).
[0051] As used herein, the terms "mismatch motif" or "mismatch
region" refers to a portion of an interfering RNA (e.g., siRNA)
sequence that does not have 100% complementarity to its target
sequence. An interfering RNA may have at least one, two, three,
four, five, six, or more mismatch regions. The mismatch regions may
be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, or more nucleotides. The mismatch motifs or regions may
comprise a single nucleotide or may comprise two, three, four,
five, or more nucleotides.
[0052] As used herein, the terms "inhibiting expression of a target
gene" refers to the ability of an interfering RNA (e.g., siRNA) to
silence, reduce, or inhibit the expression of a target gene. To
examine the extent of gene silencing, a test sample (e.g., a sample
of cells in culture expressing the target gene) or a test mammal
(e.g., a mammal such as a human or an animal model such as a rodent
(e.g., mouse) or a non-human primate (e.g., monkey) model) is
contacted with an interfering RNA (e.g., siRNA) that silences,
reduces, or inhibits expression of the target gene. Expression of
the target gene in the test sample or test animal is compared to
expression of the target gene in a control sample (e.g., a sample
of cells in culture expressing the target gene) or a control mammal
(e.g., a mammal such as a human or an animal model such as a rodent
(e.g., mouse) or non-human primate (e.g., monkey) model) that is
not contacted with or administered the interfering RNA (e.g.,
siRNA). The expression of the target gene in a control sample or a
control mammal may be assigned a value of 100%. In particular
embodiments, silencing, inhibition, or reduction of expression of a
target gene is achieved when the level of target gene expression in
the test sample or the test mammal relative to the level of target
gene expression in the control sample or the control mammal is
about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,
35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the
interfering RNA (e.g., siRNA) silences, reduces, or inhibits the
expression of a target gene by at least about 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 100% in a test sample or a test mammal relative to the
level of target gene expression in a control sample or a control
mammal not contacted with or administered the interfering RNA
(e.g., siRNA). Suitable assays for determining the level of target
gene expression include, without limitation, examination of protein
or mRNA levels using techniques known to those of skill in the art,
such as, e.g., dot blots, Northern blots, in situ hybridization,
ELISA, immunoprecipitation, enzyme function, as well as phenotypic
assays known to those of skill in the art.
[0053] As used herein, the terms "effective amount" or
"therapeutically effective amount" of an active agent or
therapeutic agent such as an interfering RNA is an amount
sufficient to produce the desired effect, e.g., an inhibition of
expression of a target sequence in comparison to the normal
expression level detected in the absence of an interfering RNA.
Inhibition of expression of a target gene or target sequence is
achieved when the value obtained with an interfering RNA relative
to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
Suitable assays for measuring expression of a target gene or target
sequence include, e.g., examination of protein or RNA levels using
techniques known to those of skill in the art such as dot blots,
northern blots, in situ hybridization, ELISA, immunoprecipitation,
enzyme function, as well as phenotypic assays known to those of
skill in the art.
[0054] As used herein, the terms "decrease," "decreasing,"
"reduce," or "reducing" of an immune response by an interfering RNA
is intended to mean a detectable decrease of an immune response to
a given interfering RNA (e.g., a modified interfering RNA). The
amount of decrease of an immune response by a modified interfering
RNA may be determined relative to the level of an immune response
in the presence of an unmodified interfering RNA. A detectable
decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more
lower than the immune response detected in the presence of the
unmodified interfering RNA. A decrease in the immune response to
interfering RNA is typically measured by a decrease in cytokine
production (e.g., IFN.gamma., IFN.alpha., TNF.alpha., IL-6, or
IL-12) by a responder cell in vitro or a decrease in cytokine
production in the sera of a mammalian subject after administration
of the interfering RNA.
[0055] As used herein, the term "responder cell" refers to a cell,
preferably a mammalian cell, that produces a detectable immune
response when contacted with an immunostimulatory interfering RNA
such as an unmodified siRNA. Exemplary responder cells include,
e.g., dendritic cells, macrophages, peripheral blood mononuclear
cells (PBMCs), splenocytes, and the like. Detectable immune
responses include, e.g., production of cytokines or growth factors
such as TNF-.alpha., IFN-.alpha., IFN-.beta., IFN-.gamma., IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, and
combinations thereof. Detectable immune responses also include,
e.g., induction of interferon-induced protein with
tetratricopeptide repeats 1 (IFIT1) mRNA.
[0056] As used herein, the terms "substantial identity" refers to a
sequence that hybridizes to a reference sequence under stringent
conditions, or to a sequence that has a specified percent identity
over a specified region of a reference sequence.
[0057] As used herein, the phrase "stringent hybridization
conditions" refers to conditions under which a nucleic acid will
hybridize to its target sequence, typically in a complex mixture of
nucleic acids, but to no other sequences. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Probes, "Overview of principles
of hybridization and the strategy of nucleic acid assays" (1993).
Generally, stringent conditions are selected to be about
5-10.degree. C. lower than the thermal melting point (T.sub.m) for
the specific sequence at a defined ionic strength pH. The T., is
the temperature (under defined ionic strength, pH, and nucleic
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at T.sub.m, 50% of the
probes are occupied at equilibrium). Stringent conditions may also
be achieved with the addition of destabilizing agents such as
formamide. For selective or specific hybridization, a positive
signal is at least two times background, preferably 10 times
background hybridization.
[0058] Exemplary stringent hybridization conditions can be as
follows: 50% formamide, 5.times.SSC, and 1% SDS, incubating at
42.degree. C., or, 5.times.SSC, 1% SDS, incubating at 65.degree.
C., with wash in 0.2.times.SSC, and 0.1% SDS at 65.degree. C. For
PCR, a temperature of about 36.degree. C. is typical for low
stringency amplification, although annealing temperatures may vary
between about 32.degree. C. and 48.degree. C. depending on primer
length. For high stringency PCR amplification, a temperature of
about 62.degree. C. is typical, although high stringency annealing
temperatures can range from about 50.degree. C. to about 65.degree.
C., depending on the primer length and specificity. Typical cycle
conditions for both high and low stringency amplifications include
a denaturation phase of 90.degree. C.-95.degree. C. for 30 sec.-2
min., an annealing phase lasting 30 sec.-2 min., and an extension
phase of about 72.degree. C. for 1-2 min. Protocols and guidelines
for low and high stringency amplification reactions are provided,
e.g., in Innis et al., PCR Protocols, A Guide to Methods and
Applications, Academic Press, Inc. N.Y. (1990).
[0059] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
references, e.g., Current Protocols in Molecular Biology, Ausubel
et al., eds.
[0060] As used herein, the terms "substantially identical" or
"substantial identity," in the context of two or more nucleic
acids, refer to two or more sequences or subsequences that are the
same or have a specified percentage of nucleotides that are the
same (i.e., at least about 60%, preferably at least about 65%, 70%,
75%, 80%, 85%, 90%, or 95% identity over a specified region), when
compared and aligned for maximum correspondence over a comparison
window, or designated region as measured using one of the following
sequence comparison algorithms or by manual alignment and visual
inspection. This definition, when the context indicates, also
refers analogously to the complement of a sequence. Preferably, the
substantial identity exists over a region that is at least about 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in
length.
[0061] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0062] As used herein, the terms "comparison window," includes
reference to a segment of any one of a number of contiguous
positions selected from the group consisting of from about 5 to
about 60, usually about 10 to about 45, more usually about 15 to
about 30, in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned. Methods of alignment of sequences
for comparison are well known in the art. Optimal alignment of
sequences for comparison can be conducted, e.g., by the local
homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482
(1981), by the homology alignment algorithm of Needleman and
Wunsch, J. Mol. Biol., 48:443 (1970), by the search for similarity
method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444
(1988), by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.),
or by manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology, Ausubel et al., eds. (1995
supplement)).
[0063] Non-limiting examples of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al.,
J. Mol. Biol., 215:403-410 (1990), respectively. BLAST and BLAST
2.0 are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids of the invention.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information. Another
example is a global alignment algorithm for determining percent
sequence identity such as the Needleman-Wunsch algorithm for
aligning protein or nucleotide (e.g., RNA) sequences.
[0064] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.2, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0065] As used herein, the term "nucleic acid" as used herein
refers to a polymer containing at least two deoxyribonucleotides or
ribonucleotides in either single- or double-stranded form and
includes DNA and RNA. DNA may be in the form of, e.g., antisense
molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors
(P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes,
chimeric sequences, chromosomal DNA, or derivatives and
combinations of these groups. RNA may be in the form of small
interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA
(shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA),
mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.
Nucleic acids include nucleic acids containing known nucleotide
analogs or modified backbone residues or linkages, which are
synthetic, naturally occurring, and non-naturally occurring, and
which have similar binding properties as the reference nucleic
acid. Examples of such analogs include, without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and
peptide-nucleic acids (PNAs). Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides that have similar binding properties as the reference
nucleic acid. Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions), alleles,
orthologs, SNPs, and complementary sequences as well as the
sequence explicitly indicated. Specifically, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes,
8:91-98 (1994)). "Nucleotides" contain a sugar deoxyribose (DNA) or
ribose (RNA), a base, and a phosphate group. Nucleotides are linked
together through the phosphate groups. "Bases" include purines and
pyrimidines, which further include natural compounds adenine,
thymine, guanine, cytosine, uracil, inosine, and natural analogs,
and synthetic derivatives of purines and pyrimidines, which
include, but are not limited to, modifications which place new
reactive groups such as, but not limited to, amines, alcohols,
thiols, carboxylates, and alkylhalides.
[0066] As used herein, the term "gene" refers to a nucleic acid
(e.g., DNA or RNA) sequence that comprises partial length or entire
length coding sequences necessary for the production of a
polypeptide or precursor polypeptide.
[0067] As used herein, the terms "gene product," refer to a product
of a gene such as an RNA transcript or a polypeptide.
[0068] As used herein, the term "lipid" refers to a group of
organic compounds that include, but are not limited to, esters of
fatty acids and are characterized by being insoluble in water, but
soluble in many organic solvents. They are usually divided into at
least three classes: (1) "simple lipids," which include fats and
oils as well as waxes; (2) "compound lipids," which include
phospholipids and glycolipids; and (3) "derived lipids" such as
steroids.
[0069] As used herein, the terms "lipid particle" includes a lipid
formulation that can be used to deliver an active agent or
therapeutic agent, such as a nucleic acid (e.g., an interfering
RNA), to a target site of interest (e.g., cell, tissue, organ, and
the like). In preferred embodiments, the lipid particle of the
invention is a nucleic acid-lipid particle, which is typically
formed from a cationic lipid, a non-cationic lipid, and optionally
a conjugated lipid that prevents aggregation of the particle. In
other preferred embodiments, the active agent or therapeutic agent,
such as a nucleic acid, may be encapsulated in the lipid portion of
the particle, thereby protecting it from enzymatic degradation.
[0070] As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle. A SNALP represents a particle made from lipids
(e.g., a cationic lipid, a non-cationic lipid, and optionally a
conjugated lipid that prevents aggregation of the particle),
wherein the nucleic acid (e.g., an interfering RNA) is fully
encapsulated within the lipid. In certain instances, SNALP are
extremely useful for systemic applications, as they can exhibit
extended circulation lifetimes following intravenous (i.v.)
injection, they can accumulate at distal sites (e.g., sites
physically separated from the administration site), and they can
mediate silencing of target gene expression at these distal sites.
The nucleic acid may be complexed with a condensing agent and
encapsulated within a SNALP as set forth in PCT Publication No. WO
00/03683, the disclosure of which is herein incorporated by
reference in its entirety for all purposes.
[0071] The lipid particles of the invention (e.g., SNALP) typically
have a mean diameter of from about 30 nm to about 150 nm, from
about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from
about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from
about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from
about 90 nm to about 100 nm, from about 70 to about 90 nm, from
about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or
about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70
nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115
nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and
are substantially non-toxic. In addition, nucleic acids, when
present in the lipid particles of the present invention, are
resistant in aqueous solution to degradation with a nuclease.
Nucleic acid-lipid particles and their method of preparation are
disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and
20070042031, the disclosures of which are herein incorporated by
reference in their entirety for all purposes.
[0072] As used herein, "lipid encapsulated" can refer to a lipid
particle that provides an active agent or therapeutic agent, such
as a nucleic acid (e.g., an interfering RNA), with full
encapsulation, partial encapsulation, or both. In a preferred
embodiment, the nucleic acid is fully encapsulated in the lipid
particle (e.g., to form a SNALP or other nucleic acid-lipid
particle).
[0073] As used herein, the term "lipid conjugate" refers to a
conjugated lipid that inhibits aggregation of lipid particles. Such
lipid conjugates include, but are not limited to, PEG-lipid
conjugates such as, e.g., PEG coupled to dialkyloxypropyls (e.g.,
PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG
conjugates), PEG coupled to cholesterol, PEG coupled to
phosphatidylethanolamines, and PEG conjugated to ceramides (see,
e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids, polyoxazoline
(POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see, e.g., U.S.
Provisional Application No. 61/294,828, filed Jan. 13, 2010, and
U.S. Provisional Application No. 61/295, 140, filed Jan. 14, 2010),
polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures
thereof. Additional examples of POZ-lipid conjugates are described
in PCT Publication No. WO 2010/006282. PEG or POZ can be conjugated
directly to the lipid or may be linked to the lipid via a linker
moiety. Any linker moiety suitable for coupling the PEG or the POZ
to a lipid can be used including, e.g., non-ester containing linker
moieties and ester-containing linker moieties. In certain preferred
embodiments, non-ester containing linker moieties, such as amides
or carbamates, are used. The disclosures of each of the above
patent documents are herein incorporated by reference in their
entirety for all purposes.
[0074] As used herein, the terms "amphipathic lipid" refers, in
part, to any suitable material wherein the hydrophobic portion of
the lipid material orients into a hydrophobic phase, while the
hydrophilic portion orients toward the aqueous phase. Hydrophilic
characteristics derive from the presence of polar or charged groups
such as carbohydrates, phosphate, carboxylic, sulfato, amino,
sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity
can be conferred by the inclusion of apolar groups that include,
but are not limited to, long-chain saturated and unsaturated
aliphatic hydrocarbon groups and such groups substituted by one or
more aromatic, cycloaliphatic, or heterocyclic group(s). Examples
of amphipathic compounds include, but are not limited to,
phospholipids, aminolipids, and sphingolipids.
[0075] Representative examples of phospholipids include, but are
not limited to, phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, phosphatidic acid,
palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and
dilinoleoylphosphatidylcholine. Other compounds lacking in
phosphorus, such as sphingolipid, glycosphingolipid families,
diacylglycerols, and .beta.-acyloxyacids, are also within the group
designated as amphipathic lipids. Additionally, the amphipathic
lipids described above can be mixed with other lipids including
triglycerides and sterols.
[0076] As used herein, the terms "neutral lipid" refer to any of a
number of lipid species that exist either in an uncharged or
neutral zwitterionic form at a selected pH. At physiological pH,
such lipids include, for example, diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,
cholesterol, cerebrosides, and diacylglycerols.
[0077] As used herein, the terms "non-cationic lipid" refer to any
amphipathic lipid as well as any other neutral lipid or anionic
lipid.
[0078] As used herein, the terms "anionic lipid" refer to any lipid
that is negatively charged at physiological pH. These lipids
include, but are not limited to, phosphatidylglycerols,
cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids,
N-dodecanoyl phosphatidylethanolamines, N-succinyl
phosphatidylethanolamines, N-glutarylphosphatidylethanolamines,
lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol
(POPG), and other anionic modifying groups joined to neutral
lipids.
[0079] As used herein, the term "hydrophobic lipid" refer to
compounds having apolar groups that include, but are not limited
to, long-chain saturated and unsaturated aliphatic hydrocarbon
groups and such groups optionally substituted by one or more
aromatic, cycloaliphatic, or heterocyclic group(s). Suitable
examples include, but are not limited to, diacylglycerol,
dialkylglycerol, N--N-dialkylamino, 1,2-diacyloxy-3-aminopropane,
and 1,2-dialkyl-3-aminopropane.
[0080] As used herein, the term "fusogenic" refers to the ability
of a lipid particle, such as a SNALP, to fuse with the membranes of
a cell. The membranes can be either the plasma membrane or
membranes surrounding organelles, e.g., endosome, nucleus, etc.
[0081] As used herein, the term "aqueous solution" refers to a
composition comprising in whole, or in part, water.
[0082] As used herein, the term "organic lipid solution" refers to
a composition comprising in whole, or in part, an organic solvent
having a lipid.
[0083] As used herein, the terms "distal site," refer to a
physically separated site, which is not limited to an adjacent
capillary bed, but includes sites broadly distributed throughout an
organism.
[0084] As used herein, the terms "serum-stable" in relation to
nucleic acid-lipid particles such as SNALP mean that the particle
is not significantly degraded after exposure to a serum or nuclease
assay that would significantly degrade free DNA or RNA. Suitable
assays include, for example, a standard serum assay, a DNAse assay,
or an RNAse assay.
[0085] As used herein, the terms "systemic delivery," refer to
delivery of lipid particles that leads to a broad biodistribution
of an active agent such as an interfering RNA (e.g., siRNA) within
an organism. Some techniques of administration can lead to the
systemic delivery of certain agents, but not others. Systemic
delivery means that a useful, preferably therapeutic, amount of an
agent is exposed to most parts of the body. To obtain broad
biodistribution generally requires a blood lifetime such that the
agent is not rapidly degraded or cleared (such as by first pass
organs (liver, lung, etc.) or by rapid, nonspecific cell binding)
before reaching a disease site distal to the site of
administration. Systemic delivery of lipid particles can be by any
means known in the art including, for example, intravenous,
subcutaneous, and intraperitoneal. In a preferred embodiment,
systemic delivery of lipid particles is by intravenous
delivery.
[0086] As used herein, the terms "local delivery," refer to
delivery of an active agent such as an interfering RNA (e.g.,
siRNA) directly to a target site within an organism. For example,
an agent can be locally delivered by direct injection into a
disease site such as a tumor or other target site such as a site of
inflammation or a target organ such as the liver, heart, pancreas,
kidney, and the like.
[0087] As used herein, the term "mammal" refers to any mammalian
species such as a human, mouse, rat, dog, cat, hamster, guinea pig,
rabbit, livestock, and the like.
[0088] As used herein, the term "cancer" refers to any member of a
class of diseases characterized by the uncontrolled growth of
aberrant cells. The term includes all known cancers and neoplastic
conditions, whether characterized as malignant, benign, soft
tissue, or solid, and cancers of all stages and grades including
pre- and post-metastatic cancers. Examples of different types of
cancer include, but are not limited to, liver cancer, lung cancer,
colon cancer, rectal cancer, anal cancer, bile duct cancer, small
intestine cancer, stomach (gastric) cancer, esophageal cancer;
gallbladder cancer, pancreatic cancer, appendix cancer, breast
cancer, ovarian cancer; cervical cancer, prostate cancer, renal
cancer (e.g., renal cell carcinoma), cancer of the central nervous
system, glioblastoma, skin cancer, lymphomas, choriocarcinomas,
head and neck cancers, osteogenic sarcomas, and blood cancers.
Non-limiting examples of specific types of liver cancer include
hepatocellular carcinoma (HCC), secondary liver cancer (e.g.,
caused by metastasis of some other non-liver cancer cell type), and
hepatoblastoma. As used herein, a "tumor" comprises one or more
cancerous cells.
[0089] The compounds of the invention may be prepared by known
organic synthesis techniques, including the methods described in
the Examples. In some embodiments, the synthesis of the cationic
lipids of the invention may require the use of protecting groups.
Protecting group methodology is well known to those skilled in the
art (see, e.g., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T.
W. et. al., Wiley-Interscience, New York City, 1999). Briefly,
protecting groups within the context of this invention are any
group that reduces or eliminates the unwanted reactivity of a
functional group. A protecting group can be added to a functional
group to mask its reactivity during certain reactions and then
removed to reveal the original functional group. In certain
instances, an "alcohol protecting group" is used. An "alcohol
protecting group" is any group which decreases or eliminates the
unwanted reactivity of an alcohol functional group. Protecting
groups can be added and removed using techniques well known in the
art.
[0090] In certain embodiments, the cationic lipids of the present
invention have at least one protonatable or deprotonatable group,
such that the lipid is positively charged at a pH at or below
physiological pH (e.g., pH 7.4), and neutral at a second pH,
preferably at or above physiological pH. It will be understood by
one of ordinary skill in the art that the addition or removal of
protons as a function of pH is an equilibrium process, and that the
reference to a charged or a neutral lipid refers to the nature of
the predominant species and does not require that all of the lipid
be present in the charged or neutral form. Lipids that have more
than one protonatable or deprotonatable group, or which are
zwiterrionic, are not excluded from use in the invention.
[0091] In certain other embodiments, protonatable lipids according
to the invention have a pK.sub.a of the protonatable group in the
range of about 4 to about 11. Most preferred is a pK.sub.a of about
4 to about 7, because these lipids will be cationic at a lower pH
formulation stage, while particles will be largely (though not
completely) surface neutralized at physiological pH of around pH
7.4. One of the benefits of this pK.sub.a is that at least some
nucleic acid associated with the outside surface of the particle
will lose its electrostatic interaction at physiological pH and be
removed by simple dialysis, thus greatly reducing the particle's
susceptibility to clearance.
[0092] As used herein, the term "isomers" refers to refers to
compounds with the same chemical formula but which are structurally
distinguishable.
[0093] The terms "optionally substituted alkyl," "optionally
substituted cyclic alkyl," "optionally substituted alkenyl,"
"optionally substituted alkynyl," "optionally substituted acyl,"
and "optionally substituted heterocycle" mean that, when
substituted, at least one hydrogen atom is replaced with a
substituent. In the case of an "oxo" substituent (.dbd.O), two
hydrogen atoms are replaced. Non-limiting examples of substituents
include oxo, --OH, halogen, heterocycle, --CN, --OR.sup.x,
--NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, R.sup.x and
R.sup.y are the same or different and are independently hydrogen,
alkyl, or heterocycle, and each of the alkyl and heterocycle
substituents may be further substituted with one or more of oxo,
halogen, --OH, --CN, alkyl, --OR.sup.x, heterocycle,
--NR.sub.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y. The term "optionally substituted," when
used before a list of substituents, means that each of the
substituents in the list may be optionally substituted as described
herein.
[0094] As used herein, the terms "quaternary amine" refers to an
amine group with four substituents bonded to the N atom.
III. Novel Cationic Lipids
[0095] The present invention provides, inter alia, novel cationic
(amino) lipids that can advantageously be used in the lipid
particles described herein for the in vitro and/or in vivo delivery
of therapeutic agents such as nucleic acids to cells. The novel
cationic lipids of the present invention have the structures set
forth in Formulas I-VI herein, and include the (R) and/or (S)
enantiomers thereof.
[0096] In some embodiments, a lipid of the present invention
comprises a racemic mixture. In other embodiments, a lipid of the
present invention comprises a mixture of one or more diastereomers.
In certain embodiments, a lipid of the present invention is
enriched in one enantiomer, such that the lipid comprises at least
about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% enantiomeric
excess. In certain other embodiments, a lipid of the present
invention is enriched in one diastereomer, such that the lipid
comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or
95% diastereomeric excess. In certain additional embodiments, a
lipid of the present invention is chirally pure (e.g., comprises a
single optical isomer). In further embodiments, a lipid of the
present invention is enriched in one optical isomer (e.g., an
optically active isomer), such that the lipid comprises at least
about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isomeric
excess. The present invention provides the synthesis of the
cationic lipids of Formulas I-VI as a racemic mixture or in
optically pure form.
[0097] As used herein, the terms "cationic lipid" and "amino lipid"
are used interchangeably to include those lipids and salts thereof
having one, two, three, or more fatty acid or fatty alkyl chains
and a pH-titratable amino head group (e.g., an alkylamino or
dialkylamino head group). The cationic lipid is typically
protonated (i.e., positively charged) at a pH below the pK.sub.a of
the cationic lipid and is substantially neutral at a pH above the
pK.sub.a. The cationic lipids of the invention may also be termed
titratable cationic lipids.
[0098] As used herein, the term "salts" includes any anionic and
cationic complex, such as the complex formed between a cationic
lipid disclosed herein and one or more anions. Non-limiting
examples of anions include inorganic and organic anions, e.g.,
hydride, fluoride, chloride, bromide, iodide, oxalate (e.g.,
hemioxalate), phosphate, phosphonate, hydrogen phosphate,
dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate,
nitrite, nitride, bisulfate, sulfide, sulfite, bisulfate, sulfate,
thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate,
citrate, tartrate, lactate, acrylate, polyacrylate, fumarate,
maleate, itaconate, glycolate, gluconate, malate, mandelate,
tiglate, ascorbate, salicylate, polymethacrylate, perchlorate,
chlorate, chlorite, hypochlorite, bromate, hypobromite, iodate, an
alkylsulfonate, an arylsulfonate, arsenate, arsenite, chromate,
dichromate, cyanide, cyanate, thiocyanate, hydroxide, peroxide,
permanganate, and mixtures thereof. In particular embodiments, the
salts of the cationic lipids disclosed herein are crystalline
salts.
[0099] The term "alkyl" includes a straight chain or branched,
noncyclic or cyclic, saturated aliphatic hydrocarbon containing
from 1 to 30 carbon atoms. Representative saturated straight chain
alkyls include, but are not limited to, methyl, ethyl, n-propyl,
n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched
alkyls include, without limitation, isopropyl, sec-butyl, isobutyl,
tert-butyl, isopentyl, and the like. Representative saturated
cyclic alkyls include, but are not limited to, the C.sub.3-8
cycloalkyls described herein, while unsaturated cyclic alkyls
include, without limitation, the C.sub.3-8 cycloalkenyls described
herein.
[0100] The term "heteroalkyl," includes a straight chain or
branched, noncyclic or cyclic, saturated aliphatic hydrocarbon as
defined above having from about 1 to about 5 heteroatoms (i.e., 1,
2, 3, 4, or 5 heteroatoms) such as, for example, O, N, Si, and/or
S, wherein the nitrogen and sulfur atoms may optionally be oxidized
and the nitrogen heteroatom may optionally be quaternized. The
heteroalkyl group can be attached to the remainder of the molecule
through a carbon atom or a heteroatom.
[0101] The term "cyclic alkyl" includes any of the substituted or
unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, and
heterocycloalkenyl groups described below.
[0102] The term "cycloalkyl" includes a substituted or
unsubstituted cyclic alkyl group having from about 3 to about 8
carbon atoms (i.e., 3, 4, 5, 6, 7, or 8 carbon atoms) as ring
vertices. Preferred cycloalkyl groups include those having from
about 3 to about 6 carbon atoms as ring vertices. Examples of
C.sub.3-8 cycloalkyl groups include, but are not limited to,
cyclopropyl, methyl-cyclopropyl, dimethyl-cyclopropyl, cyclobutyl,
methyl-cyclobutyl, cyclopentyl, methyl-cyclopentyl, cyclohexyl,
methyl-cyclohexyl, dimethyl-cyclohexyl, cycloheptyl, and
cyclooctyl, as well as other substituted C.sub.3-8 cycloalkyl
groups.
[0103] The term "heterocycloalkyl" includes a substituted or
unsubstituted cyclic alkyl group as defined above having from about
1 to about 3 heteroatoms as ring members selected from the group
consisting of O, N, Si and S, wherein the nitrogen and sulfur atoms
may optionally be oxidized and the nitrogen heteroatom may
optionally be quaternized. The heterocycloalkyl group can be
attached to the remainder of the molecule through a carbon atom or
a heteroatom.
[0104] The term "cycloalkenyl" includes a substituted or
unsubstituted cyclic alkenyl group having from about 3 to about 8
carbon atoms (i.e., 3, 4, 5, 6, 7, or 8 carbon atoms) as ring
vertices. Preferred cycloalkenyl groups are those having from about
3 to about 6 carbon atoms as ring vertices. Examples of C.sub.3-8
cycloalkenyl groups include, but are not limited to, cyclopropenyl,
methyl-cyclopropenyl, dimethyl-cyclopropenyl, cyclobutenyl,
cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl, as
well as other substituted C.sub.3-8 cycloalkenyl groups.
[0105] The term "heterocycloalkenyl" includes a substituted or
unsubstituted cyclic alkenyl group as defined above having from
about 1 to about 3 heteroatoms as ring members selected from the
group consisting of O, N, Si and S, wherein the nitrogen and sulfur
atoms may optionally be oxidized and the nitrogen heteroatom may
optionally be quaternized. The heterocycloalkenyl group can be
attached to the remainder of the molecule through a carbon atom or
a heteroatom.
[0106] The term "alkoxy" includes a group of the formula alkyl-O--,
wherein "alkyl" has the previously given definition. Non-limiting
examples of alkoxy groups include methoxy, ethoxy, n-propoxy,
iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy and tert-butoxy.
[0107] The term "alkenyl" includes an alkyl, as defined above,
containing at least one double bond between adjacent carbon atoms.
Alkenyls include both cis and trans isomers. Representative
straight chain and branched alkenyls include, but are not limited
to, ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,
1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,
2,3-dimethyl-2-butenyl, and the like. Representative cyclic
alkenyls are described above.
[0108] The term "alkynyl" includes any alkyl or alkenyl, as defined
above, which additionally contains at least one triple bond between
adjacent carbons. Representative straight chain and branched
alkynyls include, without limitation, acetylenyl, propynyl,
1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl,
and the like.
[0109] The term "aryl" includes a polyunsaturated, typically
aromatic, hydrocarbon group which can be a single ring or multiple
rings (up to three rings) which are fused together or linked
covalently, and which optionally carries one or more substituents,
such as, for example, halogen, trifluoromethyl, amino, alkyl,
alkoxy, alkylcarbonyl, cyano, carbamoyl, alkoxycarbamoyl,
methylendioxy, carboxy, alkoxycarbonyl, aminocarbonyl,
alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, nitro, and the
like. Non-limiting examples of unsubstituted aryl groups include
phenyl, naphthyl, and biphenyl. Examples of substituted aryl groups
include, but are not limited to, phenyl, chlorophenyl,
trifluoromethylphenyl, chlorofluorophenyl, and aminophenyl.
[0110] The term "acyl" includes any alkyl, alkenyl, or alkynyl
wherein the carbon at the point of attachment is substituted with
an oxo group, as defined below. The following are non-limiting
examples of acyl groups: --C(.dbd.O)alkyl, --C(.dbd.O)alkenyl, and
--C(.dbd.O)alkynyl.
[0111] The term "heterocycle" includes a 5- to 7-membered
monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which
is either saturated, unsaturated, or aromatic, and which contains
from 1 or 2 heteroatoms independently selected from nitrogen,
oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms
may be optionally oxidized, and the nitrogen heteroatom may be
optionally quaternized, including bicyclic rings in which any of
the above heterocycles are fused to a benzene ring. The heterocycle
may be attached via any heteroatom or carbon atom. Heterocycles
include, but are not limited to, heteroaryls as defined below, as
well as morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,
piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,
tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
and the like.
[0112] The term "heteroaryl" includes an aromatic 5- to 10-membered
heterocycle which contains one, two, or more heteroatoms selected
from nitrogen (N), oxygen (O), and sulfur (S). The heteroaryl can
be substituted on one or more carbon atoms with substituents such
as, for example, halogen, alkyl, alkoxy, cyano, haloalkyl (e.g.,
trifluoromethyl), heterocyclyl (e.g., morpholinyl or pyrrolidinyl),
and the like. Non-limiting examples of heteroaryls include
pyridinyl and furanyl.
[0113] The term "halogen" includes fluoro, chloro, bromo, and
iodo.
[0114] A. Exemplary Embodiments of Trialkyl Cationic Lipids
[0115] In one aspect, the present invention provides a cationic
lipid of Formula I having the following structure:
##STR00005##
or salts and isomers thereof, wherein: R.sup.1 and R.sup.2 are
independently selected from hydrogen (H), optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6
alkynyl. Alternatively, R.sup.1 and R.sup.2 may join to form an
optionally substituted heterocycloalkyl of 4 to 6 carbon atoms and
1 to 2 heteroatoms selected from nitrogen (N), oxygen (O), and
mixtures thereof. R.sup.3 is absent or is hydrogen (H) or a
C.sub.1-C.sub.6 alkyl to provide a quaternary amine R.sup.4 is
absent or is selected from --CH.sub.2--, optionally substituted
C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30 alkenyl,
C.sub.10-C.sub.30 alkynyl, C.sub.10-C.sub.30 acyl, and
NR.sup.4aR.sup.4b, wherein R.sup.4a and R.sup.4b are independently
selected from hydrogen (H), an optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6
alkynyl. R.sup.5 is selected from hydrogen (H), oxygen (O),
optionally substituted C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30
alkenyl, C.sub.10-C.sub.30 alkynyl, and OR.sup.5a, wherein R.sup.5a
is selected from hydrogen (H), an optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6
alkynyl. Alternatively, R.sup.4 and R.sup.5 may join to form a
C.sub.3-10 heterocycloalkyl having from 1 to 3 heteroatoms each
independently selected from nitrogen (N), oxygen (O), and sulfur
(S), and optionally substituted with from 1 to 3 groups each
independently selected from hydrogen (H) and C.sub.1-C.sub.6 alkyl.
R.sup.6 and R.sup.7 are independently selected from optionally
substituted C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30 alkenyl,
C.sub.10-C.sub.30 alkynyl, and C.sub.10-C.sub.30 acyl. R.sup.8 is
selected from optionally substituted C.sub.10-C.sub.30 alkyl,
C.sub.10-C.sub.30 alkenyl, C.sub.10-C.sub.30 alkynyl, and
C.sub.10-C.sub.30 acyl. R.sup.13 and R.sup.14 are independently
selected from hydrogen (H), optionally substituted C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl. X is
selected from --CH--, nitrogen (N), oxygen (O), sulfur (S),
N(R.sup.9), C(O), C(O)O, OC(O), C(O)N(R.sup.9), N(R.sup.9)C(O),
OC(O)N(R.sup.9), N(R.sup.9)C(O)O, C(O)S, SC(O), C(S)O, OC(S), S(O),
S(O)(O), and C(S), wherein R.sup.9 is selected from hydrogen (H),
optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, and
##STR00006##
wherein * represents the point of attachment to nitrogen (N). Y is
selected from --CR.sup.11R.sup.12-- and oxygen (O), wherein
R.sup.11 and R.sup.12 are independently selected from hydrogen (H),
optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, and C.sub.2-C.sub.6 alkynyl. Z.sup.1, Z.sup.2, and Z.sup.3
are independently selected from oxygen (O), C(O)O, or OC(O).
T.sup.1, T.sup.2, and T.sup.3 are independently selected from
hydrogen (H) or OR.sup.10, wherein R.sup.16 is hydrogen (H) or
optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, or C.sub.2-C.sub.6 alkynyl. Subscripts a, b, c, d, e, f,
and g are independently selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8.
Subscripts h, i, j, k, and m are independently selected from of 0
and 1. When R.sup.5 is an optionally substituted C.sub.10-C.sub.30
alkyl, C.sub.10-C.sub.30 alkenyl, C.sub.10-C.sub.30 alkynyl, and
C.sub.10-C.sub.30 acyl, then either subscript h is 0 or at least
one of Z.sup.1 and Z.sup.2 is oxygen (O). At least three of
R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are selected from
optionally substituted C.sub.10-C.sub.30 alkyl, C.sub.10-C.sub.30
alkenyl, and C.sub.10-C.sub.30 alkynyl.
[0116] A person of ordinary skill in the art will recognize that,
in some embodiments, the X linker in Formula I can be oriented in
either possible orientation. For instance, when X is an ester,
i.e., C(O)O or OC(O), X can connect to the rest of Formula I with
the single-bonded oxygen (O) atom bonded to either the carbon with
subscript a or the carbon with subscript c. Similarly, when X is a
carbamate, i.e., OC(O)N(R.sup.9) or N(R.sup.9)C(O)O, X can connect
to the rest of Formula I with the single-bonded oxygen (O) atom
bonded to either the carbon with subscript a or with subscript
c.
[0117] In some embodiments, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl (e.g., C.sub.1-C.sub.2 alkyl, C.sub.1-C.sub.3
alkyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.5 alkyl,
C.sub.2-C.sub.3 alkyl, C.sub.2-C.sub.4 alkyl, C.sub.2-C.sub.5
alkyl, C.sub.2-C.sub.6 alkyl, C.sub.3-C.sub.4 alkyl,
C.sub.3-C.sub.5 alkyl, C.sub.3-C.sub.6 alkyl, C.sub.4-C.sub.5
alkyl, C.sub.4-C.sub.6 alkyl, C.sub.5-C.sub.6 alkyl, or C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl),
C.sub.2-C.sub.6 alkenyl (e.g., C.sub.2-C.sub.3 alkenyl,
C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.6
alkenyl, C.sub.3-C.sub.4 alkenyl, C.sub.3-C.sub.5 alkenyl,
C.sub.3-C.sub.6 alkenyl, C.sub.4-C.sub.5 alkenyl, C.sub.4-C.sub.6
alkenyl, C.sub.5-C.sub.6 alkenyl, or C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6 alkenyl), or C.sub.2-C.sub.6 alkynyl (e.g.,
C.sub.2-C.sub.3 alkynyl, C.sub.2-C.sub.4 alkynyl, C.sub.2-C.sub.5
alkynyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.4 alkynyl,
C.sub.3-C.sub.5 alkynyl, C.sub.3-C.sub.6 alkynyl, C.sub.4-C.sub.5
alkynyl, C.sub.4-C.sub.6 alkynyl, or C.sub.5-C.sub.6 alkynyl, or
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkynyl). In other
embodiments, R.sup.1 and R.sup.2 are joined to form a heterocyclic
ring of 5 carbon atoms and 1 nitrogen atom, wherein the
heterocyclic ring can be substituted with a substituent such as a
hydroxyl (--OH) group at the ortho, meta, and/or para positions. In
particular embodiments, R.sup.1 and R.sup.2 are both methyl groups
(i.e., C.sub.1 alkyls). In certain instances, R.sup.3 is absent
when the pH is above the pK.sub.a of the cationic lipid and R.sup.3
is hydrogen (H) when the pH is below the pK.sub.a of the cationic
lipid such that the amino head group is protonated. In certain
other instances, R.sup.3 is an optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl group to provide a quaternary amine
[0118] In certain embodiments, at least three, four, or all five of
R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are independently
selected from an optionally substituted C.sub.10-C.sub.24,
C.sub.12-C.sub.24, C.sub.12-C.sub.22, C.sub.12-C.sub.20,
C.sub.14-C.sub.24, C.sub.14-C.sub.22, C.sub.14-C.sub.20,
C.sub.16-C.sub.24, C.sub.16-C.sub.22, or C.sub.16-C.sub.20 alkyl or
acyl group (i.e., C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14,
C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19, C.sub.20,
C.sub.21, C.sub.22, C.sub.23, or C.sub.24 alkyl or acyl group). In
other embodiments, at least one, two, three, four, or all five of
R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 independently
comprise at least 1, 2, 3, 4, 5, or 6 sites of unsaturation (e.g.,
1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6) or a branched alkyl
or acyl group. In certain instances, the unsaturated side-chain may
comprise a myristoleyl moiety, a palmitoleyl moiety, an oleyl
moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienyl
moiety, or an acyl derivative thereof (e.g., linoleoyl, linolenoyl,
.gamma.-linolenoyl, etc.). In some instances, the octadecadienyl
moiety is a linoleyl moiety. In particular embodiments, at least
three, four, or all five of R.sup.4, R.sup.5, R.sup.6, R.sup.7, and
R.sup.8 are linoleyl moieties. In other instances, the
octadecatrienyl moiety is a linolenyl moiety or a .gamma.-linolenyl
moiety. In particular embodiments, at least three, four, or all
five of R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are
linolenyl moieties or .gamma.-linolenyl moieties. In embodiments
where at least one, two, three, four, or all five of R.sup.4,
R.sup.5, R.sup.6, R.sup.7, and R.sup.8 independently comprise a
branched alkyl or acyl group (e.g., a substituted alkyl or acyl
group), the branched alkyl or acyl group may comprise a
C.sub.12-C.sub.24 alkyl or acyl having at least 1-6 (e.g., 1, 2, 3,
4, 5, 6, or more) C.sub.1-C.sub.6 alkyl substituents. In particular
embodiments, the branched alkyl or acyl group comprises a
C.sub.12-C.sub.20 or C.sub.14-C.sub.22 alkyl or acyl with 1-6
(e.g., 1, 2, 3, 4, 5, 6) C.sub.1-C.sub.4 alkyl (e.g., methyl,
ethyl, propyl, or butyl) substituents. In some embodiments, the
branched alkyl group comprises a phytanyl
(3,7,11,15-tetramethyl-hexadecanyl) moiety and the branched acyl
group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)
moiety. In particular embodiments, at least one, two, three, four,
or all five of R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are
phytanyl moieties.
[0119] In other embodiments, at least one, two, three, four, or all
five of R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8
independently comprise at least 1, 2, 3, 4, 5, 6, or more
optionally substituted C.sub.1-6 cyclic alkyl groups (e.g., 1-2,
1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6). In certain instances,
at least one, two, three, four, or all five of R.sup.4, R.sup.5,
R.sup.6, R.sup.7, and R.sup.8 independently comprise an optionally
substituted C.sub.10-C.sub.24, C.sub.12-C.sub.24,
C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl, alkenyl, alkynyl, or
acyl group, wherein at least one, two, three, four, or all five of
R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 independently
comprise at least 1, 2, 3, 4, 5, or 6 optionally substituted cyclic
alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or
2-6).
[0120] In particular embodiments, one or more of the optionally
substituted cyclic alkyl groups present in R.sup.4, R.sup.5,
R.sup.6, R.sup.7, and/or R.sup.8 are independently selected from
the group consisting of an optionally substituted saturated cyclic
alkyl group, an optionally substituted unsaturated cyclic alkyl
group, and combinations thereof. In certain instances, the
optionally substituted saturated cyclic alkyl group comprises an
optionally substituted C.sub.3-8 cycloalkyl group (e.g.,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, etc.). In preferred embodiments, the optionally
substituted saturated cyclic alkyl group comprises a cyclopropyl
group, optionally containing one or more substituents and/or
heteroatoms. In other instances, the optionally substituted
unsaturated cyclic alkyl group comprises an optionally substituted
C.sub.3-8 cycloalkenyl group (e.g., cyclopropenyl, cyclobutenyl,
cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,
etc.).
[0121] In particular embodiments, at least one, two, three, four,
or all five of R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are
independently selected from C.sub.10-C.sub.24, C.sub.12-C.sub.24,
C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl groups having at
least 1, 2, 3, 4, 5, or 6 optionally substituted cyclic alkyl
groups. In preferred embodiments, at least one, two, three, four,
or all five of R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are
C.sub.18 alkyl groups having at least one, two, three, or more
optionally substituted cyclic alkyl groups such as, for example, an
optionally substituted C.sub.3-8 cycloalkyl group (e.g., a
cyclopropyl group, optionally containing one or more substituents
and/or heteroatoms). In certain embodiments, each of the optionally
substituted cyclic alkyl groups is independently selected and can
be the same cyclic alkyl group (e.g., all cyclopropyl groups) or
different cyclic alkyl groups (e.g., cyclopropyl and other
cycloalkyl, heterocycloalkyl, cycloalkenyl, and/or
heterocycloalkenyl groups).
[0122] In preferred embodiments, the optionally substituted cyclic
alkyl groups present in R.sup.4, R.sup.5, R.sup.6, R.sup.7, and/or
R.sup.8 are located at the site(s) of unsaturation in the
corresponding unsaturated side-chain. As a non-limiting example, at
least one, two, three, four, or all five of R.sup.4, R.sup.5,
R.sup.6, R.sup.7, and R.sup.8 are C.sub.18 alkyl groups having 1,
2, or 3 optionally substituted cyclic alkyl groups, wherein the
optionally substituted cyclic alkyl groups (e.g., independently
selected cyclopropyl groups) are located at one or more (e.g., all)
of the sites of unsaturation present in a corresponding oleyl
moiety, linoleyl moiety, linolenyl moiety, or .gamma.-linolenyl
moiety.
[0123] In some embodiments, R.sup.5 is hydrogen (H) when subscript
m is 1. In other embodiments, R.sup.5 is oxygen (O) when subscript
m is 1 and R.sup.4 and R.sup.5 join to form a C.sub.3-10
heterocycloalkyl. In certain other embodiments, R.sup.5 is selected
from an optionally substituted C.sub.10-C.sub.30 alkyl,
C.sub.10-C.sub.30 alkenyl, and C.sub.10-C.sub.30 alkynyl wherein
subscript m is 0. In further embodiments, R.sup.4 is --CH.sub.2--;
R.sup.5 and Y are both 0; X is --CH--; subscripts b and c are 0;
subscript h is 1; when R.sup.4 and R.sup.5 are joined to form a
C.sub.5 heterocycloalkyl.
[0124] In certain embodiments X is O, subscript b is 0 and R.sup.4
is absent. In some other embodiments, X is either C(O)O or OC(O),
and subscript b is 0 and R.sup.4 is absent. In some other
embodiments, X is either OC(O)N(R.sup.9) or N(R.sup.9)C(O)O,
subscript b is 0 and R.sup.4 is absent, wherein R.sup.9 is selected
from the group consisting of hydrogen (H), optionally substituted
C.sub.1-C.sub.6 alkyl (e.g., C.sub.1-C.sub.2 alkyl, C.sub.1-C.sub.3
alkyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.5 alkyl,
C.sub.2-C.sub.3 alkyl, C.sub.2-C.sub.4 alkyl, C.sub.2-C.sub.5
alkyl, C.sub.2-C.sub.6 alkyl, C.sub.3-C.sub.4 alkyl,
C.sub.3-C.sub.5 alkyl, C.sub.3-C.sub.6 alkyl, C.sub.4-C.sub.5
alkyl, C.sub.4-C.sub.6 alkyl, C.sub.5-C.sub.6 alkyl, or C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl),
C.sub.2-C.sub.6 alkenyl (e.g., C.sub.2-C.sub.3 alkenyl,
C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.6
alkenyl, C.sub.3-C.sub.4 alkenyl, C.sub.3-C.sub.5 alkenyl,
C.sub.3-C.sub.6 alkenyl, C.sub.4-C.sub.5 alkenyl, C.sub.4-C.sub.6
alkenyl, C.sub.5-C.sub.6 alkenyl, or C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6 alkenyl), C.sub.2-C.sub.6 alkynyl (e.g.,
C.sub.2-C.sub.3 alkynyl, C.sub.2-C.sub.4 alkynyl, C.sub.2-C.sub.5
alkynyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.4 alkynyl,
C.sub.3-C.sub.5 alkynyl, C.sub.3-C.sub.6 alkynyl, C.sub.4-C.sub.5
alkynyl, C.sub.4-C.sub.6 alkynyl, or C.sub.5-C.sub.6 alkynyl, or
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkynyl). In some
other embodiments, X is either OC(O)N(R.sup.9) or N(R.sup.9)C(O)O,
wherein R.sup.9 is selected from the group consisting of hydrogen
(H), optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, and
##STR00007##
wherein * represents the point of attachment to N. In some
instances, R.sup.9 is methyl. R.sup.4 is as described above.
[0125] In some other embodiments Y is --CR.sup.1lR.sup.12-- and
R.sup.11 and R.sup.12 are independently selected from hydrogen (H)
and C.sub.1-C.sub.6 alkyl (e.g., C.sub.1-C.sub.2 alkyl,
C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.5
alkyl, C.sub.2-C.sub.3 alkyl, C.sub.2-C.sub.4 alkyl,
C.sub.2-C.sub.5 alkyl, C.sub.2-C.sub.6 alkyl, C.sub.3-C.sub.4
alkyl, C.sub.3-C.sub.5 alkyl, C.sub.3-C.sub.6 alkyl,
C.sub.4-C.sub.5 alkyl, C.sub.4-C.sub.6 alkyl, C.sub.2-C.sub.6
alkyl, or C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6
alkyl), C.sub.2-C.sub.6 alkenyl (e.g., C.sub.2-C.sub.3 alkenyl,
C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.6
alkenyl, C.sub.3-C.sub.4 alkenyl, C.sub.3-C.sub.5 alkenyl,
C.sub.3-C.sub.6 alkenyl, C.sub.4-C.sub.5 alkenyl, C.sub.4-C.sub.6
alkenyl, C.sub.5-C.sub.6 alkenyl, or C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6 alkenyl), or C.sub.2-C.sub.6 alkynyl (e.g.,
C.sub.2-C.sub.3 alkynyl, C.sub.2-C.sub.4 alkynyl, C.sub.2-C.sub.5
alkynyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.4 alkynyl,
C.sub.3-C.sub.5 alkynyl, C.sub.3-C.sub.6 alkynyl, C.sub.4-C.sub.5
alkynyl, C.sub.4-C.sub.6 alkynyl, or C.sub.5-C.sub.6 alkynyl, or
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkynyl). In some
embodiments, Y is --CR.sup.11R.sup.12--, R.sup.11 and R.sup.12 are
both either hydrogen (H) or methyl, and subscript c is 1.
[0126] In some embodiments, at least one, two, or three of Z.sup.1,
Z.sup.2, and Z.sup.3 are independently selected from oxygen (O),
C(O)O, or OC(O). In certain embodiments both Z.sup.2 and Z.sup.3
are either (O), C(O)O, or OC(O) and subscript i is 0. In certain
other embodiments, Z.sup.1 is either (O), C(O)O, or OC(O) and
subscripts j and k are both 0. In certain other embodiments,
Z.sup.2 is either (O), C(O)O, or OC(O) and subscripts i and k are
both 0. In certain other embodiments, Z.sup.3 is either (O), C(O)O,
or OC(O) and subscripts i and j are both 0.
[0127] In some embodiments at least one, two, or three of T.sup.1,
T.sup.2, and T.sup.3 are independently selected from hydrogen (H)
or OR.sup.10, wherein R.sup.10 is hydrogen (H) or optionally
substituted C.sub.1-C.sub.6 alkyl (e.g., C.sub.1-C.sub.2 alkyl,
C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.5
alkyl, C.sub.2-C.sub.3 alkyl, C.sub.2-C.sub.4 alkyl,
C.sub.2-C.sub.5 alkyl, C.sub.2-C.sub.6 alkyl, C.sub.3-C.sub.4
alkyl, C.sub.3-C.sub.5 alkyl, C.sub.3-C.sub.6 alkyl,
C.sub.4-C.sub.5 alkyl, C.sub.4-C.sub.6 alkyl, C.sub.5-C.sub.6
alkyl, or C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6
alkyl), C.sub.2-C.sub.6 alkenyl (e.g., C.sub.2-C.sub.3 alkenyl,
C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.6
alkenyl, C.sub.3-C.sub.4 alkenyl, C.sub.3-C.sub.5 alkenyl,
C.sub.3-C.sub.6 alkenyl, C.sub.4-C.sub.5 alkenyl, C.sub.4-C.sub.6
alkenyl, C.sub.5-C.sub.6 alkenyl, or C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6 alkenyl), or C.sub.2-C.sub.6 alkynyl (e.g.,
C.sub.2-C.sub.3 alkynyl, C.sub.2-C.sub.4 alkynyl, C.sub.2-C.sub.5
alkynyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.4 alkynyl,
C.sub.3-C.sub.5 alkynyl, C.sub.3-C.sub.6 alkynyl, C.sub.4-C.sub.5
alkynyl, C.sub.4-C.sub.6 alkynyl, or C.sub.5-C.sub.6 alkynyl, or
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkynyl). In some
preferred embodiments, T.sup.1 is OR.sup.10, R.sup.10 is hydrogen
(H), and subscript g is 0. In some other preferred embodiments,
T.sup.2 is OR.sup.10, R.sup.10 is hydrogen (H), and both T.sup.1
and T.sup.3 are hydrogen (H). In some other preferred embodiments,
both T.sup.2 and T.sup.3 are OR.sup.10, R.sup.10 is hydrogen (H),
and T.sup.1 is hydrogen (H). In some other preferred embodiments,
T.sup.1 and T.sup.2 are OR.sup.10, R.sup.10 is hydrogen (H), and
T.sup.3 is hydrogen (H). In some other preferred embodiments,
T.sup.1, T.sup.2 and T.sup.3 are OR.sup.10, R.sup.10 is hydrogen
(H).
[0128] In some embodiments, subscripts a, b, c, d, e, f, and g are
independently selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8. More
preferably, subscript a is selected from 1, 2, 3, 4, and 5. Even
more preferably, subscript a is selected from 2, 3, or 4. Subscript
b is preferably selected from 0 or 1. More preferably, subscript b
is O, Subscript c is preferably selected from 0, 1, or 2. Subscript
d is preferably 0 or 1. In certain instances, subscripts c and d
and both 1. Subscript e is preferably 0 or 1. Subscript f is
preferably 0 or 1. More preferably, subscript f is 0. Subscript g
is preferably 0 or 1.
[0129] In some embodiments, subscripts h, i, j, k, and m are
independently selected from 0 and 1. In some instances, subscript h
is 1. In other instances, subscript h is 0. In some embodiments,
subscript i is 0 and subscripts j and k are 1. In some embodiments,
subscript i is 1 and subscripts j and k are 0. In some embodiments,
subscript m is 1. In some other embodiments, subscripts h, i, j,
and k are all 0. In some other embodiments, subscripts i, j, and k
are all 0. In some other embodiments, subscripts h, i, j, and k are
all 1.
[0130] In some embodiments, the cationic lipid of Formula I forms a
salt (preferably a crystalline salt) with one or more anions. In
one particular embodiment, the cationic lipid of Formula I is the
oxalate (e.g., hemioxalate) salt thereof, which is preferably a
crystalline salt.
[0131] In a related aspect, the present invention provides a
cationic lipid of Formula II having the following structure:
##STR00008##
or salts and isomers thereof,
[0132] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
Y, Z.sup.1, Z.sup.2, Z.sup.3, T.sup.1, T.sup.2, T.sup.3, and
subscripts a, b, c, d, e, f, g, h, i, j, k, and m are the same as
described above for Formula I.
[0133] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In yet another embodiment, subscript c is 0, 1, or 2 and
subscript d is 0. In one particular embodiment, subscripts c and d
are both 0. In still yet another embodiment, R.sup.5 is hydrogen
(H). In a further embodiment, subscripts e, f, and g are all 0. In
another embodiment, subscripts i, j, and k are all 0. In yet
another embodiment, T.sup.1 is hydrogen (H) or OR.sup.10, wherein
R.sup.10 is H or optionally substituted C.sub.1-C.sub.4 alkyl. In
one particular embodiment, T.sup.1 is H or OH. In still yet another
embodiment, subscript m is 1 and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from an optionally substituted
C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24 alkenyl, and
C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6, R.sup.7, and
R.sup.8 independently comprise an optionally substituted
C.sub.12-C.sub.24, C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl
group. In other instances, at least one, two, or all three of
R.sup.6, R.sup.7, and R.sup.8 independently comprises at least 1,
2, 3, 4, 5, or 6 sites of unsaturation or a substituted alkyl group
such as a phytanyl moiety. The unsaturated side-chains may
independently comprise a myristoleyl moiety, a palmitoleyl moiety,
an oleyl moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, or an
icosatrienyl moiety. In some instances, the octadecadienyl moiety
is a linoleyl moiety. In particular embodiments, R.sup.6, R.sup.7,
and R.sup.8 are all linoleyl moieties. In other embodiments,
R.sup.7 and R.sup.8 are the same and R.sup.6 is different. In
certain instances, R.sup.6 is an oleyl moiety and R.sup.7 and
R.sup.8 are linoleyl moieties. In certain other instances, R.sup.6
is a linoleyl moiety and R.sup.7 and R.sup.8 are oleyl moieties.
One of skill in the art will appreciate that the compounds of
Formula II may comprise a combination of two or more of the
features described in this paragraph. As a non-limiting example, in
certain preferred embodiments, subscripts e, f, g, i, j, and k are
all 0, m is 1, and R.sup.6, R.sup.7, and R.sup.8 are independently
selected C.sub.18 alkyl groups (e.g., R.sup.6, R.sup.7, and R.sup.8
are all linoleyl moieties). Exemplary methods of making the
ester-containing lipids of Formula II described in this paragraph
are illustrated for Compound 4 of Scheme 1 and Compound 8 of Scheme
2 below.
[0134] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
subscripts c and d are both 0; R.sup.5 is hydrogen (H); subscripts
e, f, g, i, j, and k are all 0; T.sup.1 is H or OH; subscript m is
1; and R.sup.6, R.sup.7, and R.sup.8 are independently selected
from C.sub.16-C.sub.20 (e.g., C.sub.18) alkenyl groups (e.g.,
R.sup.6, R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl
moieties; at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl
moieties and the other is a linoleyl moiety; or at least two of
R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties and the other
is an oleyl moiety).
[0135] In particularly preferred embodiments, the cationic lipid of
Formula II has one of the following structures:
##STR00009##
[0136] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, or 5. In a preferred embodiment, subscript a is 2, 3, or 4.
In yet another embodiment, subscript c is 0, 1, or 2 and subscript
d is 0 or 1. In one particular embodiment, subscript c is 2 and
subscript d is 0. In another particular embodiment, subscripts c
and d are both 1. In yet another embodiment, R.sup.5 is hydrogen
(H). In a further embodiment, subscripts e, f, and g are all 0. In
another embodiment, subscript i is either 0 or 1 and subscripts j
and k are both 0. In a further embodiment, Z.sup.1 is oxygen (O).
In yet another embodiment, T.sup.1 is hydrogen (H) or OR.sup.10,
wherein R.sup.10 is H or optionally substituted C.sub.1-C.sub.4
alkyl. In one particular embodiment, T.sup.1 is H or OH. In still
yet another embodiment, subscript m is 1 and R.sup.6, R.sup.7, and
R.sup.8 are independently selected from an optionally substituted
C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24 alkenyl, and
C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6, R.sup.7, and
R.sup.8 independently comprise an optionally substituted
C.sub.12-C.sub.24, C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl
group. In other instances, at least one, two, or all three of
R.sup.6, R.sup.7, and R.sup.8 independently comprises at least 1,
2, 3, 4, 5, or 6 sites of unsaturation or a substituted alkyl group
such as a phytanyl moiety. The unsaturated side-chains may
independently comprise a myristoleyl moiety, a palmitoleyl moiety,
an oleyl moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, or an
icosatrienyl moiety. In some instances, the octadecadienyl moiety
is a linoleyl moiety. In particular embodiments, R.sup.6, R.sup.7,
and R.sup.8 are all linoleyl moieties. In other embodiments,
R.sup.7 and R.sup.8 are the same and R.sup.6 is different. In
certain instances, R.sup.6 is an oleyl moiety and R.sup.7 and
R.sup.8 are linoleyl moieties. In certain other instances, R.sup.6
is a linoleyl moiety and R.sup.7 and R.sup.8 are oleyl moieties.
One of skill in the art will appreciate that the compounds of
Formula II may comprise a combination of two or more of the
features described in this paragraph. As a non-limiting example, in
certain preferred embodiments, subscripts i and m are both 1;
subscripts e, f, g, j, and k are all 0; and R.sup.6, R.sup.7, and
R.sup.8 are independently selected C.sub.18 alkyl groups (e.g.,
R.sup.6, R.sup.7, and R.sup.8 are all linoleyl moieties). An
exemplary method of making the ester-containing lipids of Formula
II described in this paragraph is illustrated for Compound 12 in
Scheme 3 below.
[0137] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
subscripts c and d are both 1; Y is --CR.sup.11R.sup.12-, wherein
R.sup.11 and R.sup.12 are independently selected from H, CH.sub.3,
and ethyl; R.sup.5 is hydrogen (H); subscripts e, f, g, j, and k
are all 0; subscript i is 1 and Z.sup.1 is 0; T.sup.1 is OH;
subscript m is 1; and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from C.sub.16-C.sub.20 (e.g., C.sub.18)
alkyl groups (e.g., R.sup.6, R.sup.7, and R.sup.8 are all linoleyl
moieties or oleyl moieties; at least two of R.sup.6, R.sup.7, and
R.sup.8 are oleyl moieties and the other is a linoleyl moiety; or
at least two of R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties
and the other is an oleyl moiety).
[0138] In particularly preferred embodiments, the cationic lipid of
Formula II has one of the following structures:
##STR00010##
[0139] In another embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In yet another embodiment, subscript c is 0, 1, or 2 and
subscript d is 0 or 1. In a preferred embodiment subscript c is 0.
In yet another embodiment, R.sup.5 is hydrogen (H). In a further
embodiment, subscript f is 1 and subscripts e and g are both 0. In
another embodiment, subscript i is 0, subscripts j and k are both
1, and Z.sup.2 and Z.sup.3 are independently selected from oxygen
(O), C(O)O, and OC(O). In a preferred embodiment, Z.sup.2 and
Z.sup.3 are both oxygen (O). In yet another embodiment, T.sup.1 is
hydrogen (H) or OR.sup.10, wherein R.sup.10 is H or optionally
substituted C.sub.1-C.sub.4 alkyl. In one particular embodiment,
T.sup.1 is H or OH. In still yet another embodiment, subscript m is
1 and R.sup.6, R.sup.2, and R.sup.8 are independently selected from
an optionally substituted C.sub.10-C.sub.24 alkyl,
C.sub.10-C.sub.24 alkenyl, and C.sub.10-C.sub.24 alkynyl. In some
instances, R.sup.6, R.sup.2, and R.sup.8 independently comprise an
optionally substituted C.sub.12-C.sub.24, C.sub.14-C.sub.24, or
C.sub.16-C.sub.20 alkyl group. In other instances, at least one,
two, or all three of R.sup.6, R.sup.2, and R.sup.8 independently
comprises at least 1, 2, 3, 4, 5, or 6 sites of unsaturation or a
substituted alkyl group such as a phytanyl moiety. The unsaturated
side-chains may independently comprise a myristoleyl moiety, a
palmitoleyl moiety, an oleyl moiety, a dodecadienyl moiety, a
tetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienyl
moiety, an icosadienyl moiety, a dodecatrienyl moiety, a
tetradectrienyl moiety, a hexadecatrienyl moiety, an
octadecatrienyl moiety, or an icosatrienyl moiety. In some
instances, the octadecadienyl moiety is a linoleyl moiety. In
particular embodiments, R.sup.6, R.sup.2, and R.sup.8 are all
linoleyl moieties. In other embodiments, R.sup.2 and R.sup.8 are
the same and R.sup.6 is different. In certain instances, R.sup.6 is
an oleyl moiety and R.sup.2 and R.sup.8 are linoleyl moieties. In
certain other instances, R.sup.6 is a linoleyl moiety and R.sup.2
and R.sup.8 are oleyl moieties. One of skill in the art will
appreciate that the compounds of Formula II may comprise a
combination of two or more of the features described in this
paragraph. As a non-limiting example, in certain preferred
embodiments, subscripts f, j, k and m are all 1; subscripts e, g,
and i are all 0; and R.sup.6, R.sup.2, and R.sup.8 are
independently selected C.sub.18 alkyl groups (e.g., R.sup.6,
R.sup.2, and R.sup.8 are all linoleyl moieties). An exemplary
method of making the ester-containing lipids of Formula II
described in this paragraph is illustrated for Compound 16 of
Scheme 4 below.
[0140] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
subscripts c and d are both 0; R.sup.5 is hydrogen (H); subscripts
f, j, and k are all 1; subscripts e, g, and i are all 0; Z.sup.2
and Z.sup.3 are both oxygen (O); T.sup.1 is hydrogen (H); subscript
m is 1; and R.sup.6, R.sup.7, and R.sup.8 are independently
selected from C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups
(e.g., R.sup.6, R.sup.7, and R.sup.8 are all linoleyl moieties or
oleyl moieties; at least two of R.sup.6, R.sup.7, and R.sup.8 are
oleyl moieties and the other is a linoleyl moiety; or at least two
of R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties and the
other is an oleyl moiety).
[0141] In particularly preferred embodiments, the cationic lipid of
Formula II has one of the following structures:
##STR00011##
[0142] In a related aspect, the present invention provides a
cationic lipid of Formula III having the following structure:
##STR00012##
or salts and isomers thereof, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10,
R.sup.11, R.sup.12, Y, Z.sup.1, Z.sup.2, Z.sup.3, T.sup.1, T.sup.2,
T.sup.3, and subscripts a, b, c, d, e, f, g, h, i, j, k, and m are
the same as described above for Formula I.
[0143] In another embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, a is 2, 3, or 4. In yet
another embodiment, subscript c is 0, 1, or 2 and d is 0 or 1. In a
preferred embodiment subscripts c and d are both 0. In yet another
embodiment, R.sup.5 is hydrogen (H). In a further embodiment,
subscripts e, f, and g are 0. In another embodiment, subscripts i,
j and k are 0. In one particular embodiment, R.sup.9 is selected
from hydrogen (H), optionally substituted C.sub.1-C.sub.6 alkyl
(e.g., C.sub.1-C.sub.2 alkyl, C.sub.1-C.sub.3 alkyl,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.3
alkyl, C.sub.2-C.sub.4 alkyl, C.sub.2-C.sub.5 alkyl,
C.sub.2-C.sub.6 alkyl, C.sub.3-C.sub.4 alkyl, C.sub.3-C.sub.5
alkyl, C.sub.3-C.sub.6 alkyl, C.sub.4-C.sub.5 alkyl,
C.sub.4-C.sub.6 alkyl, C.sub.5-C.sub.6 alkyl, or C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl), C.sub.2-C.sub.6
alkenyl (e.g., C.sub.2-C.sub.3 alkenyl, C.sub.2-C.sub.4 alkenyl,
C.sub.2-C.sub.5 alkenyl, C.sub.2-C.sub.6 alkenyl, C.sub.3-C.sub.4
alkenyl, C.sub.3-C.sub.5 alkenyl, C.sub.3-C.sub.6 alkenyl,
C.sub.4-C.sub.5 alkenyl, C.sub.4-C.sub.6 alkenyl, C.sub.5-C.sub.6
alkenyl, or C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6
alkenyl), C.sub.2-C.sub.6 alkynyl (e.g., C.sub.2-C.sub.3 alkynyl,
C.sub.2-C.sub.4 alkynyl, C.sub.2-C.sub.5 alkynyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.4 alkynyl, C.sub.3-C.sub.5 alkynyl,
C.sub.3-C.sub.6 alkynyl, C.sub.4-C.sub.5 alkynyl, C.sub.4-C.sub.6
alkynyl, or C.sub.5-C.sub.6 alkynyl, or C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6 alkynyl), and
##STR00013##
wherein * represents the point of attachment to N. R.sup.4 is as
described above. In some instances, R.sup.9 is methyl. In some
other instances, R.sup.9 is
##STR00014##
subscript b is 0, and R.sup.4 is a linoleyl moiety. In further
instances, R.sup.9 is
##STR00015##
subscript b is 1, 2, or 3, and R.sup.4 is NR.sup.4aR.sup.4b wherein
R.sup.4a and R.sup.4b are independently selected from hydrogen (H),
an optionally substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, and C.sub.2-C.sub.6 alkynyl. In preferred embodiments,
subscript b is 2 and R.sup.4a and R.sup.4b are both methyl. In yet
another embodiment, T.sup.1 is hydrogen (H) or OR.sup.10, wherein
R.sup.10 is H or optionally substituted C.sub.1-C.sub.4 alkyl. In
one particular embodiment, T.sup.1 is H or OH. In still yet another
embodiment, subscript m is 1 and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from an optionally substituted
C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24 alkenyl, and
C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6, R.sup.7, and
R.sup.8 independently comprise an optionally substituted
C.sub.12-C.sub.24, C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl
group. In other instances, at least one, two, or all three of
R.sup.6, R.sup.7, and R.sup.8 independently comprises at least 1,
2, 3, 4, 5, or 6 sites of unsaturation or a substituted alkyl group
such as a phytanyl moiety. The unsaturated side-chains may
independently comprise a myristoleyl moiety, a palmitoleyl moiety,
an oleyl moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, or an
icosatrienyl moiety. In some instances, the octadecadienyl moiety
is a linoleyl moiety. In particular embodiments, R.sup.6, R.sup.7,
and R.sup.8 are all linoleyl moieties. In other embodiments,
R.sup.7 and R.sup.8 are the same and R.sup.6 is different. In
certain instances, R.sup.6 is an oleyl moiety and R.sup.7 and
R.sup.8 are linoleyl moieties. In certain other instances, R.sup.6
is a linoleyl moiety and R.sup.7 and R.sup.8 are oleyl moieties.
One of skill in the art will appreciate that the compounds of
Formula III may comprise a combination of two or more of the
features described in this paragraph. As a non-limiting example, in
certain preferred embodiments, subscript m is 1; subscripts e, f,
g, i, j, and k are all 0; and R.sup.6, R.sup.7, and R.sup.8 are
independently selected C.sub.18 alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties). Exemplary methods
of making the carbamate-containing lipids of Formula III described
in this paragraph are illustrated for Compound 2 of Scheme 1,
Compound 7 of Scheme 2, and Example 7 below.
[0144] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
subscripts c and d are both 0; R.sup.5 is hydrogen (H); subscripts
e, f, g, i, j, k are all 0; T.sup.1 is hydrogen (H) or OH;
subscript m is 1; R.sup.6, R.sup.7, and R.sup.8 are independently
selected from C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups
(e.g., R.sup.6, R.sup.7, and R.sup.8 are all linoleyl moieties or
oleyl moieties; at least two of R.sup.6, R.sup.7, and R.sup.8 are
oleyl moieties and the other is a linoleyl moiety; or at least two
of R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties and the
other is an oleyl moiety); and R.sup.9 is selected from methyl,
##STR00016##
wherein subscript b is 0 and R.sup.4 is a linoleyl moiety, and
##STR00017##
wherein R.sup.4 is NR.sup.4aR.sup.4b, subscript b is 2, and
R.sup.4a and R.sup.4b are both methyl.
[0145] In particularly preferred embodiments, the cationic lipid of
Formula III has one of the following structures:
##STR00018##
[0146] In another embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In yet another embodiment, subscript c is 0, 1, or 2 and
subscript d is 0 or 1. In a preferred embodiment subscripts c and d
are both 1. In yet another embodiment, R.sup.5 is hydrogen (H). In
a further embodiment, subscripts e, f, and g are all 0. In another
embodiment, subscripts i is 1 and j and k are both 0. In a further
embodiment, Z.sup.1 is 0. In one particular embodiment, R.sup.9 is
selected from hydrogen (H), optionally substituted C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, and
##STR00019##
wherein * represents the point of attachment to N. R.sup.4 is as
described above. In some instances, R.sup.9 is methyl. In some
other instances, R.sup.9 is
##STR00020##
subscript b is 0, and R.sup.4 is a linoleyl moiety. In further
instances, R.sup.9 is
##STR00021##
subscript b is 1, 2, or 3, and R.sup.4 is NR.sup.4aR.sup.4b,
wherein R.sup.4a and R.sup.4b are independently selected from
hydrogen (H), an optionally substituted C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl. In preferred
embodiments, subscript b is 2 and R.sup.4a and R.sup.4b are both
methyl. In yet another embodiment, T.sup.1 is hydrogen (H) or
OR.sup.10, wherein R.sup.10 is H or optionally substituted
C.sub.1-C.sub.4 alkyl. In one particular embodiment, T.sup.1 is H
or OH. In still yet another embodiment, subscript m is 1 and
R.sup.6, R.sup.7, and R.sup.8 are independently selected from an
optionally substituted C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24
alkenyl, and C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6,
R.sup.7, and R.sup.8 independently comprise an optionally
substituted C.sub.12-C.sub.24, C.sub.14-C.sub.24, or
C.sub.16-C.sub.20 alkyl group. In other instances, at least one,
two, or all three of R.sup.6, R.sup.7, and R.sup.8 independently
comprises at least 1, 2, 3, 4, 5, or 6 sites of unsaturation or a
substituted alkyl group such as a phytanyl moiety. The unsaturated
side-chains may independently comprise a myristoleyl moiety, a
palmitoleyl moiety, an oleyl moiety, a dodecadienyl moiety, a
tetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienyl
moiety, an icosadienyl moiety, a dodecatrienyl moiety, a
tetradectrienyl moiety, a hexadecatrienyl moiety, an
octadecatrienyl moiety, or an icosatrienyl moiety. In some
instances, the octadecadienyl moiety is a linoleyl moiety. In
particular embodiments, R.sup.6, R.sup.7, and R.sup.8 are all
linoleyl moieties. In other embodiments, R.sup.7 and R.sup.8 are
the same and R.sup.6 is different. In certain instances, R.sup.6 is
an oleyl moiety and R.sup.7 and R.sup.8 are linoleyl moieties. In
certain other instances, R.sup.6 is a linoleyl moiety and R.sup.7
and R.sup.8 are oleyl moieties. One of skill in the art will
appreciate that the compounds of Formula III may comprise a
combination of two or more of the features described in this
paragraph. As a non-limiting example, in certain preferred
embodiments, subscripts i and m are both 1; subscripts e, f, g, j,
and k are all 0, and R.sup.6, R.sup.7, and R.sup.8 are
independently selected C.sub.18 alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties). An exemplary
method of making the carbamate-containing lipids of Formula III is
illustrated for Compound 11 of Scheme 3 below.
[0147] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3. or 4;
subscripts c and d are both 1; Y is --CR.sup.11R.sup.12--. R.sup.11
and R.sup.12 are independently selected from hydrogen, methyl, and
ethyl; R.sup.5 is hydrogen (H); subscript i is 1; subscripts e, f,
g, j, and k are all 0; Z.sup.1 is 0; T.sup.1 is OH; subscript m is
1; R.sup.6, R.sup.7, and R.sup.8 are independently selected from
C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl moieties;
at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl moieties
and the other is a linoleyl moiety; or at least two of R.sup.6,
R.sup.7, and R.sup.8 are linoleyl moieties and the other is an
oleyl moiety); and R.sup.9 is selected from methyl,
##STR00022##
wherein subscript b is 0 and R.sup.4 is a linoleyl moiety, and
##STR00023##
wherein R.sup.4 is NR.sup.4aR.sup.4b, subscript b is 2, and
R.sup.4a and R.sup.4b are both methyl.
[0148] In particularly preferred embodiments, the cationic lipid of
Formula III has the following structure:
##STR00024##
wherein subscript a is 2, 3, or 4; R.sup.11 and R.sup.12 are
independently selected from hydrogen (H) and methyl; and "lin"
refers to a linoleyl moiety.
[0149] In another embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In yet another embodiment, subscript c is 0, 1, or 2 and
subscript d is 0 or 1. In a preferred embodiment subscripts c and d
are both 0. In yet another embodiment, R.sup.5 is hydrogen (H). In
a further embodiment, subscript f is 1 and subscripts e and g are
both 0. In another embodiment, subscript i is 0, subscripts j and k
are both 1, and Z.sup.2 and Z.sup.3 are independently selected from
oxygen (O), C(O)O, and OC(O). In a preferred embodiment, Z.sup.2
and Z.sup.3 are both O. In further embodiments, R.sup.9 is selected
from hydrogen (H), optionally substituted C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, and
##STR00025##
wherein * represents the point of attachment to N. R.sup.4 is as
described above. In some instances, R.sup.9 is methyl. In some
other instances, R.sup.9 is
##STR00026##
subscript b is 0, and R.sup.4 is a linoleyl moiety. In further
instances, R.sup.9 is
##STR00027##
subscript b is 1, 2, or 3, and R.sup.4 is NR.sup.4aR.sup.4b,
wherein R.sup.4a and R.sup.4b are independently selected from
hydrogen (H), an optionally substituted C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl. In preferred
embodiments, subscript b is 2 and R.sup.4a and R.sup.4b are both
methyl. In yet another embodiment, T.sup.1 is hydrogen (H) or
OR.sup.10, wherein R.sup.10 is H or optionally substituted
C.sub.1-C.sub.4 alkyl. In one particular embodiment, T.sup.1 is H
or OH. In still yet another embodiment, subscript m is 1 and
R.sup.6, R.sup.7, and R.sup.8 are independently selected from an
optionally substituted C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24
alkenyl, and C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6,
R.sup.7, and R.sup.8 independently comprise an optionally
substituted C.sub.12-C.sub.24, C.sub.14-C.sub.24, or
C.sub.16-C.sub.20 alkyl group. In other instances, at least one,
two, or all three of R.sup.6, R.sup.7, and R.sup.8 independently
comprises at least 1, 2, 3, 4, 5, or 6 sites of unsaturation or a
substituted alkyl group such as a phytanyl moiety. The unsaturated
side-chains may independently comprise a myristoleyl moiety, a
palmitoleyl moiety, an oleyl moiety, a dodecadienyl moiety, a
tetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienyl
moiety, an icosadienyl moiety, a dodecatrienyl moiety, a
tetradectrienyl moiety, a hexadecatrienyl moiety, an
octadecatrienyl moiety, or an icosatrienyl moiety. In some
instances, the octadecadienyl moiety is a linoleyl moiety. In
particular embodiments, R.sup.6, R.sup.7, and R.sup.8 are all
linoleyl moieties. In other embodiments, R.sup.7 and R.sup.8 are
the same and R.sup.6 is different. In certain instances, R.sup.6 is
an oleyl moiety and R.sup.7 and R.sup.8 are linoleyl moieties. In
certain other instances, R.sup.6 is a linoleyl moiety and R.sup.7
and R.sup.8 are oleyl moieties. One of skill in the art will
appreciate that the compounds of Formula III may comprise a
combination of two or more of the features described in this
paragraph. As a non-limiting example, in certain preferred
embodiments, subscripts f, j, k are all 1; subscripts e, g, and i,
are all 0; and R.sup.6, R.sup.7, and R.sup.8 are independently
selected C.sub.18 alkyl groups (e.g., R.sup.6, R.sup.7, and R.sup.8
are all linoleyl moieties). An exemplary method of making the
carbamate-containing lipids of Formula III described in this
paragraph is illustrated for Compound 15 of Scheme 4 below. In
preferred embodiments, R.sup.1 and R.sup.2 are each independently
methyl or ethyl groups; subscript a is 2, 3, or 4; subscripts c and
d are both 0; R.sup.5 is hydrogen (H); subscripts f, j, and k are
all 1; subscripts e, g, and i are all 0; Z.sup.2 and Z.sup.3 are
both oxygen (O). T.sup.1 is hydrogen (H); subscript m is 1;
R.sup.6, R.sup.7, and R.sup.8 are independently selected from
C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl moieties;
at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl moieties
and the other is a linoleyl moiety; or at least two of R.sup.6,
R.sup.7, and R.sup.8 are linoleyl moieties and the other is an
oleyl moiety); and R.sup.9 is selected from methyl,
##STR00028##
wherein subscript b is 0 and R.sup.4 is a linoleyl moiety, and
##STR00029##
wherein R.sup.4 is NR.sup.4aR.sup.4b, subscript b is 2, and
R.sup.4a and R.sup.4b are both methyl.
[0150] In particularly preferred embodiments, the cationic lipid of
Formula II has the following structure:
##STR00030##
[0151] In a related aspect, the present invention provides a
cationic lipid of Formula IV having the following structure:
##STR00031##
[0152] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
Y, Z.sup.1, Z.sup.2, Z.sup.3, T.sup.1, T.sup.2, T.sup.3, and
subscripts a, b, c, d, e, f, g, h, i, j, k, and m are the same as
described above for Formula I.
[0153] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In yet another embodiment, subscript c is 0, 1, or 2 and
subscript d is 0. In one particular embodiment, c and d are both 0.
In still yet another embodiment, R.sup.5 is hydrogen (H). In a
further embodiment, subscripts e, f, and g are all 0. In another
embodiment, subscripts i, j, and k are all 0. In yet another
embodiment, T.sup.1 is hydrogen (H) or OR.sup.10, wherein R.sup.10
is H or optionally substituted C.sub.1-C.sub.4 alkyl. In one
particular embodiment, T.sup.1 is H or OH. In still yet another
embodiment, subscript m is 1 and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from an optionally substituted
C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24 alkenyl, and
C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6, R.sup.7, and
R.sup.8 independently comprise an optionally substituted
C.sub.12-C.sub.24, C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl
group. In other instances, at least one, two, or all three of
R.sup.6, R.sup.7, and R.sup.8 independently comprises at least 1,
2, 3, 4, 5, or 6 sites of unsaturation or a substituted alkyl group
such as a phytanyl moiety. The unsaturated side-chains may
independently comprise a myristoleyl moiety, a palmitoleyl moiety,
an oleyl moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, or an
icosatrienyl moiety. In some instances, the octadecadienyl moiety
is a linoleyl moiety. In particular embodiments, R.sup.6, R.sup.7,
and R.sup.8 are all linoleyl moieties. In other embodiments,
R.sup.7 and R.sup.8 are the same and R.sup.6 is different. In
certain instances, R.sup.6 is an oleyl moiety and R.sup.7 and
R.sup.8 are linoleyl moieties. In certain other instances, R.sup.6
is a linoleyl moiety and R.sup.7 and R.sup.8 are oleyl moieties.
One of skill in the art will appreciate that the compounds of
Formula IV may comprise a combination of two or more of the
features described in this paragraph. As a non-limiting example, in
certain preferred embodiments, subscripts e, f, g, i, j, and k are
all 0, subscript m is 1, and R.sup.6, R.sup.7, and R.sup.8 are
independently selected C.sub.18 alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties). Exemplary methods
of making the ether-containing lipids of Formula IV are illustrated
for compound 3 of Scheme 1 and for compound 6 of Scheme 2
below.
[0154] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
subscripts c and d are both 0; R.sup.5 is hydrogen (H); subscripts
e, f, g, i, j, and k are all 0; T.sup.1 is H or OH; subscript m is
1; and R.sup.6, R.sup.7, and R.sup.8 are independently selected
from C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups (e.g.,
R.sup.6, R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl
moieties; at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl
moieties and the other is a linoleyl moiety; or at least two of
R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties and the other
is an oleyl moiety).
[0155] In particularly preferred embodiments, the cationic lipid of
Formula IV has the structure of compound 3 of Scheme 1 and compound
6 of Scheme 2.
[0156] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In yet another embodiment, subscript c is 0, 1, or 2 and
subscript d is 0. In one particular embodiment, subscripts c and d
are both 1. In still yet another embodiment, R.sup.5 is hydrogen
(H). In a further embodiment, subscripts e, f, and g are all 0. In
another embodiment, subscript i is 1 and subscripts j and k are
both 0. In another embodiment, Z.sup.1 is oxygen (O). In yet
another embodiment, T.sup.1 is hydrogen (H) or OR.sup.10, wherein
R.sup.10 is H or optionally substituted C.sub.1-C.sub.4 alkyl. In
one particular embodiment, T.sup.1 is H or OH. In still yet another
embodiment, subscript m is 1 and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from an optionally substituted
C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24 alkenyl, and
C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6, R.sup.7, and
R.sup.8 independently comprise an optionally substituted
C.sub.12-C.sub.24, C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl
group. In other instances, at least one, two, or all three of
R.sup.6, R.sup.7, and R.sup.8 independently comprises at least 1,
2, 3, 4, 5, or 6 sites of unsaturation or a substituted alkyl group
such as a phytanyl moiety. The unsaturated side-chains may
independently comprise a myristoleyl moiety, a palmitoleyl moiety,
an oleyl moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, or an
icosatrienyl moiety. In some instances, the octadecadienyl moiety
is a linoleyl moiety. In particular embodiments, R.sup.6, R.sup.7,
and R.sup.8 are all linoleyl moieties. In other embodiments,
R.sup.7 and R.sup.8 are the same and R.sup.6 is different. In
certain instances, R.sup.6 is an oleyl moiety and R.sup.7 and
R.sup.8 are linoleyl moieties. In certain other instances, R.sup.6
is a linoleyl moiety and R.sup.7 and R.sup.8 are oleyl moieties.
One of skill in the art will appreciate that the compounds of
Formula IV may comprise a combination of two or more of the
features described in this paragraph. As a non-limiting example, in
certain preferred embodiments, subscripts e, f, g, i, j, and k are
all 0, subscript m is 1, and R.sup.6, R.sup.7, and R.sup.8 are
independently selected C.sub.18 alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties). Exemplary methods
of making the ether-containing lipids of Formula IV are illustrated
for compound 10 in Scheme 3 below.
[0157] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
subscripts c and d are both 1; Y is --CR.sup.11R.sup.12; R.sup.11
and R.sup.12 are independently selected from hydrogen (H), methyl,
or ethyl. R.sup.5 is hydrogen (H); subscript i is 1 and subscripts
e, f, g, j, and k are all 0; T.sup.1 is H or OH; subscript m is 1;
and R.sup.6, R.sup.7, and R.sup.8 are independently selected from
C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl moieties;
at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl moieties
and the other is a linoleyl moiety; or at least two of R.sup.6,
R.sup.7, and R.sup.8 are linoleyl moieties and the other is an
oleyl moiety).
[0158] In particularly preferred embodiments, the cationic lipid of
Formula IV has the structure of compound 10 in Scheme 3.
[0159] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In yet another embodiment, subscript c is 0, 1, or 2 and
subscript d is 0. In one particular embodiment, subscripts c and d
are both 1. In still yet another embodiment, R.sup.5 is hydrogen
(H). In a further embodiment, subscripts e, f, and g are all 0. In
another embodiment, subscript i is 0 and subscripts j and k are
both 1. Z.sup.2 and Z.sup.3 are O. In yet another embodiment,
T.sup.1 is hydrogen (H) or OR.sup.10, wherein R.sup.10 is H or
optionally substituted C.sub.1-C.sub.4 alkyl. In one particular
embodiment, T.sup.1 is H or OH. In still yet another embodiment,
subscript m is 1 and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from an optionally substituted
C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24 alkenyl, and
C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6, R.sup.7, and
R.sup.8 independently comprise an optionally substituted
C.sub.12-C.sub.24, C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl
group. In other instances, at least one, two, or all three of
R.sup.6, R.sup.7, and R.sup.8 independently comprises at least 1,
2, 3, 4, 5, or 6 sites of unsaturation or a substituted alkyl group
such as a phytanyl moiety. The unsaturated side-chains may
independently comprise a myristoleyl moiety, a palmitoleyl moiety,
an oleyl moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, or an
icosatrienyl moiety. In some instances, the octadecadienyl moiety
is a linoleyl moiety. In particular embodiments, R.sup.6, R.sup.7,
and R.sup.8 are all linoleyl moieties. In other embodiments,
R.sup.7 and R.sup.8 are the same and R.sup.6 is different. In
certain instances, R.sup.6 is an oleyl moiety and R.sup.7 and
R.sup.8 are linoleyl moieties. In certain other instances, R.sup.6
is a linoleyl moiety and R.sup.7 and R.sup.8 are oleyl moieties.
One of skill in the art will appreciate that the compounds of
Formula IV may comprise a combination of two or more of the
features described in this paragraph. As a non-limiting example, in
certain preferred embodiments, e, f, g, i, j, and k are all 0, m is
1, and R.sup.6, R.sup.7, and R.sup.8 are independently selected
C.sub.18 alkyl groups (e.g., R.sup.6, R.sup.7, and R.sup.8 are all
linoleyl moieties). Exemplary methods of making the
ether-containing lipids of Formula IV are illustrated for compound
14 in Scheme 4 below.
[0160] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
subscripts c and d are both 0; R.sup.5 is hydrogen (H); subscripts
e, f, and g are all 0; subscript i is 0 and subscripts j and k are
both 1; Z.sup.2 and Z.sup.3 are 0; T.sup.1 is H or OH; subscript m
is 1; and R.sup.6, R.sup.7, and R.sup.8 are independently selected
from C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups (e.g.,
R.sup.6, R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl
moieties; at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl
moieties and the other is a linoleyl moiety; or at least two of
R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties and the other
is an oleyl moiety).
[0161] In particularly preferred embodiments, the cationic lipid of
Formula IV has the structure of compound 14 in Scheme 4.
[0162] In a related aspect, the present invention provides a
cationic lipid of Formula V having the following structure:
##STR00032##
[0163] or salts and isomers thereof,
[0164] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
Y, Z.sup.1, Z.sup.2, Z.sup.3, T.sup.1, T.sup.2, T.sup.3, and
subscripts a, b, c, d, e, f, g, h, i, j, k, and m are the same as
described above for Formula I.
[0165] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In a further embodiment, subscripts e, f, and g are all 0. In
another embodiment, subscripts i, j, and k are all 0. In yet
another embodiment, T.sup.1 is hydrogen (H) or OR.sup.10, wherein
R.sup.10 is H or optionally substituted C.sub.1-C.sub.4 alkyl. In
one particular embodiment, T.sup.1 is H or OH. In still yet another
embodiment, subscript m is 1 and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from an optionally substituted
C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24 alkenyl, and
C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6, R.sup.7, and
R.sup.8 independently comprise an optionally substituted
C.sub.12-C.sub.24, C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl
group. In other instances, at least one, two, or all three of
R.sup.6, R.sup.7, and R.sup.8 independently comprises at least 1,
2, 3, 4, 5, or 6 sites of unsaturation or a substituted alkyl group
such as a phytanyl moiety. The unsaturated side-chains may
independently comprise a myristoleyl moiety, a palmitoleyl moiety,
an oleyl moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, or an
icosatrienyl moiety. In some instances, the octadecadienyl moiety
is a linoleyl moiety. In particular embodiments, R.sup.6, R.sup.7,
and R.sup.8 are all linoleyl moieties. In other embodiments,
R.sup.7 and R.sup.8 are the same and R.sup.6 is different. In
certain instances, R.sup.6 is an oleyl moiety and R.sup.7 and
R.sup.8 are linoleyl moieties. In certain other instances, R.sup.6
is a linoleyl moiety and R.sup.7 and R.sup.8 are oleyl moieties.
One of skill in the art will appreciate that the compounds of
Formula V may comprise a combination of two or more of the features
described in this paragraph. As a non-limiting example, in certain
preferred embodiments, subscripts e, f, g, i, j, and k are all 0,
subscript m is 1, and R.sup.6, R.sup.7, and R.sup.8 are
independently selected C.sub.18 alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties). Exemplary methods
of making the dioxylanyl-containing lipids of Formula V are
illustrated for compound 1 of Scheme 1 and for compound 5 of Scheme
2 below.
[0166] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4; e,
f, and g are all 0; subscripts i, j, and k are all 0; T.sup.1 is H
or OH; subscript m is 1; and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from C.sub.16-C.sub.20 (e.g., C.sub.18)
alkyl groups (e.g., R.sup.6, R.sup.7, and R.sup.8 are all linoleyl
moieties or oleyl moieties; at least two of R.sup.6, R.sup.7, and
R.sup.8 are oleyl moieties and the other is a linoleyl moiety; or
at least two of R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties
and the other is an oleyl moiety).
[0167] In particularly preferred embodiments, the cationic lipid of
Formula V has the structure of compound 1 of Scheme 1 and compound
5 of Scheme 2.
[0168] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In a preferred embodiment, subscript a is 2, 3, or
4. In a further embodiment, subscript f is 1 and subscripts e and g
are both 0. In another embodiment, subscript j is 1 and subscripts
i and k are both 0. In another embodiment Z.sup.2 is 0. In yet
another embodiment, T.sup.1 is hydrogen (H) or OR.sup.10, wherein
R.sup.10 is H or optionally substituted C.sub.1-C.sub.4 alkyl. In
one particular embodiment, T.sup.1 is H or OH. In still yet another
embodiment, subscript m is 1 and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from an optionally substituted
C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24 alkenyl, and
C.sub.10-C.sub.24 alkynyl. In some instances, R.sup.6, R.sup.7, and
R.sup.8 independently comprise an optionally substituted
C.sub.12-C.sub.24, C.sub.14-C.sub.24, or C.sub.16-C.sub.20 alkyl
group. In other instances, at least one, two, or all three of
R.sup.6, R.sup.7, and R.sup.8 independently comprises at least 1,
2, 3, 4, 5, or 6 sites of unsaturation or a substituted alkyl group
such as a phytanyl moiety. The unsaturated side-chains may
independently comprise a myristoleyl moiety, a palmitoleyl moiety,
an oleyl moiety, a dodecadienyl moiety, a tetradecadienyl moiety, a
hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl
moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a
hexadecatrienyl moiety, an octadecatrienyl moiety, or an
icosatrienyl moiety. In some instances, the octadecadienyl moiety
is a linoleyl moiety. In particular embodiments, R.sup.6, R.sup.7,
and R.sup.8 are all linoleyl moieties. In other embodiments,
R.sup.7 and R.sup.8 are the same and R.sup.6 is different. In
certain instances, R.sup.6 is an oleyl moiety and R.sup.7 and
R.sup.8 are linoleyl moieties. In certain other instances, R.sup.6
is a linoleyl moiety and R.sup.7 and R.sup.8 are oleyl moieties.
One of skill in the art will appreciate that the compounds of
Formula V may comprise a combination of two or more of the features
described in this paragraph. As a non-limiting example, in certain
preferred embodiments, subscripts f and j are both 1; subscripts e,
g, i, and k are all 0; subscript m is 1, and R.sup.6, R.sup.7, and
R.sup.8 are independently selected from C.sub.18 alkyl groups
(e.g., R.sup.6, R.sup.7, and R.sup.8 are all linoleyl moieties).
Exemplary methods of making the dioxylanyl-containing lipids of
Formula V are illustrated for compound 13 in Scheme 4 below.
[0169] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
subscripts f and j are both 1; Z.sup.2 is oxygen (O); subscripts e,
g, and k are all 0; T.sup.1 is H or OH; subscript m is 1; and
R.sup.6, R.sup.7, and R.sup.8 are independently selected from
C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl moieties;
at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl moieties
and the other is a linoleyl moiety; or at least two of R.sup.6,
R.sup.7, and R.sup.8 are linoleyl moieties and the other is an
oleyl moiety).
[0170] In particularly preferred embodiments, the cationic lipid of
Formula V has the structure of compound 13 in Scheme 4.
[0171] In particularly preferred embodiments, the cationic lipid of
Formula VI has one of the following structures:
##STR00033##
[0172] or salts and isomers thereof,
[0173] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
Y, Z.sup.1, Z.sup.2, Z.sup.3, T.sup.1, T.sup.2, T.sup.3, and
subscripts a, b, c, d, e, f, g, h, i, j, k, and m are the same as
described above for Formula I.
[0174] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In another embodiment, subscript d is 0, 1, 2, or 3.
In a preferred embodiment, subscript a is 2, 3, or 4. In another
preferred embodiment, subscript d is 0. In still yet another
embodiment, R.sup.5 is hydrogen (H). In a further embodiment,
subscripts e, f, and g are all 0. In another embodiment, subscript
g is 1 and subscripts e and f are both 0. In another embodiment,
subscripts i, j and k are all 0. In yet another embodiment, T.sup.1
is hydrogen (H) or OR.sup.10, wherein R.sup.10 is H or optionally
substituted C.sub.1-C.sub.4 alkyl. In one particular embodiment,
T.sup.1 is H or OH. In still yet another embodiment, subscript m is
1 and R.sup.6, R.sup.7, and R.sup.8 are independently selected from
an optionally substituted C.sub.10-C.sub.24 alkyl,
C.sub.10-C.sub.24 alkenyl, and C.sub.10-C.sub.24 alkynyl. In some
instances, R.sup.6, R.sup.7, and R.sup.8 independently comprise an
optionally substituted C.sub.12-C.sub.24, C.sub.14-C.sub.24, or
C.sub.16-C.sub.20 alkyl group. In other instances, at least one,
two, or all three of R.sup.6, R.sup.7, and R.sup.8 independently
comprises at least 1, 2, 3, 4, 5, or 6 sites of unsaturation or a
substituted alkyl group such as a phytanyl moiety. The unsaturated
side-chains may independently comprise a myristoleyl moiety, a
palmitoleyl moiety, an oleyl moiety, a dodecadienyl moiety, a
tetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienyl
moiety, an icosadienyl moiety, a dodecatrienyl moiety, a
tetradectrienyl moiety, a hexadecatrienyl moiety, an
octadecatrienyl moiety, or an icosatrienyl moiety. In some
instances, the octadecadienyl moiety is a linoleyl moiety. In
particular embodiments, R.sup.6, R.sup.7, and R.sup.8 are all
linoleyl moieties. In other embodiments, R.sup.7 and R.sup.8 are
the same and R.sup.6 is different. In certain instances, R.sup.6 is
an oleyl moiety and R.sup.7 and R.sup.8 are linoleyl moieties. In
certain other instances, R.sup.6 is a linoleyl moiety and R.sup.7
and R.sup.8 are oleyl moieties. In some other instances, R.sup.6 is
an C.sub.19 alkenyl optionally substituted with at least one
substituent selected from methyl, ethyl, or hydroxyl. One of skill
in the art will appreciate that the compounds of Formula VI may
comprise a combination of two or more of the features described in
this paragraph. As a non-limiting example, in certain preferred
embodiments, subscript i is 1 and subscripts e, f, g, j, and k are
all 0, Z.sup.1 is oxygen (O), subscript m is 1, and R.sup.6,
R.sup.7, and R.sup.8 are independently selected optionally
substituted C.sub.18 alkyl groups (e.g., R.sup.6, R.sup.7, and
R.sup.8 are all linoleyl moieties). Exemplary methods of making the
lipids of Formula VI are illustrated for compound 17 of Scheme
5.
[0175] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
R.sup.5 is hydrogen (H); subscript g is 1; subscripts e, f, i, j,
and k are all 0; T.sup.1 is H or OH; subscript m is 1; and R.sup.6,
R.sup.7, and R.sup.8 are independently selected from
C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups (e.g., R.sup.6,
R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl moieties;
at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl moieties
and the other is a linoleyl moiety; or at least two of R.sup.6,
R.sup.7, and R.sup.8 are linoleyl moieties and the other is an
oleyl moiety). In some instances, R.sup.6 is optionally substituted
with groups selected from hydroxyl and C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkenyl, or C.sub.1-C.sub.6 alkynyl.
[0176] In another preferred embodiment, R.sup.1 and R.sup.2 are
each independently methyl or ethyl groups; subscript a is 2, 3, or
4; R.sup.5 is hydrogen (H); subscripts e and g are both 1;
subscripts f, i, j, and k are all 0; R.sup.13 is selected from
methyl or ethyl; R.sup.14 is selected from hydroxyl; T.sup.1 is H
or OH; subscript m is 1; and R.sup.6, R.sup.7, and R.sup.8 are
independently selected from C.sub.16-C.sub.20 (e.g., C.sub.18)
alkyl groups (e.g., R.sup.6, R.sup.7, and R.sup.8 are all linoleyl
moieties or oleyl moieties; at least two of R.sup.6, R.sup.7, and
R.sup.8 are oleyl moieties and the other is a linoleyl moiety; or
at least two of R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties
and the other is an oleyl moiety). In some instances, R.sup.6 is
optionally substituted with groups selected from hydroxyl and
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl, or C.sub.1-C.sub.6
alkynyl.
[0177] In particularly preferred embodiments, the cationic lipid of
Formula VI has one of the structures in compound 17 of Scheme
5.
[0178] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In another embodiment, subscript d is 0, 1, 2, or 3.
In a preferred embodiment, subscript a is 2, 3, or 4. In another
preferred embodiment, subscript d is 1 and Y is
--CR.sup.11R.sup.12-- and R.sup.11 and R.sup.12 are independently
selected from hydrogen (H), C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkenyl, C.sub.1-C.sub.6 alkynyl. In still another preferred
embodiment, R.sup.11 and R.sup.12 are hydrogen (H) or methyl. In
still yet another embodiment, R.sup.5 is hydrogen (H). In a further
embodiment, subscripts e, f, and g are all 0. In another
embodiment, subscript i is 1 and subscripts j and k are both 0.
Z.sup.1 is oxygen (O). In yet another embodiment, T.sup.1 is
hydrogen (H) or OR.sup.10, wherein R.sup.10 is H or optionally
substituted C.sub.1-C.sub.4 alkyl. In one particular embodiment,
T.sup.1 is H or OH. In still yet another embodiment, subscript m is
1 and R.sup.6, R.sup.7, and R.sup.8 are independently selected from
an optionally substituted C.sub.10-C.sub.24 alkyl,
C.sub.10-C.sub.24 alkenyl, and C.sub.10-C.sub.24 alkynyl. In some
instances, R.sup.6, R.sup.7, and R.sup.8 independently comprise an
optionally substituted C.sub.12-C.sub.24, C.sub.14-C.sub.24, or
C.sub.16-C.sub.20 alkyl group. In other instances, at least one,
two, or all three of R.sup.6, R.sup.7, and R.sup.8 independently
comprises at least 1, 2, 3, 4, 5, or 6 sites of unsaturation or a
substituted alkyl group such as a phytanyl moiety. The unsaturated
side-chains may independently comprise a myristoleyl moiety, a
palmitoleyl moiety, an oleyl moiety, a dodecadienyl moiety, a
tetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienyl
moiety, an icosadienyl moiety, a dodecatrienyl moiety, a
tetradectrienyl moiety, a hexadecatrienyl moiety, an
octadecatrienyl moiety, or an icosatrienyl moiety. In some
instances, the octadecadienyl moiety is a linoleyl moiety. In
particular embodiments, R.sup.6, R.sup.7, and R.sup.8 are all
linoleyl moieties. In other embodiments, R.sup.7 and R.sup.8 are
the same and R.sup.6 is different. In certain instances, R.sup.6 is
an oleyl moiety and R.sup.7 and R.sup.8 are linoleyl moieties. In
certain other instances, R.sup.6 is a linoleyl moiety and R.sup.7
and R.sup.8 are oleyl moieties. One of skill in the art will
appreciate that the compounds of Formula VI may comprise a
combination of two or more of the features described in this
paragraph. As a non-limiting example, in certain preferred
embodiments, subscript i is 1 and subscripts e, f, g, j, and k are
all 0, Z.sup.1 is oxygen (O), subscript m is 1, and R.sup.6,
R.sup.7, and R.sup.8 are independently selected optionally
substituted C.sub.18 alkyl groups (e.g., R.sup.6, R.sup.7, and
R.sup.8 are all linoleyl moieties). Exemplary methods of making the
lipids of Formula VI are illustrated for compound 9 of Scheme
3.
[0179] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
R.sup.5 is hydrogen (H); subscript d is 1; Y is
--CR.sup.11R.sup.12--; R.sup.11 and R.sup.12 are hydrogen (H),
methyl, or ethyl; subscript i is 1 and subscripts e, f, g, j, and k
are all 0; Z.sup.1 is oxygen (O); T.sup.1 is H or OH; subscript m
is 1; and R.sup.6, R.sup.7, and R.sup.8 are independently selected
from C.sub.16-C.sub.20 (e.g., C.sub.18) alkyl groups (e.g.,
R.sup.6, R.sup.7, and R.sup.8 are all linoleyl moieties or oleyl
moieties; at least two of R.sup.6, R.sup.7, and R.sup.8 are oleyl
moieties and the other is a linoleyl moiety; or at least two of
R.sup.6, R.sup.7, and R.sup.8 are linoleyl moieties and the other
is an oleyl moiety). In some instances, R.sup.6 is optionally
substituted with groups selected from hydroxyl and C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkenyl, or C.sub.1-C.sub.6 alkynyl.
[0180] In particularly preferred embodiments, the cationic lipid of
Formula VI has one of the structures in compound 9 in Scheme 3.
[0181] In one embodiment, R.sup.1 and R.sup.2 are each
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl (e.g.,
methyl, ethyl, propyl, butyl). In particular embodiments, R.sup.1
and R.sup.2 are both methyl groups (i.e., C.sub.1 alkyls). In
certain instances, R.sup.3 is absent when the pH is above the
pK.sub.a of the cationic lipid and R.sup.3 is hydrogen (H) when the
pH is below the pK.sub.a of the cationic lipid such that the amino
head group is protonated. In another embodiment, subscript a is 2,
3, 4, 5, or 6. In another embodiment, subscript d is 0, 1, 2, or 3.
In a preferred embodiment, subscript a is 2, 3, or 4. In another
preferred embodiment, subscript d is 0. In still yet another
embodiment, R.sup.5 is hydrogen (H). In a further embodiment,
subscripts e, f, and g are all 0. In another embodiment, subscripts
i, j and k are all 0. In yet another embodiment, T.sup.1 is
hydrogen (H) or OR.sup.10, wherein R.sup.10 is H or optionally
substituted C.sub.1-C.sub.4 alkyl. In one particular embodiment,
T.sup.1 is H or OH. In still yet another embodiment, subscript m is
1 and R.sup.6, R.sup.7, and R.sup.8 are independently selected from
an optionally substituted C.sub.10-C.sub.24 alkyl,
C.sub.10-C.sub.24 alkenyl, and C.sub.10-C.sub.24 alkynyl. In some
instances, R.sup.6, R.sup.7, and R.sup.8 independently comprise an
optionally substituted C.sub.12-C.sub.24, C.sub.14-C.sub.24, or
C.sub.16-C.sub.20 alkyl group. In other instances, at least one,
two, or all three of R.sup.6, R.sup.7, and R.sup.8 independently
comprises at least 1, 2, 3, 4, 5, or 6 sites of unsaturation or a
substituted alkyl group such as a phytanyl moiety. The unsaturated
side-chains may independently comprise a myristoleyl moiety, a
palmitoleyl moiety, an oleyl moiety, a dodecadienyl moiety, a
tetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienyl
moiety, an icosadienyl moiety, a dodecatrienyl moiety, a
tetradectrienyl moiety, a hexadecatrienyl moiety, an
octadecatrienyl moiety, or an icosatrienyl moiety. In some
instances, the octadecadienyl moiety is a linoleyl moiety. In
particular embodiments, R.sup.6, R.sup.7, and R.sup.8 are all
linoleyl moieties. In other embodiments, R.sup.7 and R.sup.8 are
the same and R.sup.6 is different. In certain instances, R.sup.6 is
an oleyl moiety and R.sup.7 and R.sup.8 are linoleyl moieties. In
certain other instances, R.sup.6 is a linoleyl moiety and R.sup.7
and R.sup.8 are oleyl moieties. One of skill in the art will
appreciate that the compounds of Formula VI may comprise a
combination of two or more of the features described in this
paragraph. As a non-limiting example, in certain preferred
embodiments, subscript i is 1 and subscripts e, f, g, j, and k are
all 0, Z.sup.1 is oxygen (O), subscript m is 1, and R.sup.6,
R.sup.7, and R.sup.8 are independently selected optionally
substituted C.sub.18 alkyl groups (e.g., R.sup.6, R.sup.7, and
R.sup.8 are all linoleyl moieties). Exemplary methods of making the
lipids of Formula VI are illustrated for compound 18 in Scheme
6.
[0182] In preferred embodiments, R.sup.1 and R.sup.2 are each
independently methyl or ethyl groups; subscript a is 2, 3, or 4;
R.sup.5 is a linoleyl moiety; subscripts j and k are both 1 and
subscripts e, f, g, and i, are all 0; Z.sup.1 and Z.sup.2 are
oxygen (O); T.sup.1 is H or OH; subscript m is 0; and R.sup.6 and
R.sup.7 are independently selected from C.sub.16-C.sub.20 (e.g.,
C.sub.18) alkyl groups (e.g., R.sup.6 and R.sup.7 are linoleyl
moieties or oleyl moieties;
[0183] In particularly preferred embodiments, the cationic lipid of
Formula VI has one of the structures in compound 18 in Scheme
6.
[0184] B. Methods of Making Trialkyl Cationic Lipids
[0185] In the below schemes, the term "Lin" refers to linoleyl.
[0186] Non-limiting exemplified synthetic preparations of the
trialkyl cationic lipids of the present invention are illustrated
in Scheme 1 through Scheme 6 below:
[0187] Scheme 1 demonstrates a synthetic procedure to prepare
tri-linoleyl cationic lipids with varying linking groups, including
a dioxylanyl linker, a carbamate linker, an ether linker, and an
ester linker. In part (i), the reagents used are MsCl, TEA, and
CH.sub.2Cl.sub.2. In part (ii), the reagents used are NaCN and DMF.
In part (iii), the reagents used are DIBAL and hexane. In part
(iv), the reagents used are LinMgBr and THF. In part (v), the
reagents used are LinMgBr and THF then H.sub.2O. In part (vi), the
reagents used are dimethylamino-diol, PPTS, and toluene. In part
(vii), the reagents used are dimethylamino-carboxylic acid, EDC,
DIPEA, and CH.sub.2Cl.sub.2. In part (viii), the reagents used are
first, MsCl, TEA, and CH.sub.2Cl.sub.2, and then second,
Dimethylamino-alcohol, NaH, benzene. In part (ix), the reagents
used are first, Phosgene and CH.sub.2Cl.sub.2, and then,
Dimethylamino-amine and CH.sub.2Cl.sub.2.
##STR00034##
[0188] Scheme 2 demonstrates a synthetic procedure to prepare
hydroxyl-substituted tri-linoleyl cationic lipids with varying
linking groups, including a dioxylanyl linker, a carbamate linker,
an ether linker, and an ester linker.
##STR00035##
[0189] Scheme 3 demonstrates a synthetic procedure to prepare
hydroxyl-substituted tri-linoleyl cationic lipids, wherein one
linoleyl moiety is bonded to the rest of the molecule through an
ether linkage, with varying linking groups, including an alkyl
linker, a carbamate linker, an ether linker, and an ester
linker.
##STR00036##
[0190] Scheme 4 demonstrates a synthetic procedure to prepare
hydroxyl-substituted tri-linoleyl cationic lipids, wherein two
linoleyl moieties are bonded to the rest of the molecule through
ether linkages, with varying linking groups, including a dioxylanyl
linker, a carbamate linker, an ether linker, and an ester
linker.
##STR00037##
[0191] Scheme 5 demonstrates a synthetic procedure to prepare
hydroxyl-substituted tri-linoleyl cationic lipids, with an alkyl
linker group.
##STR00038##
[0192] Scheme 6 demonstrates a synthetic procedure to prepare
hydroxyl-substituted tri-linoleyl cationic lipids, wherein two
linoleyl moieties are bonded to the rest of the molecule through
ether linkages, with an alkyl linker moiety.
##STR00039##
[0193] Description of compounds of the present invention are
limited by principles of chemical bonding known to those skilled in
the art. Accordingly, where a group may be substituted by one or
more of a number of substituents, such substitutions are selected
so as to comply with principles of chemical bonding and to give
compounds which are not inherently unstable and/or would be known
to one of ordinary skill in the art as likely to be unstable under
ambient conditions, such as aqueous, neutral, or physiological
conditions.
[0194] The compounds of the invention can be synthesized by a
variety of methods known to one of skill in the art (see
Comprehensive Organic Transformations Richard C. Larock, 1989) or
by an appropriate combination of generally well known synthetic
methods. Techniques useful in synthesizing the compounds of the
invention are both readily apparent and accessible to those of
skill in the relevant art. The discussion above is offered to
illustrate certain of the diverse methods available for use in
assembling the compounds of the invention. However, the discussion
is not intended to define the scope of reactions or reaction
sequences that are useful in preparing the compounds of the present
invention. One of skill in the art will appreciate that other
methods of making the compounds are useful in the present
invention. Although some compounds in Scheme 1 through Scheme 6 may
indicate relative stereochemistry, the compounds may exist as a
racemic mixture or as either enantiomer.
[0195] Linkers useful in the present invention includes those
possessing one or more different reactive functional groups that
allow for covalent attachment of moieties. Suitable linkers
include, without limitation, those described herein as well as
those available from Pierce Biotechnology, Inc. (Rockford, Ill.).
Additional linkers can be found in Bioconjugate Techniques, Greg T.
Hermanson, Academic Press, 2d ed., 2008 (incorporated by reference
in its entirety herein).
IV. Active Agents
[0196] Active agents (e.g., therapeutic agents) include any
molecule or compound capable of exerting a desired effect on a
cell, tissue, organ, or subject. Such effects may be, e.g.,
biological, physiological, and/or cosmetic. Active agents may be
any type of molecule or compound including, but not limited to,
nucleic acids, peptides, polypeptides, small molecules, and
mixtures thereof. Non-limiting examples of nucleic acids include
interfering RNA molecules (e.g., siRNA, Dicer-substrate dsRNA,
shRNA, aiRNA, and/or miRNA), antisense oligonucleotides, plasmids,
ribozymes, immunostimulatory oligonucleotides, and mixtures
thereof. Examples of peptides or polypeptides include, without
limitation, antibodies (e.g., polyclonal antibodies, monoclonal
antibodies, antibody fragments; humanized antibodies, recombinant
antibodies, recombinant human antibodies, and/or Primatized.TM.
antibodies), cytokines, growth factors, apoptotic factors,
differentiation-inducing factors, cell-surface receptors and their
ligands, hormones, and mixtures thereof. Examples of small
molecules include, but are not limited to, small organic molecules
or compounds such as any conventional agent or drug known to those
of skill in the art.
[0197] In some embodiments, the active agent is a therapeutic
agent, or a salt or derivative thereof. Therapeutic agent
derivatives may be therapeutically active themselves or they may be
prodrugs, which become active upon further modification. Thus, in
one embodiment, a therapeutic agent derivative retains some or all
of the therapeutic activity as compared to the unmodified agent,
while in another embodiment, a therapeutic agent derivative is a
prodrug that lacks therapeutic activity, but becomes active upon
further modification.
[0198] A. Nucleic Acids
[0199] In certain embodiments, lipid particles of the present
invention are associated with a nucleic acid, resulting in a
nucleic acid-lipid particle (e.g., SNALP). In some embodiments, the
nucleic acid is fully encapsulated in the lipid particle. As used
herein, the term "nucleic acid" includes any oligonucleotide or
polynucleotide, with fragments containing up to 60 nucleotides
generally termed oligonucleotides, and longer fragments termed
polynucleotides. In particular embodiments, oligonucleotides of the
invention are from about 15 to about 60 nucleotides in length.
Nucleic acid may be administered alone in the lipid particles of
the invention, or in combination (e.g., co-administered) with lipid
particles of the invention comprising peptides, polypeptides, or
small molecules such as conventional drugs.
[0200] In the context of this invention, the terms "polynucleotide"
and "oligonucleotide" refer to a polymer or oligomer of nucleotide
or nucleoside monomers consisting of naturally-occurring bases,
sugars and intersugar (backbone) linkages. The terms
"polynucleotide" and "oligonucleotide" also include polymers or
oligomers comprising non-naturally occurring monomers, or portions
thereof, which function similarly. Such modified or substituted
oligonucleotides are often preferred over native forms because of
properties such as, for example, enhanced cellular uptake, reduced
immunogenicity, and increased stability in the presence of
nucleases.
[0201] Oligonucleotides are generally classified as
deoxyribooligonucleotides or ribooligonucleotides. A
deoxyribooligonucleotide consists of a 5-carbon sugar called
deoxyribose joined covalently to phosphate at the 5' and 3' carbons
of this sugar to form an alternating, unbranched polymer. A
ribooligonucleotide consists of a similar repeating structure where
the 5-carbon sugar is ribose.
[0202] The nucleic acid that is present in a nucleic acid-lipid
particle according to this invention includes any form of nucleic
acid that is known. The nucleic acids used herein can be
single-stranded DNA or RNA, or double-stranded DNA or RNA, or
DNA-RNA hybrids. Examples of double-stranded DNA are described
herein and include, e.g., structural genes, genes including control
and termination regions, and self-replicating systems such as viral
or plasmid DNA. Examples of double-stranded RNA are described
herein and include, e.g., siRNA and other RNAi agents such as
Dicer-substrate dsRNA, shRNA, aiRNA, and pre-miRNA. Single-stranded
nucleic acids include, e.g., antisense oligonucleotides, ribozymes,
mature miRNA, and triplex-forming oligonucleotides.
[0203] Nucleic acids of the invention may be of various lengths,
generally dependent upon the particular form of nucleic acid. For
example, in particular embodiments, plasmids or genes may be from
about 1,000 to about 100,000 nucleotide residues in length. In
particular embodiments, oligonucleotides may range from about 10 to
about 100 nucleotides in length. In various related embodiments,
oligonucleotides, both single-stranded, double-stranded, and
triple-stranded, may range in length from about 10 to about 60
nucleotides, from about 15 to about 60 nucleotides, from about 20
to about 50 nucleotides, from about 15 to about 30 nucleotides, or
from about 20 to about 30 nucleotides in length.
[0204] In particular embodiments, an oligonucleotide (or a strand
thereof) of the invention specifically hybridizes to or is
complementary to a target polynucleotide sequence. The terms
"specifically hybridizable" and "complementary" as used herein
indicate a sufficient degree of complementarity such that stable
and specific binding occurs between the DNA or RNA target and the
oligonucleotide. It is understood that an oligonucleotide need not
be 100% complementary to its target nucleic acid sequence to be
specifically hybridizable. In preferred embodiments, an
oligonucleotide is specifically hybridizable when binding of the
oligonucleotide to the target sequence interferes with the normal
function of the target sequence to cause a loss of utility or
expression therefrom, and there is a sufficient degree of
complementarity to avoid non-specific binding of the
oligonucleotide to non-target sequences under conditions in which
specific binding is desired, i.e., under physiological conditions
in the case of in vivo assays or therapeutic treatment, or, in the
case of in vitro assays, under conditions in which the assays are
conducted. Thus, the oligonucleotide may include 1, 2, 3, or more
base substitutions as compared to the region of a gene or mRNA
sequence that it is targeting or to which it specifically
hybridizes.
[0205] 1. siRNA
[0206] The siRNA component of the nucleic acid-lipid particles of
the present invention is capable of silencing the expression of a
target gene of interest. Each strand of the siRNA duplex is
typically about 15 to about 60 nucleotides in length, preferably
about 15 to about 30 nucleotides in length. In certain embodiments,
the siRNA comprises at least one modified nucleotide. The modified
siRNA is generally less immunostimulatory than a corresponding
unmodified siRNA sequence and retains RNAi activity against the
target gene of interest. In some embodiments, the modified siRNA
contains at least one 2'OMe purine or pyrimidine nucleotide such as
a 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-adenosine, and/or
2'OMe-cytosine nucleotide. The modified nucleotides can be present
in one strand (i.e., sense or antisense) or both strands of the
siRNA. In some preferred embodiments, one or more of the uridine
and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in
one strand (i.e., sense or antisense) or both strands of the siRNA.
In these embodiments, the modified siRNA can further comprise one
or more modified (e.g., 2'OMe-modified) adenosine and/or modified
(e.g., 2'OMe-modified) cytosine nucleotides. In other preferred
embodiments, only uridine and/or guanosine nucleotides are modified
(e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or
both strands of the siRNA. The siRNA sequences may have overhangs
(e.g., 3' or 5' overhangs as described in Elbashir et al., Genes
Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)), or
may lack overhangs (i.e., have blunt ends).
[0207] In particular embodiments, the selective incorporation of
modified nucleotides such as 2'OMe uridine and/or guanosine
nucleotides into the double-stranded region of either or both
strands of the siRNA reduces or completely abrogates the immune
response to that siRNA molecule. In certain instances, the
immunostimulatory properties of specific siRNA sequences and their
ability to silence gene expression can be balanced or optimized by
the introduction of minimal and selective 2'OMe modifications
within the double-stranded region of the siRNA duplex. This can be
achieved at therapeutically viable siRNA doses without cytokine
induction, toxicity, and off-target effects associated with the use
of unmodified siRNA.
[0208] The modified siRNA generally comprises from about 1% to
about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,
24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in
the double-stranded region of the siRNA duplex. In certain
embodiments, one, two, three, four, five, six, seven, eight, nine,
ten, or more of the nucleotides in the double-stranded region of
the siRNA comprise modified nucleotides. In certain other
embodiments, some or all of the modified nucleotides in the
double-stranded region of the siRNA are 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more nucleotides apart from each other. In one preferred
embodiment, none of the modified nucleotides in the double-stranded
region of the siRNA are adjacent to each other (e.g., there is a
gap of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified
nucleotides between each modified nucleotide).
[0209] In some embodiments, less than about 50% (e.g., less than
about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%,
37%, or 36%, preferably less than about 35%, 34%, 33%, 32%, 31%, or
30%) of the nucleotides in the double-stranded region of the siRNA
comprise modified (e.g., 2'OMe) nucleotides. In one aspect of these
embodiments, less than about 50% of the uridine and/or guanosine
nucleotides in the double-stranded region of one or both strands of
the siRNA are selectively (e.g., only) modified. In another aspect
of these embodiments, less than about 50% of the nucleotides in the
double-stranded region of the siRNA comprise 2'OMe nucleotides,
wherein the siRNA comprises 2'OMe nucleotides in both strands of
the siRNA, wherein the siRNA comprises at least one 2'OMe-guanosine
nucleotide and at least one 2'OMe-uridine nucleotide, and wherein
2'OMe-guanosine nucleotides and 2'OMe-uridine nucleotides are the
only 2'OMe nucleotides present in the double-stranded region. In
yet another aspect of these embodiments, less than about 50% of the
nucleotides in the double-stranded region of the siRNA comprise
2'OMe nucleotides, wherein the siRNA comprises 2'OMe nucleotides in
both strands of the modified siRNA, wherein the siRNA comprises
2'OMe nucleotides selected from the group consisting of
2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides,
2'OMe-adenosine nucleotides, and mixtures thereof, and wherein the
siRNA does not comprise 2'OMe-cytosine nucleotides in the
double-stranded region. In a further aspect of these embodiments,
less than about 50% of the nucleotides in the double-stranded
region of the siRNA comprise 2'OMe nucleotides, wherein the siRNA
comprises 2'OMe nucleotides in both strands of the siRNA, wherein
the siRNA comprises at least one 2'OMe-guanosine nucleotide and at
least one 2'OMe-uridine nucleotide, and wherein the siRNA does not
comprise 2'OMe-cytosine nucleotides in the double-stranded region.
In another aspect of these embodiments, less than about 50% of the
nucleotides in the double-stranded region of the siRNA comprise
2'OMe nucleotides, wherein the siRNA comprises 2'OMe nucleotides in
both strands of the modified siRNA, wherein the siRNA comprises
2'OMe nucleotides selected from the group consisting of
2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides,
2'OMe-adenosine nucleotides, and mixtures thereof, and wherein the
2'OMe nucleotides in the double-stranded region are not adjacent to
each other.
[0210] In other embodiments, from about 1% to about 50% (e.g., from
about 5%-50%, 10%-50%, 15%-50%, 20%-50%, 25%-50%, 30%-50%, 35%-50%,
40%-50%, 45%-50%, 5%-45%, 10%-45%, 15%-45%, 20%-45%, 25%-45%,
30%-45%, 35%-45%, 40%-45%, 5%-40%, 10%-40%, 15%-40%, 20%-40%,
25%-40%, 25%-39%, 25%-38%, 25%-37%, 25%-36%, 26%-39%, 26%-38%,
26%-37%, 26%-36%, 27%-39%, 27%-38%, 27%-37%, 27%-36%, 28%-39%,
28%-38%, 28%-37%, 28%-36%, 29%-39%, 29%-38%, 29%-37%, 29%-36%,
30%-40%, 30%-39%, 30%-38%, 30%-37%, 30%-36%, 31%-39%, 31%-38%,
31%-37%, 31%-36%, 32%-39%, 32%-38%, 32%-37%, 32%-36%, 33%-39%,
33%-38%, 33%-37%, 33%-36%, 34%-39%, 34%-38%, 34%-37%, 34%-36%,
35%-40%, 5%-35%, 10%-35%, 15%-35%, 20%-35%, 21%-35%, 22%-35%,
23%-35%, 24%-35%, 25%-35%, 26%-35%, 27%-35%, 28%-35%, 29%-35%,
30%-35%, 31%-35%, 32%-35%, 33%-35%, 34%-35%, 30%-34%, 31%-34%,
32%-34%, 33%-34%, 30%-33%, 31%-33%, 32%-33%, 30%-32%, 31%-32%,
25%-34%, 25%-33%, 25%-32%, 25%-31%, 26%-34%, 26%-33%, 26%-32%,
26%-31%, 27%-34%, 27%-33%, 27%-32%, 27%-31%, 28%-34%, 28%-33%,
28%-32%, 28%-31%, 29%-34%, 29%-33%, 29%-32%, 29%-31%, 5%-30%,
10%-30%, 15%-30%, 20%-34%, 20%-33%, 20%-32%, 20%-31%, 20%-30%,
21%-30%, 22%-30%, 23%-30%, 24%-30%, 25%-30%, 25%-29%, 25%-28%,
25%-27%, 25%-26%, 26%-30%, 26%-29%, 26%-28%, 26%-27%, 27%-30%,
27%-29%, 27%-28%, 28%-30%, 28%-29%, 29%-30%, 5%-25%, 10%-25%,
15%-25%, 20%-29%, 20%-28%, 20%-27%, 20%-26%, 20%-25%, 5%-20%,
10%-20%, 15%-20%, 5%-15%, 10%-15%, or 5%-10%) of the nucleotides in
the double-stranded region of the siRNA comprise modified
nucleotides. In one aspect of these embodiments, from about 1% to
about 50% of the uridine and/or guanosine nucleotides in the
double-stranded region of one or both strands of the siRNA are
selectively (e.g., only) modified. In another aspect of these
embodiments, from about 1% to about 50% of the nucleotides in the
double-stranded region of the siRNA comprise 2'OMe nucleotides,
wherein the siRNA comprises 2'OMe nucleotides in both strands of
the siRNA, wherein the siRNA comprises at least one 2'OMe-guanosine
nucleotide and at least one 2'OMe-uridine nucleotide, and wherein
2'OMe-guanosine nucleotides and 2'OMe-uridine nucleotides are the
only 2'OMe nucleotides present in the double-stranded region. In
yet another aspect of these embodiments, from about 1% to about 50%
of the nucleotides in the double-stranded region of the siRNA
comprise 2'OMe nucleotides, wherein the siRNA comprises 2'OMe
nucleotides in both strands of the modified siRNA, wherein the
siRNA comprises 2'OMe nucleotides selected from the group
consisting of 2'OMe-guanosine nucleotides, 2'OMe-uridine
nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, and
wherein the siRNA does not comprise 2'OMe-cytosine nucleotides in
the double-stranded region. In a further aspect of these
embodiments, from about 1% to about 50% of the nucleotides in the
double-stranded region of the siRNA comprise 2'OMe nucleotides,
wherein the siRNA comprises 2'OMe nucleotides in both strands of
the siRNA, wherein the siRNA comprises at least one 2'OMe-guanosine
nucleotide and at least one 2'OMe-uridine nucleotide, and wherein
the siRNA does not comprise 2'OMe-cytosine nucleotides in the
double-stranded region. In another aspect of these embodiments,
from about 1% to about 50% of the nucleotides in the
double-stranded region of the siRNA comprise 2'OMe nucleotides,
wherein the siRNA comprises 2'OMe nucleotides in both strands of
the modified siRNA, wherein the siRNA comprises 2'OMe nucleotides
selected from the group consisting of 2'OMe-guanosine nucleotides,
2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and
mixtures thereof, and wherein the 2'OMe nucleotides in the
double-stranded region are not adjacent to each other.
[0211] Additional ranges, percentages, and patterns of
modifications that may be introduced into siRNA are described in
U.S. Patent Publication No. 20070135372, the disclosure of which is
herein incorporated by reference in its entirety for all
purposes.
[0212] a) Selection of siRNA Sequences
[0213] Suitable siRNA sequences can be identified using any means
known in the art. Typically, the methods described in Elbashir et
al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J.,
20:6877-6888 (2001) are combined with rational design rules set
forth in Reynolds et al., Nature Biotech., 22(3):326-330
(2004).
[0214] As a non-limiting example, the nucleotide sequence 3' of the
AUG start codon of a transcript from the target gene of interest
may be scanned for dinucleotide sequences (e.g., AA, NA, CC, GG, or
UU, wherein N.dbd.C, G, or U) (see, e.g., Elbashir et al., EMBO J.,
20:6877-6888 (2001)). The nucleotides immediately 3' to the
dinucleotide sequences are identified as potential siRNA sequences
(i.e., a target sequence or a sense strand sequence). Typically,
the 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides
immediately 3' to the dinucleotide sequences are identified as
potential siRNA sequences. In some embodiments, the dinucleotide
sequence is an AA or NA sequence and the 19 nucleotides immediately
3' to the AA or NA dinucleotide are identified as potential siRNA
sequences. siRNA sequences are usually spaced at different
positions along the length of the target gene. To further enhance
silencing efficiency of the siRNA sequences, potential siRNA
sequences may be analyzed to identify sites that do not contain
regions of homology to other coding sequences, e.g., in the target
cell or organism. For example, a suitable siRNA sequence of about
21 base pairs typically will not have more than 16-17 contiguous
base pairs of homology to coding sequences in the target cell or
organism. If the siRNA sequences are to be expressed from an RNA
Pol III promoter, siRNA sequences lacking more than 4 contiguous
A's or T's are selected.
[0215] Once a potential siRNA sequence has been identified, a
complementary sequence (i.e., an antisense strand sequence) can be
designed. A potential siRNA sequence can also be analyzed using a
variety of criteria known in the art. For example, to enhance their
silencing efficiency, the siRNA sequences may be analyzed by a
rational design algorithm to identify sequences that have one or
more of the following features: (1) G/C content of about 25% to
about 60% G/C; (2) at least 3 A/Us at positions 15-19 of the sense
strand; (3) no internal repeats; (4) an A at position 19 of the
sense strand; (5) an A at position 3 of the sense strand; (6) a U
at position 10 of the sense strand; (7) no G/C at position 19 of
the sense strand; and (8) no G at position 13 of the sense strand.
siRNA design tools that incorporate algorithms that assign suitable
values of each of these features and are useful for selection of
siRNA can be found at, e.g.,
http://ihome.ust.hk/.about.bokcmho/siRNA/siRNA.html. One of skill
in the art will appreciate that sequences with one or more of the
foregoing characteristics may be selected for further analysis and
testing as potential siRNA sequences.
[0216] Additionally, potential siRNA sequences with one or more of
the following criteria can often be eliminated as siRNA: (1)
sequences comprising a stretch of 4 or more of the same base in a
row; (2) sequences comprising homopolymers of Gs (i.e., to reduce
possible non-specific effects due to structural characteristics of
these polymers; (3) sequences comprising triple base motifs (e.g.,
GGG, CCC, AAA, or TTT); (4) sequences comprising stretches of 7 or
more G/Cs in a row; and (5) sequences comprising direct repeats of
4 or more bases within the candidates resulting in internal
fold-back structures. However, one of skill in the art will
appreciate that sequences with one or more of the foregoing
characteristics may still be selected for further analysis and
testing as potential siRNA sequences.
[0217] In some embodiments, potential siRNA sequences may be
further analyzed based on siRNA duplex asymmetry as described in,
e.g., Khvorova et al., Cell, 115:209-216 (2003); and Schwarz et
al., Cell, 115:199-208 (2003). In other embodiments, potential
siRNA sequences may be further analyzed based on secondary
structure at the target site as described in, e.g., Luo et al.,
Biophys. Res. Commun., 318:303-310 (2004). For example, secondary
structure at the target site can be modeled using the Mfold
algorithm (available at http://mfold.burnet.edu.au/rna_form) to
select siRNA sequences which favor accessibility at the target site
where less secondary structure in the form of base-pairing and
stem-loops is present.
[0218] Once a potential siRNA sequence has been identified, the
sequence can be analyzed for the presence of any immunostimulatory
properties, e.g., using an in vitro cytokine assay or an in vivo
animal model. Motifs in the sense and/or antisense strand of the
siRNA sequence such as GU-rich motifs (e.g.,
5'-GU-3',5'-UGU-3',5'-GUGU-3',5'-UGUGU-3', etc.) can also provide
an indication of whether the sequence may be immunostimulatory.
Once an siRNA molecule is found to be immunostimulatory, it can
then be modified to decrease its immunostimulatory properties as
described herein. As a non-limiting example, an siRNA sequence can
be contacted with a mammalian responder cell under conditions such
that the cell produces a detectable immune response to determine
whether the siRNA is an immunostimulatory or a
non-immunostimulatory siRNA. The mammalian responder cell may be
from a naive mammal (i.e., a mammal that has not previously been in
contact with the gene product of the siRNA sequence). The mammalian
responder cell may be, e.g., a peripheral blood mononuclear cell
(PBMC), a macrophage, and the like. The detectable immune response
may comprise production of a cytokine or growth factor such as,
e.g., TNF-.alpha., IFN-.alpha., IFN-.beta., IFN-.gamma., IL-6,
IL-12, or a combination thereof. An siRNA molecule identified as
being immunostimulatory can then be modified to decrease its
immunostimulatory properties by replacing at least one of the
nucleotides on the sense and/or antisense strand with modified
nucleotides. For example, less than about 30% (e.g., less than
about 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in the
double-stranded region of the siRNA duplex can be replaced with
modified nucleotides such as 2'OMe nucleotides. The modified siRNA
can then be contacted with a mammalian responder cell as described
above to confirm that its immunostimulatory properties have been
reduced or abrogated.
[0219] Suitable in vitro assays for detecting an immune response
include, but are not limited to, the double monoclonal antibody
sandwich immunoassay technique of David et al. (U.S. Pat. No.
4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et
al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and
S. Livingstone, Edinburgh (1970)); the "Western blot" method of
Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of
labeled ligand (Brown et al., J. Biol. Chem., 255:4980-4983
(1980)); enzyme-linked immunosorbent assays (ELISA) as described,
for example, by Raines et al., J. Biol. Chem., 257:5154-5160
(1982); immunocytochemical techniques, including the use of
fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980));
and neutralization of activity (Bowen-Pope et al., Proc. Natl.
Acad. Sci. USA, 81:2396-2400 (1984)). In addition to the
immunoassays described above, a number of other immunoassays are
available, including those described in U.S. Pat. Nos. 3,817,827;
3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074;
and 4,098,876. The disclosures of these references are herein
incorporated by reference in their entirety for all purposes.
[0220] A non-limiting example of an in vivo model for detecting an
immune response includes an in vivo mouse cytokine induction assay
as described in, e.g., Judge et al., Mol. Ther., 13:494-505 (2006).
In certain embodiments, the assay that can be performed as follows:
(1) siRNA can be administered by standard intravenous injection in
the lateral tail vein; (2) blood can be collected by cardiac
puncture about 6 hours after administration and processed as plasma
for cytokine analysis; and (3) cytokines can be quantified using
sandwich ELISA kits according to the manufacturer's instructions
(e.g., mouse and human IFN-.alpha. (PBL Biomedical; Piscataway,
N.J.); human IL-6 and TNF-.alpha. (eBioscience; San Diego, Calif.);
and mouse IL-6, TNF-.alpha., and IFN-.gamma. (BD Biosciences; San
Diego, Calif.)).
[0221] Monoclonal antibodies that specifically bind cytokines and
growth factors are commercially available from multiple sources and
can be generated using methods known in the art (see, e.g., Kohler
et al., Nature, 256: 495-497 (1975) and Harlow and Lane,
ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication,
New York (1999)). Generation of monoclonal antibodies has been
previously described and can be accomplished by any means known in
the art (Buhring et al., in Hybridoma, Vol. 10, No. 1, pp. 77-78
(1991)). In some methods, the monoclonal antibody is labeled (e.g.,
with any composition detectable by spectroscopic, photochemical,
biochemical, electrical, optical, or chemical means) to facilitate
detection.
[0222] b) Generating siRNA Molecules
[0223] siRNA can be provided in several forms including, e.g., as
one or more isolated small-interfering RNA (siRNA) duplexes, as
longer double-stranded RNA (dsRNA), or as siRNA or dsRNA
transcribed from a transcriptional cassette in a DNA plasmid. In
some embodiments, siRNA may be produced enzymatically or by
partial/total organic synthesis, and modified ribonucleotides can
be introduced by in vitro enzymatic or organic synthesis. In
certain instances, each strand is prepared chemically. Methods of
synthesizing RNA molecules are known in the art, e.g., the chemical
synthesis methods as described in Verma and Eckstein (1998) or as
described herein.
[0224] An RNA population can be used to provide long precursor
RNAs, or long precursor RNAs that have substantial or complete
identity to a selected target sequence can be used to make the
siRNA. The RNAs can be isolated from cells or tissue, synthesized,
and/or cloned according to methods well known to those of skill in
the art. The RNA can be a mixed population (obtained from cells or
tissue, transcribed from cDNA, subtracted, selected, etc.), or can
represent a single target sequence. RNA can be naturally occurring
(e.g., isolated from tissue or cell samples), synthesized in vitro
(e.g., using T7 or SP6 polymerase and PCR products or a cloned
cDNA), or chemically synthesized.
[0225] To form a long dsRNA, for synthetic RNAs, the complement is
also transcribed in vitro and hybridized to form a dsRNA. If a
naturally occurring RNA population is used, the RNA complements are
also provided (e.g., to form dsRNA for digestion by E. coli RNAse
III or Dicer), e.g., by transcribing cDNAs corresponding to the RNA
population, or by using RNA polymerases. The precursor RNAs are
then hybridized to form double stranded RNAs for digestion. The
dsRNAs can be directly administered to a subject or can be digested
in vitro prior to administration.
[0226] Methods for isolating RNA, synthesizing RNA, hybridizing
nucleic acids, making and screening cDNA libraries, and performing
PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene,
25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra),
as are PCR methods (see, U.S. Pat. Nos. 4,683,195 and 4,683,202;
PCR Protocols: A Guide to Methods and Applications (Innis et al.,
eds, 1990)). Expression libraries are also well known to those of
skill in the art. Additional basic texts disclosing the general
methods of use in this invention include Sambrook et al., Molecular
Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene
Transfer and Expression: A Laboratory Manual (1990); and Current
Protocols in Molecular Biology (Ausubel et al., eds., 1994). The
disclosures of these references are herein incorporated by
reference in their entirety for all purposes.
[0227] Preferably, siRNA are chemically synthesized. The
oligonucleotides that comprise the siRNA molecules of the invention
can be synthesized using any of a variety of techniques known in
the art, such as those described in Usman et al., J. Am. Chem.
Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res., 18:5433
(1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995); and
Wincott et al., Methods Mol. Bio., 74:59 (1997). The synthesis of
oligonucleotides makes use of common nucleic acid protecting and
coupling groups, such as dimethoxytrityl at the 5'-end and
phosphoramidites at the 3'-end. As a non-limiting example, small
scale syntheses can be conducted on an Applied Biosystems
synthesizer using a 0.2 .mu.mol scale protocol. Alternatively,
syntheses at the 0.2 .mu.mol scale can be performed on a 96-well
plate synthesizer from Protogene (Palo Alto, Calif.). However, a
larger or smaller scale of synthesis is also within the scope of
this invention. Suitable reagents for oligonucleotide synthesis,
methods for RNA deprotection, and methods for RNA purification are
known to those of skill in the art.
[0228] siRNA molecules can also be synthesized via a tandem
synthesis technique, wherein both strands are synthesized as a
single continuous oligonucleotide fragment or strand separated by a
cleavable linker that is subsequently cleaved to provide separate
fragments or strands that hybridize to form the siRNA duplex. The
linker can be a polynucleotide linker or a non-nucleotide linker.
The tandem synthesis of siRNA can be readily adapted to both
multiwell/multiplate synthesis platforms as well as large scale
synthesis platforms employing batch reactors, synthesis columns,
and the like. Alternatively, siRNA molecules can be assembled from
two distinct oligonucleotides, wherein one oligonucleotide
comprises the sense strand and the other comprises the antisense
strand of the siRNA. For example, each strand can be synthesized
separately and joined together by hybridization or ligation
following synthesis and/or deprotection. In certain other
instances, siRNA molecules can be synthesized as a single
continuous oligonucleotide fragment, where the self-complementary
sense and antisense regions hybridize to form an siRNA duplex
having hairpin secondary structure.
[0229] c) Modifying siRNA Sequences
[0230] In certain aspects, siRNA molecules comprise a duplex having
two strands and at least one modified nucleotide in the
double-stranded region, wherein each strand is about 15 to about 60
nucleotides in length. Advantageously, the modified siRNA is less
immunostimulatory than a corresponding unmodified siRNA sequence,
but retains the capability of silencing the expression of a target
sequence. In preferred embodiments, the degree of chemical
modifications introduced into the siRNA molecule strikes a balance
between reduction or abrogation of the immunostimulatory properties
of the siRNA and retention of RNAi activity. As a non-limiting
example, an siRNA molecule that targets a gene of interest can be
minimally modified (e.g., less than about 30%, 25%, 20%, 15%, 10%,
or 5% modified) at selective uridine and/or guanosine nucleotides
within the siRNA duplex to eliminate the immune response generated
by the siRNA while retaining its capability to silence target gene
expression.
[0231] Examples of modified nucleotides suitable for use in the
invention include, but are not limited to, ribonucleotides having a
2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy,
5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or
2'-C-allyl group. Modified nucleotides having a Northern
conformation such as those described in, e.g., Saenger, Principles
of Nucleic Acid Structure, Springer-Verlag Ed. (1984), are also
suitable for use in siRNA molecules. Such modified nucleotides
include, without limitation, locked nucleic acid (LNA) nucleotides
(e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides),
2'-O-(2-methoxyethyl) (MOE) nucleotides, 2'-methyl-thio-ethyl
nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides,
2'-deoxy-2'-chloro (2'Cl) nucleotides, and 2'-azido nucleotides. In
certain instances, the siRNA molecules described herein include one
or more G-clamp nucleotides. A G-clamp nucleotide refers to a
modified cytosine analog wherein the modifications confer the
ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a
complementary guanine nucleotide within a duplex (see, e.g., Lin et
al., J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition,
nucleotides having a nucleotide base analog such as, for example,
C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole
carboxamides, and nitroazole derivatives such as 3-nitropyrrole,
4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes,
Nucl. Acids Res., 29:2437-2447 (2001)) can be incorporated into
siRNA molecules.
[0232] In certain embodiments, siRNA molecules may further comprise
one or more chemical modifications such as terminal cap moieties,
phosphate backbone modifications, and the like. Examples of
terminal cap moieties include, without limitation, inverted deoxy
abasic residues, glyceryl modifications, 4',5'-methylene
nucleotides, 1-(.beta.-D-erythrofuranosyl) nucleotides, 4'-thio
nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol
nucleotides, L-nucleotides, .alpha.-nucleotides, modified base
nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seco
nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic
3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide
moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide
moieties, 3'-2'-inverted abasic moieties, 5'-5'-inverted nucleotide
moieties, 5'-5'-inverted abasic moieties, 3'-5'-inverted deoxy
abasic moieties, 5'-amino-alkyl phosphate, 1,3-diamino-2-propyl
phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate,
1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol
phosphate, 3'-phosphoramidate, 5'-phosphoramidate, hexylphosphate,
aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioate,
5'-phosphorothioate, phosphorodithioate, and bridging or
non-bridging methylphosphonate or 5'-mercapto moieties (see, e.g.,
U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron 49:1925
(1993)). Non-limiting examples of phosphate backbone modifications
(i.e., resulting in modified internucleotide linkages) include
phosphorothioate, phosphorodithioate, methylphosphonate,
phosphotriester, morpholino, amidate, carbamate, carboxymethyl,
acetamidate, polyamide, sulfonate, sulfonamide, sulfamate,
formacetal, thioformacetal, and alkylsilyl substitutions (see,
e.g., Hunziker et al., Nucleic Acid Analogues: Synthesis and
Properties, in Modern Synthetic Methods, VCH, 331-417 (1995);
Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides,
in Carbohydrate Modifications in Antisense Research, ACS, 24-39
(1994)). Such chemical modifications can occur at the 5'-end and/or
3'-end of the sense strand, antisense strand, or both strands of
the siRNA. The disclosures of these references are herein
incorporated by reference in their entirety for all purposes.
[0233] In some embodiments, the sense and/or antisense strand of
the siRNA molecule can further comprise a 3'-terminal overhang
having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2'-deoxy
ribonucleotides, modified (e.g., 2'OMe) and/or unmodified uridine
ribonucleotides, and/or any other combination of modified (e.g.,
2'OMe) and unmodified nucleotides.
[0234] Additional examples of modified nucleotides and types of
chemical modifications that can be introduced into siRNA molecules
are described, e.g., in UK Patent No. GB 2,397,818 B and U.S.
Patent Publication Nos. 20040192626, 20050282188, and 20070135372,
the disclosures of which are herein incorporated by reference in
their entirety for all purposes.
[0235] The siRNA molecules described herein can optionally comprise
one or more non-nucleotides in one or both strands of the siRNA. As
used herein, the term "non-nucleotide" refers to any group or
compound that can be incorporated into a nucleic acid chain in the
place of one or more nucleotide units, including sugar and/or
phosphate substitutions, and allows the remaining bases to exhibit
their activity. The group or compound is abasic in that it does not
contain a commonly recognized nucleotide base such as adenosine,
guanine, cytosine, uracil, or thymine and therefore lacks a base at
the l'-position.
[0236] In other embodiments, chemical modification of the siRNA
comprises attaching a conjugate to the siRNA molecule. The
conjugate can be attached at the 5' and/or 3'-end of the sense
and/or antisense strand of the siRNA via a covalent attachment such
as, e.g., a biodegradable linker. The conjugate can also be
attached to the siRNA, e.g., through a carbamate group or other
linking group (see, e.g., U.S. Patent Publication Nos. 20050074771,
20050043219, and 20050158727). In certain instances, the conjugate
is a molecule that facilitates the delivery of the siRNA into a
cell. Examples of conjugate molecules suitable for attachment to
siRNA include, without limitation, steroids such as cholesterol,
glycols such as polyethylene glycol (PEG), human serum albumin
(HSA), fatty acids, carotenoids, terpenes, bile acids, folates
(e.g., folic acid, folate analogs and derivatives thereof), sugars
(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,
mannose, fructose, fucose, etc.), phospholipids, peptides, ligands
for cellular receptors capable of mediating cellular uptake, and
combinations thereof (see, e.g., U.S. Patent Publication Nos.
20030130186, 20040110296, and 20040249178; U.S. Pat. No.
6,753,423). Other examples include the lipophilic moiety, vitamin,
polymer, peptide, protein, nucleic acid, small molecule,
oligosaccharide, carbohydrate cluster, intercalator, minor groove
binder, cleaving agent, and cross-linking agent conjugate molecules
described in U.S. Patent Publication Nos. 20050119470 and
20050107325. Yet other examples include the 2'-O-alkyl amine,
2'-.beta.-alkoxyalkyl amine, polyamine, C5-cationic modified
pyrimidine, cationic peptide, guanidinium group, amidininium group,
cationic amino acid conjugate molecules described in U.S. Patent
Publication No. 20050153337. Additional examples include the
hydrophobic group, membrane active compound, cell penetrating
compound, cell targeting signal, interaction modifier, and steric
stabilizer conjugate molecules described in U.S. Patent Publication
No. 20040167090. Further examples include the conjugate molecules
described in U.S. Patent Publication No. 20050239739. The type of
conjugate used and the extent of conjugation to the siRNA molecule
can be evaluated for improved pharmacokinetic profiles,
bioavailability, and/or stability of the siRNA while retaining RNAi
activity. As such, one skilled in the art can screen siRNA
molecules having various conjugates attached thereto to identify
ones having improved properties and full RNAi activity using any of
a variety of well-known in vitro cell culture or in vivo animal
models. The disclosures of the above-described patent documents are
herein incorporated by reference in their entirety for all
purposes.
[0237] d) Target Genes
[0238] The siRNA component of the nucleic acid-lipid particles
described herein can be used to downregulate or silence the
translation (i.e., expression) of a gene of interest. Genes of
interest include, but are not limited to, genes associated with
viral infection and survival, genes associated with metabolic
diseases and disorders (e.g., liver diseases and disorders), genes
associated with tumorigenesis or cell transformation (e.g.,
cancer), angiogenic genes, immunomodulator genes such as those
associated with inflammatory and autoimmune responses, receptor
ligand genes, and genes associated with neurodegenerative
disorders.
[0239] In particular embodiments, the present invention provides a
cocktail of two, three, four, five, six, seven, eight, nine, ten,
or more siRNA molecules that silences the expression of multiple
genes of interest. In some embodiments, the cocktail of siRNA
molecules is fully encapsulated in a lipid particle such as a
nucleic acid-lipid particle (e.g., SNALP). The siRNA molecules may
be co-encapsulated in the same lipid particle, or each siRNA
species present in the cocktail may be formulated in separate
particles.
[0240] Genes associated with viral infection and survival include
those expressed by a host (e.g., a host factor such as tissue
factor (TF)) or a virus in order to bind, enter, and replicate in a
cell. Of particular interest are viral sequences associated with
chronic viral diseases. Viral sequences of particular interest
include sequences of Filoviruses such as Ebola virus and Marburg
virus (see, e.g., Geisbert et al., J. Infect. Dis., 193:1650-1657
(2006)); Arenaviruses such as Lassa virus, Junin virus, Machupo
virus, Guanarito virus, and Sabia virus (Buchmeier et al.,
Arenaviridae: the viruses and their replication, In: FIELDS
VIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven,
Philadelphia, (2001)); Influenza viruses such as Influenza A, B,
and C viruses, (see, e.g., Steinhauer et al., Annu Rev Genet.,
36:305-332 (2002); and Neumann et al., J Gen Virol., 83:2635-2662
(2002)); Hepatitis viruses (see, e.g., Hamasaki et al., FEBS Lett.,
543:51 (2003); Yokota et al., EMBO Rep., 4:602 (2003); Schlomai et
al., Hepatology, 37:764 (2003); Wilson et al., Proc. Natl. Acad.
Sci. USA, 100:2783 (2003); Kapadia et al., Proc. Natl. Acad. Sci.
USA, 100:2014 (2003); and FIELDS VIROLOGY, Knipe et al. (eds.), 4th
ed., Lippincott-Raven, Philadelphia (2001)); Human Immunodeficiency
Virus (HIV) (Banerjea et al., Mol. Ther., 8:62 (2003); Song et al.,
J. Virol., 77:7174 (2003); Stephenson, JAMA, 289:1494 (2003); Qin
et al., Proc. Natl. Acad. Sci. USA, 100:183 (2003)); Herpes viruses
(Jia et al., J. Virol., 77:3301 (2003)); and Human Papilloma
Viruses (HPV) (Hall et al., J. Virol., 77:6066 (2003); Jiang et
al., Oncogene, 21:6041 (2002)).
[0241] Exemplary Filovirus nucleic acid sequences that can be
silenced include, but are not limited to, nucleic acid sequences
encoding structural proteins (e.g., VP30, VP35, nucleoprotein (NP),
polymerase protein (L-pol)) and membrane-associated proteins (e.g.,
VP40, glycoprotein (GP), VP24). Complete genome sequences for Ebola
virus are set forth in, e.g., Genbank Accession Nos.
NC.sub.--002549; AY769362; NC.sub.--006432; NC.sub.--004161;
AY729654; AY354458; AY142960; AB050936; AF522874; AF499101;
AF272001; and AF086833. Ebola virus VP24 sequences are set forth
in, e.g., Genbank Accession Nos. U77385 and AY058897. Ebola virus
L-pol sequences are set forth in, e.g., Genbank Accession No.
X67110. Ebola virus VP40 sequences are set forth in, e.g., Genbank
Accession No. AY058896. Ebola virus NP sequences are set forth in,
e.g., Genbank Accession No. AY058895. Ebola virus GP sequences are
set forth in, e.g., Genbank Accession No. AY058898; Sanchez et al.,
Virus Res., 29:215-240 (1993); Will et al., J. Viral., 67:1203-1210
(1993); Volchkov et al., FEBS Lett., 305:181-184 (1992); and U.S.
Pat. No. 6,713,069. Additional Ebola virus sequences are set forth
in, e.g., Genbank Accession Nos. L11365 and X61274. Complete genome
sequences for Marburg virus are set forth in, e.g., Genbank
Accession Nos. NC.sub.--001608; AY430365; AY430366; and AY358025.
Marburg virus GP sequences are set forth in, e.g., Genbank
Accession Nos. AF005734; AF005733; and AF005732. Marburg virus VP35
sequences are set forth in, e.g., Genbank Accession Nos. AF005731
and AF005730. Additional Marburg virus sequences are set forth in,
e.g., Genbank Accession Nos. X64406; Z29337; AF005735; and Z12132.
Non-limiting examples of siRNA molecules targeting Ebola virus and
Marburg virus nucleic acid sequences include those described in
U.S. Patent Publication No. 20070135370 and U.S. application Ser.
No. 12/840,225, filed Jul. 20, 2010, the disclosures of which are
herein incorporated by reference in their entirety for all
purposes.
[0242] Exemplary Arenavirus nucleic acid sequences that can be
silenced include, but are not limited to, nucleic acid sequences
encoding nucleoprotein (NP), glycoprotein (GP), L-polymerase (L),
and Z protein (Z). Complete genome sequences for Lassa virus are
set forth in, e.g., Genbank Accession Nos. NC.sub.--004296 (LASV
segment S) and NC.sub.--004297 (LASV segment L). Non-limiting
examples of siRNA molecules targeting Lassa virus nucleic acid
sequences include those described in U.S. Provisional Application
No. 61/319,855, filed Mar. 31, 2010, the disclosure of which is
herein incorporated by reference in its entirety for all
purposes.
[0243] Exemplary host nucleic acid sequences that can be silenced
include, but are not limited to, nucleic acid sequences encoding
host factors such as tissue factor (TF) that are known to play a
role in the pathogenisis of hemorrhagic fever viruses. The mRNA
sequence of TF is set forth in Genbank Accession No.
NM.sub.--001993. Those of skill in the art will appreciate that TF
is also known as F3, coagulation factor III, thromboplastin, and
CD142. Non-limiting examples of siRNA molecules targeting TF
nucleic acid sequences include those described in U.S. Provisional
Application No. 61/319,855, filed Mar. 31, 2010, the disclosure of
which is herein incorporated by reference in its entirety for all
purposes.
[0244] Exemplary Influenza virus nucleic acid sequences that can be
silenced include, but are not limited to, nucleic acid sequences
encoding nucleoprotein (NP), matrix proteins (M1 and M2),
nonstructural proteins (NS1 and NS2), RNA polymerase (PA, PB1,
PB2), neuraminidase (NA), and haemagglutinin (HA). Influenza A NP
sequences are set forth in, e.g., Genbank Accession Nos.
NC.sub.--004522; AY818138; AB166863; AB188817; AB189046; AB189054;
AB189062; AY646169; AY646177; AY651486; AY651493; AY651494;
AY651495; AY651496; AY651497; AY651498; AY651499; AY651500;
AY651501; AY651502; AY651503; AY651504; AY651505; AY651506;
AY651507; AY651509; AY651528; AY770996; AY790308; AY818138; and
AY818140. Influenza A PA sequences are set forth in, e.g., Genbank
Accession Nos. AY818132; AY790280; AY646171; AY818132; AY818133;
AY646179; AY818134; AY551934; AY651613; AY651610; AY651620;
AY651617; AY651600; AY651611; AY651606; AY651618; AY651608;
AY651607; AY651605; AY651609; AY651615; AY651616; AY651640;
AY651614; AY651612; AY651621; AY651619; AY770995; and AY724786.
Non-limiting examples of siRNA molecules targeting Influenza virus
nucleic acid sequences include those described in U.S. Patent
Publication No. 20070218122, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
[0245] Exemplary hepatitis virus nucleic acid sequences that can be
silenced include, but are not limited to, nucleic acid sequences
involved in transcription and translation (e.g., En1, En2, X, P)
and nucleic acid sequences encoding structural proteins (e.g., core
proteins including C and C-related proteins, capsid and envelope
proteins including S, M, and/or L proteins, or fragments thereof)
(see, e.g., FIELDS VIROLOGY, supra). Exemplary Hepatits C virus
(HCV) nucleic acid sequences that can be silenced include, but are
not limited to, the 5'-untranslated region (5'-UTR), the
3'-untranslated region (3'-UTR), the polyprotein translation
initiation codon region, the internal ribosome entry site (IRES)
sequence, and/or nucleic acid sequences encoding the core protein,
the E1 protein, the E2 protein, the p7 protein, the NS2 protein,
the NS3 protease/helicase, the NS4A protein, the NS4B protein, the
NS5A protein, and/or the NS5B RNA-dependent RNA polymerase. HCV
genome sequences are set forth in, e.g., Genbank Accession Nos.
NC.sub.--004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b),
NC.sub.--009823 (HCV genotype 2), NC.sub.--009824 (HCV genotype 3),
NC.sub.--009825 (HCV genotype 4), NC.sub.--009826 (HCV genotype 5),
and NC.sub.--009827 (HCV genotype 6). Hepatitis A virus nucleic
acid sequences are set forth in, e.g., Genbank Accession No.
NC.sub.--001489; Hepatitis B virus nucleic acid sequences are set
forth in, e.g., Genbank Accession No. NC.sub.--003977; Hepatitis D
virus nucleic acid sequence are set forth in, e.g., Genbank
Accession No. NC.sub.--001653; Hepatitis E virus nucleic acid
sequences are set forth in, e.g., Genbank Accession No.
NC.sub.--001434; and Hepatitis G virus nucleic acid sequences are
set forth in, e.g., Genbank Accession No. NC.sub.--001710.
Silencing of sequences that encode genes associated with viral
infection and survival can conveniently be used in combination with
the administration of conventional agents used to treat the viral
condition. Non-limiting examples of siRNA molecules targeting
hepatitis virus nucleic acid sequences include those described in
U.S. Patent Publication Nos. 20060281175, 20050058982, and
20070149470; U.S. Pat. No. 7,348,314; and PCT Application No.
PCT/CA2010/000444, entitled "Compositions and Methods for Silencing
Hepatitis C Virus Expression," filed Mar. 19, 2010, bearing
Attorney Docket No. 020801-008910PC, the disclosures of which are
herein incorporated by reference in their entirety for all
purposes.
[0246] Genes associated with metabolic diseases and disorders
(e.g., disorders in which the liver is the target and liver
diseases and disorders) include, but are not limited to, genes
expressed in dyslipidemia, such as, e.g., apolipoprotein B (APOB)
(Genbank Accession No. NM.sub.--000384), apolipoprotein CIII
(APOC3) (Genbank Accession Nos. NM.sub.--000040 and NG.sub.--008949
REGION: 5001 . . . 8164), apolipoprotein E (APOE) (Genbank
Accession Nos. NM.sub.--000041 and NG.sub.--007084 REGION: 5001 . .
. 8612), proprotein convertase subtilisin/kexin type 9 (PCSK9)
(Genbank Accession No. NM.sub.--174936), diacylglycerol
O-acyltransferase type 1 (DGAT1) (Genbank Accession No.
NM.sub.--012079), diacylglyerol O-acyltransferase type 2 (DGAT2)
(Genbank Accession No. NM.sub.--032564), liver X receptors such as
LXR.alpha. and LXR.beta. (Genback Accession No. NM.sub.--007121),
farnesoid X receptors (FXR) (Genbank Accession No.
NM.sub.--005123), sterol-regulatory element binding protein
(SREBP), site-1 protease (SIP), 3-hydroxy-3-methylglutaryl
coenzyme-A reductase (HMG coenzyme-A reductase); and genes
expressed in diabetes, such as, e.g., glucose 6-phosphatase (see,
e.g., Forman et al., Cell, 81:687 (1995); Seol et al., Mol.
Endocrinol., 9:72 (1995), Zavacki et al., Proc. Natl. Acad. Sci.
USA, 94:7909 (1997); Sakai et al., Cell, 85:1037-1046 (1996);
Duncan et al., J. Biol. Chem., 272:12778-12785 (1997); Willy et
al., Genes Dev., 9:1033-1045 (1995); Lehmann et al., J. Biol.
Chem., 272:3137-3140 (1997); Janowski et al., Nature, 383:728-731
(1996); and Peet et al., Cell, 93:693-704 (1998)).
[0247] One of skill in the art will appreciate that genes
associated with metabolic diseases and disorders (e.g., diseases
and disorders in which the liver is a target and liver diseases and
disorders) include genes that are expressed in the liver itself as
well as and genes expressed in other organs and tissues. Silencing
of sequences that encode genes associated with metabolic diseases
and disorders can conveniently be used in combination with the
administration of conventional agents used to treat the disease or
disorder. Non-limiting examples of siRNA molecules targeting the
APOB gene include those described in U.S. Patent Publication Nos.
20060134189, 20060105976, and 20070135372, and PCT Publication No.
WO 04/091515, the disclosures of which are herein incorporated by
reference in their entirety for all purposes. Non-limiting examples
of siRNA molecules targeting the APOC3 gene include those described
in PCT Application No. PCT/CA2010/000120, filed Jan. 26, 2010, the
disclosure of which is herein incorporated by reference in its
entirety for all purposes. Non-limiting examples of siRNA molecules
targeting the PCSK9 gene include those described in U.S. Patent
Publication Nos. 20070173473, 20080113930, and 20080306015, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes. Exemplary siRNA molecules targeting the
DGAT1 gene may be designed using the antisense compounds described
in U.S. Patent Publication No. 20040185559, the disclosure of which
is herein incorporated by reference in its entirety for all
purposes. Exemplary siRNA molecules targeting the DGAT2 gene may be
designed using the antisense compounds described in U.S. Patent
Publication No. 20050043524, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
[0248] Genes associated with tumorigenesis or cell transformation
(e.g., cancer or other neoplasia) include, for example, genes
involved in p53 ubiquitination, c-Jun ubiquitination, histone
deacetylation, cell cycle regulation, transcriptional regulation,
and combinations thereof. Non-limiting examples of gene sequences
associated with tumorigenesis or cell transformation include
serine/threonine kinases such as polo-like kinase 1 (PLK-1)
(Genbank Accession No. NM.sub.--005030; Barr et al., Nat. Rev. Mol.
Cell. Biol., 5:429-440 (2004)) and cyclin-dependent kinase 4 (CDK4)
(Genbank Accession No. NM.sub.--000075); ubiquitin ligases such as
COP1 (RFWD2; Genbank Accession Nos. NM.sub.--022457 and
NM.sub.--001001740) and ring-box 1 (RBX1) (ROC1; Genbank Accession
No. NM.sub.--014248); tyrosine kinases such as WEE1 (Genbank
Accession Nos. NM.sub.--003390 and NM.sub.--001143976); mitotic
kinesins such as Eg5 (KSP, KIF11; Genbank Accession No.
NM.sub.--004523); transcription factors such as forkhead box M1
(FOXM1) (Genbank Accession Nos. NM.sub.--202002, NM.sub.--021953,
and NM.sub.--202003) and RAM2 (R1 or CDCA7L; Genbank Accession Nos.
NM.sub.--018719, NM.sub.--001127370, and NM.sub.--001127371);
inhibitors of apoptosis such as XIAP (Genbank Accession No.
NM.sub.--001167); COPS signalosome subunits such as CSN1, CSN2,
CSN3, CSN4, CSN5 (JAB1; Genbank Accession No. NM.sub.--006837);
CSN6, CSN7A, CSN7B, and CSN8; and histone deacetylases such as
HDAC1, HDAC2 (Genbank Accession No. NM.sub.--001527), HDAC3, HDAC4,
HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc.
[0249] Non-limiting examples of siRNA molecules targeting the PLK-1
gene include those described in U.S. Patent Publication Nos.
20050107316 and 20070265438; and PCT Publication No. WO 09/082,817,
the disclosures of which are herein incorporated by reference in
their entirety for all purposes. Non-limiting examples of siRNA
molecules targeting the Eg5 and XIAP genes include those described
in U.S. Patent Publication No. 20090149403, the disclosure of which
is herein incorporated by reference in its entirety for all
purposes. Non-limiting examples of siRNA molecules targeting the
CSN5 gene include those described in PCT Publication No. WO
09/129,319, the disclosure of which is herein incorporated by
reference in its entirety for all purposes. Non-limiting examples
of siRNA molecules targeting the COP1, CSN5, RBX1, HDAC2, CDK4,
WEE1, FOXM1, and RAM2 genes include those described in U.S.
Provisional Application No. 61/245,143, filed Sep. 23, 2009, the
disclosure of which is herein incorporated by reference in its
entirety for all purposes.
[0250] Additional examples of gene sequences associated with
tumorigenesis or cell transformation include translocation
sequences such as MLL fusion genes, BCR-ABL (Wilda et al.,
Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)),
TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and
AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003));
overexpressed sequences such as multidrug resistance genes (Nieth
et al., FEBS Lett., 545:144 (2003); Wu et al, Cancer Res. 63:1515
(2003)), cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et
al., Genes Dev., 16:2923 (2002)), beta-catenin (Verma et al., Clin
Cancer Res., 9:1291 (2003)), telomerase genes (Kosciolek et al.,
Mol Cancer Ther., 2:209 (2003)), c-MYC, N-MYC, BCL-2, growth factor
receptors (e.g., EGFR/ErbB 1 (Genbank Accession Nos.
NM.sub.--005228, NM.sub.--201282, NM.sub.--201283, and
NM.sub.--201284; see also, Nagy et al. Exp. Cell Res., 285:39-49
(2003)), ErbB2/HER-2 (Genbank Accession Nos. NM.sub.--004448 and
NM.sub.--001005862), ErbB3 (Genbank Accession Nos. NM.sub.--001982
and NM.sub.--001005915), and ErbB4 (Genbank Accession Nos.
NM.sub.--005235 and NM.sub.--001042599)), and mutated sequences
such as RAS (Tuschl and Borkhardt, Mol. Interventions, 2:158
(2002)). Non-limiting examples of siRNA molecules targeting the
EGFR gene include those described in U.S. Patent Publication No.
20090149403, the disclosure of which is herein incorporated by
reference in its entirety for all purposes. siRNA molecules that
target VEGFR genes are set forth in, e.g., GB 2396864; U.S. Patent
Publication No. 20040142895; and CA 2456444, the disclosures of
which are herein incorporated by reference in their entirety for
all purposes.
[0251] Silencing of sequences that encode DNA repair enzymes find
use in combination with the administration of chemotherapeutic
agents (Collis et al., Cancer Res., 63:1550 (2003)). Genes encoding
proteins associated with tumor migration are also target sequences
of interest, for example, integrins, selectins, and
metalloproteinases. The foregoing examples are not exclusive. Those
of skill in the art will understand that any whole or partial gene
sequence that facilitates or promotes tumorigenesis or cell
transformation, tumor growth, or tumor migration can be included as
a template sequence.
[0252] Angiogenic genes are able to promote the formation of new
vessels. Angiogenic genes of particular interest include, but are
not limited to, vascular endothelial growth factor (VEGF) (Reich et
al., Mol. Vis., 9:210 (2003)), placental growth factor (PGF),
VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and the like. siRNA molecules
that target VEGFR genes are set forth in, e.g., GB 2396864; U.S.
Patent Publication No. 20040142895; and CA 2456444, the disclosures
of which are herein incorporated by reference in their entirety for
all purposes.
[0253] Immunomodulator genes are genes that modulate one or more
immune responses. Examples of immunomodulator genes include,
without limitation, growth factors (e.g., TGF-.alpha., TGF-.beta.,
EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins
(e.g., IL-2, IL-4, IL-12 (Hill et al., J. Immunol., 171:691
(2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g.,
IFN-.alpha., IFN-.beta., IFN-.gamma., etc.), and TNF. Fas and Fas
ligand genes are also immunomodulator target sequences of interest
(Song et al., Nat. Med., 9:347 (2003)). Genes encoding secondary
signaling molecules in hematopoietic and lymphoid cells are also
included in the present invention, for example, Tec family kinases
such as Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS
Lett., 527:274 (2002)).
[0254] Cell receptor ligand genes include ligands that are able to
bind to cell surface receptors (e.g., cytokine receptors, growth
factor receptors, receptors with tyrosine kinase activity,
G-protein coupled receptors, insulin receptor, EPO receptor, etc.)
to modulate (e.g., inhibit) the physiological pathway that the
receptor is involved in (e.g., cell proliferation, tumorigenesis,
cell transformation, mitogenesis, etc.). Non-limiting examples of
cell receptor ligand genes include cytokines (e.g., TNF-.alpha.,
interferons such as IFN-.alpha., IFN-.beta., and IFN-.gamma.,
interleukins such as IL-1.alpha., IL-1.beta., IL-2, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23,
IL-27, chemokines, etc.), growth factors (e.g., EGF, HB-EGF, VEGF,
PEDF, SDGF, bFGF, HGF, TGF-.alpha., TGF-.beta., BMP1-BMP15, PDGF,
IGF, NGF, .beta.-NGF, BDNF, NT3, NT4, GDF-9, CGF, G-CSF, GM-CSF,
GDF-8, EPO, TPO, etc.), insulin, glucagon, G-protein coupled
receptor ligands, etc.
[0255] Templates coding for an expansion of trinucleotide repeats
(e.g., CAG repeats) find use in silencing pathogenic sequences in
neurodegenerative disorders caused by the expansion of
trinucleotide repeats, such as spinobulbular muscular atrophy and
Huntington's Disease (Caplen et al., Hum. Mol. Genet., 11:175
(2002)).
[0256] In addition to its utility in silencing the expression of
any of the above-described genes for therapeutic purposes, the
siRNA described herein are also useful in research and development
applications as well as diagnostic, prophylactic, prognostic,
clinical, and other healthcare applications. As a non-limiting
example, the siRNA can be used in target validation studies
directed at testing whether a gene of interest has the potential to
be a therapeutic target. The siRNA can also be used in target
identification studies aimed at discovering genes as potential
therapeutic targets.
[0257] e) Exemplary siRNA Embodiments
[0258] In some embodiments, each strand of the siRNA molecule
comprises from about 15 to about 60 nucleotides in length (e.g.,
about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in
length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
nucleotides in length). In one particular embodiment, the siRNA is
chemically synthesized. The siRNA molecules of the invention are
capable of silencing the expression of a target sequence in vitro
and/or in vivo.
[0259] In other embodiments, the siRNA comprises at least one
modified nucleotide. In certain embodiments, the siRNA comprises
one, two, three, four, five, six, seven, eight, nine, ten, or more
modified nucleotides in the double-stranded region. In particular
embodiments, less than about 50% (e.g., less than about 50%, 45%,
40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in the
double-stranded region of the siRNA comprise modified nucleotides.
In preferred embodiments, from about 1% to about 50% (e.g., from
about 5%-50%, 10%-50%, 15%-50%, 20%-50%, 25%-50%, 30%-50%, 35%-50%,
40%-50%, 45%-50%, 5%-45%, 10%-45%, 15%-45%, 20%-45%, 25%-45%,
30%-45%, 35%-45%, 40%-45%, 5%-40%, 10%-40%, 15%-40%, 20%-40%,
25%-40%, 30%-40%, 35%-40%, 5%-35%, 10%-35%, 15%-35%, 20%-35%,
25%-35%, 30%-35%, 5%-30%, 10%-30%, 15%-30%, 20%-30%, 25%-30%,
5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20%,
5%-15%, 10%-15%, or 5%-10%) of the nucleotides in the
double-stranded region of the siRNA comprise modified
nucleotides.
[0260] In further embodiments, the siRNA comprises modified
nucleotides including, but not limited to, 2'-O-methyl (2'OMe)
nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy
nucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, locked
nucleic acid (LNA) nucleotides, and mixtures thereof. In preferred
embodiments, the siRNA comprises 2'OMe nucleotides (e.g., 2'OMe
purine and/or pyrimidine nucleotides) such as, e.g.,
2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides,
2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, or
mixtures thereof. In one particular embodiment, the siRNA comprises
at least one 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide,
or mixtures thereof. In certain instances, the siRNA does not
comprise 2'OMe-cytosine nucleotides. In other embodiments, the
siRNA comprises a hairpin loop structure.
[0261] In certain embodiments, the siRNA comprises modified
nucleotides in one strand (i.e., sense or antisense) or both
strands of the double-stranded region of the siRNA molecule.
Preferably, uridine and/or guanosine nucleotides are modified at
selective positions in the double-stranded region of the siRNA
duplex. With regard to uridine nucleotide modifications, at least
one, two, three, four, five, six, or more of the uridine
nucleotides in the sense and/or antisense strand can be a modified
uridine nucleotide such as a 2'OMe-uridine nucleotide. In some
embodiments, every uridine nucleotide in the sense and/or antisense
strand is a 2'OMe-uridine nucleotide. With regard to guanosine
nucleotide modifications, at least one, two, three, four, five,
six, or more of the guanosine nucleotides in the sense and/or
antisense strand can be a modified guanosine nucleotide such as a
2'OMe-guanosine nucleotide. In some embodiments, every guanosine
nucleotide in the sense and/or antisense strand is a
2'OMe-guanosine nucleotide.
[0262] In certain embodiments, at least one, two, three, four,
five, six, seven, or more 5'-GU-3' motifs in an siRNA sequence may
be modified, e.g., by introducing mismatches to eliminate the
5'-GU-3' motifs and/or by introducing modified nucleotides such as
2'OMe nucleotides. The 5'-GU-3' motif can be in the sense strand,
the antisense strand, or both strands of the siRNA sequence. The
5'-GU-3' motifs may be adjacent to each other or, alternatively,
they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or
more nucleotides.
[0263] In some embodiments, a modified siRNA molecule is less
immunostimulatory than a corresponding unmodified siRNA sequence.
In such embodiments, the modified siRNA molecule with reduced
immunostimulatory properties advantageously retains RNAi activity
against the target sequence. In another embodiment, the
immunostimulatory properties of the modified siRNA molecule and its
ability to silence target gene expression can be balanced or
optimized by the introduction of minimal and selective 2'OMe
modifications within the siRNA sequence such as, e.g., within the
double-stranded region of the siRNA duplex. In certain instances,
the modified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less
immunostimulatory than the corresponding unmodified siRNA. It will
be readily apparent to those of skill in the art that the
immunostimulatory properties of the modified siRNA molecule and the
corresponding unmodified siRNA molecule can be determined by, for
example, measuring INF-.alpha. and/or IL-6 levels from about two to
about twelve hours after systemic administration in a mammal or
transfection of a mammalian responder cell using an appropriate
lipid-based delivery system (such as the SNALP delivery system
disclosed herein).
[0264] In other embodiments, a modified siRNA molecule has an
IC.sub.50 (i.e., half-maximal inhibitory concentration) less than
or equal to ten-fold that of the corresponding unmodified siRNA
(i.e., the modified siRNA has an IC.sub.50 that is less than or
equal to ten-times the IC.sub.50 of the corresponding unmodified
siRNA). In other embodiments, the modified siRNA has an IC.sub.50
less than or equal to three-fold that of the corresponding
unmodified siRNA sequence. In yet other embodiments, the modified
siRNA has an IC.sub.50 less than or equal to two-fold that of the
corresponding unmodified siRNA. It will be readily apparent to
those of skill in the art that a dose-response curve can be
generated and the IC.sub.50 values for the modified siRNA and the
corresponding unmodified siRNA can be readily determined using
methods known to those of skill in the art.
[0265] In another embodiment, an unmodified or modified siRNA
molecule is capable of silencing at least about 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the
expression of the target sequence relative to a negative control
(e.g., buffer only, an siRNA sequence that targets a different
gene, a scrambled siRNA sequence, etc.).
[0266] In yet another embodiment, a modified siRNA molecule is
capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the expression of the
target sequence relative to the corresponding unmodified siRNA
sequence.
[0267] In some embodiments, the siRNA molecule does not comprise
phosphate backbone modifications, e.g., in the sense and/or
antisense strand of the double-stranded region. In other
embodiments, the siRNA comprises one, two, three, four, or more
phosphate backbone modifications, e.g., in the sense and/or
antisense strand of the double-stranded region. In preferred
embodiments, the siRNA does not comprise phosphate backbone
modifications.
[0268] In further embodiments, the siRNA does not comprise 2'-deoxy
nucleotides, e.g., in the sense and/or antisense strand of the
double-stranded region. In yet further embodiments, the siRNA
comprises one, two, three, four, or more 2'-deoxy nucleotides,
e.g., in the sense and/or antisense strand of the double-stranded
region. In preferred embodiments, the siRNA does not comprise
2'-deoxy nucleotides.
[0269] In certain instances, the nucleotide at the 3'-end of the
double-stranded region in the sense and/or antisense strand is not
a modified nucleotide. In certain other instances, the nucleotides
near the 3'-end (e.g., within one, two, three, or four nucleotides
of the 3'-end) of the double-stranded region in the sense and/or
antisense strand are not modified nucleotides.
[0270] The siRNA molecules described herein may have 3' overhangs
of one, two, three, four, or more nucleotides on one or both sides
of the double-stranded region, or may lack overhangs (i.e., have
blunt ends) on one or both sides of the double-stranded region. In
certain embodiments, the 3' overhang on the sense and/or antisense
strand independently comprises one, two, three, four, or more
modified nucleotides such as 2'OMe nucleotides and/or any other
modified nucleotide described herein or known in the art.
[0271] In particular embodiments, siRNAs are administered using a
carrier system such as a nucleic acid-lipid particle. In a
preferred embodiment, the nucleic acid-lipid particle comprises:
(a) one or more siRNA molecules; (b) a cationic lipid of Formula I
or a salt thereof; and (c) a non-cationic lipid (e.g., DPPC, DSPC,
DSPE, and/or cholesterol). In certain instances, the nucleic
acid-lipid particle may further comprise a conjugated lipid that
prevents aggregation of particles (e.g., PEG-DAA and/or
POZ-DAA).
[0272] 2. Dicer-Substrate dsRNA
[0273] As used herein, the term "Dicer-substrate dsRNA" or
"precursor RNAi molecule" is intended to include any precursor
molecule that is processed in vivo by Dicer to produce an active
siRNA which is incorporated into the RISC complex for RNA
interference of a target gene.
[0274] In one embodiment, the Dicer-substrate dsRNA has a length
sufficient such that it is processed by Dicer to produce an siRNA.
According to this embodiment, the Dicer-substrate dsRNA comprises
(i) a first oligonucleotide sequence (also termed the sense strand)
that is between about 25 and about 60 nucleotides in length (e.g.,
about 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30
nucleotides in length), preferably between about 25 and about 30
nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides
in length), and (ii) a second oligonucleotide sequence (also termed
the antisense strand) that anneals to the first sequence under
biological conditions, such as the conditions found in the
cytoplasm of a cell. The second oligonucleotide sequence may be
between about 25 and about 60 nucleotides in length (e.g., about
25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in
length), and is preferably between about 25 and about 30
nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides
in length). In addition, a region of one of the sequences,
particularly of the antisense strand, of the Dicer-substrate dsRNA
has a sequence length of at least about 19 nucleotides, for
example, from about 19 to about 60 nucleotides (e.g., about 19-60,
19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25 nucleotides),
preferably from about 19 to about 23 nucleotides (e.g., 19, 20, 21,
22, or 23 nucleotides) that are sufficiently complementary to a
nucleotide sequence of the RNA produced from the target gene to
trigger an RNAi response.
[0275] In a second embodiment, the Dicer-substrate dsRNA has
several properties which enhance its processing by Dicer. According
to this embodiment, the dsRNA has a length sufficient such that it
is processed by Dicer to produce an siRNA and has at least one of
the following properties: (i) the dsRNA is asymmetric, e.g., has a
3'-overhang on the antisense strand; and/or (ii) the dsRNA has a
modified 3'-end on the sense strand to direct orientation of Dicer
binding and processing of the dsRNA to an active siRNA. According
to this latter embodiment, the sense strand comprises from about 22
to about 28 nucleotides and the antisense strand comprises from
about 24 to about 30 nucleotides.
[0276] In one embodiment, the Dicer-substrate dsRNA has an overhang
on the 3'-end of the antisense strand. In another embodiment, the
sense strand is modified for Dicer binding and processing by
suitable modifiers located at the 3'-end of the sense strand.
Suitable modifiers include nucleotides such as
deoxyribonucleotides, acyclonucleotides, and the like, and
sterically hindered molecules such as fluorescent molecules and the
like. When nucleotide modifiers are used, they replace
ribonucleotides in the dsRNA such that the length of the dsRNA does
not change. In another embodiment, the Dicer-substrate dsRNA has an
overhang on the 3'-end of the antisense strand and the sense strand
is modified for Dicer processing. In another embodiment, the 5'-end
of the sense strand has a phosphate. In another embodiment, the
5'-end of the antisense strand has a phosphate. In another
embodiment, the antisense strand or the sense strand or both
strands have one or more 2'-.beta.-methyl (2'OMe) modified
nucleotides. In another embodiment, the antisense strand contains
2'OMe modified nucleotides. In another embodiment, the antisense
stand contains a 3'-overhang that is comprised of 2'OMe modified
nucleotides. The antisense strand could also include additional
2'OMe modified nucleotides. The sense and antisense strands anneal
under biological conditions, such as the conditions found in the
cytoplasm of a cell. In addition, a region of one of the sequences,
particularly of the antisense strand, of the Dicer-substrate dsRNA
has a sequence length of at least about 19 nucleotides, wherein
these nucleotides are in the 21-nucleotide region adjacent to the
3'-end of the antisense strand and are sufficiently complementary
to a nucleotide sequence of the RNA produced from the target gene.
Further, in accordance with this embodiment, the Dicer-substrate
dsRNA may also have one or more of the following additional
properties: (a) the antisense strand has a right shift from the
typical 21-mer (i.e., the antisense strand includes nucleotides on
the right side of the molecule when compared to the typical
21-mer); (b) the strands may not be completely complementary, i.e.,
the strands may contain simple mismatch pairings; and (c) base
modifications such as locked nucleic acid(s) may be included in the
5'-end of the sense strand.
[0277] In a third embodiment, the sense strand comprises from about
25 to about 28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides),
wherein the 2 nucleotides on the 3'-end of the sense strand are
deoxyribonucleotides. The sense strand contains a phosphate at the
5'-end. The antisense strand comprises from about 26 to about 30
nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides) and contains
a 3'-overhang of 1-4 nucleotides. The nucleotides comprising the
3'-overhang are modified with 2'OMe modified ribonucleotides. The
antisense strand contains alternating 2'OMe modified nucleotides
beginning at the first monomer of the antisense strand adjacent to
the 3'-overhang, and extending 15-19 nucleotides from the first
monomer adjacent to the 3'-overhang. For example, for a
27-nucleotide antisense strand and counting the first base at the
5'-end of the antisense strand as position number 1, 2'OMe
modifications would be placed at bases 9, 11, 13, 15, 17, 19, 21,
23, 25, 26, and 27. In one embodiment, the Dicer-substrate dsRNA
has the following structure:
TABLE-US-00001 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3'
3'-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'
[0278] wherein "X"=RNA, "p"=a phosphate group, "X"=2'OMe RNA, "Y"
is an overhang domain comprised of 1, 2, 3, or 4 RNA monomers that
are optionally 2'OMe RNA monomers, and "D"=DNA. The top strand is
the sense strand, and the bottom strand is the antisense
strand.
[0279] In a fourth embodiment, the Dicer-substrate dsRNA has
several properties which enhance its processing by Dicer. According
to this embodiment, the dsRNA has a length sufficient such that it
is processed by Dicer to produce an siRNA and at least one of the
following properties: (i) the dsRNA is asymmetric, e.g., has a
3'-overhang on the sense strand; and (ii) the dsRNA has a modified
3'-end on the antisense strand to direct orientation of Dicer
binding and processing of the dsRNA to an active siRNA. According
to this embodiment, the sense strand comprises from about 24 to
about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30
nucleotides) and the antisense strand comprises from about 22 to
about 28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28
nucleotides). In one embodiment, the Dicer-substrate dsRNA has an
overhang on the 3'-end of the sense strand. In another embodiment,
the antisense strand is modified for Dicer binding and processing
by suitable modifiers located at the 3'-end of the antisense
strand. Suitable modifiers include nucleotides such as
deoxyribonucleotides, acyclonucleotides, and the like, and
sterically hindered molecules such as fluorescent molecules and the
like. When nucleotide modifiers are used, they replace
ribonucleotides in the dsRNA such that the length of the dsRNA does
not change. In another embodiment, the dsRNA has an overhang on the
3'-end of the sense strand and the antisense strand is modified for
Dicer processing. In one embodiment, the antisense strand has a
5'-phosphate. The sense and antisense strands anneal under
biological conditions, such as the conditions found in the
cytoplasm of a cell. In addition, a region of one of the sequences,
particularly of the antisense strand, of the dsRNA has a sequence
length of at least 19 nucleotides, wherein these nucleotides are
adjacent to the 3'-end of antisense strand and are sufficiently
complementary to a nucleotide sequence of the RNA produced from the
target gene. Further, in accordance with this embodiment, the
Dicer-substrate dsRNA may also have one or more of the following
additional properties: (a) the antisense strand has a left shift
from the typical 21-mer (i.e., the antisense strand includes
nucleotides on the left side of the molecule when compared to the
typical 21-mer); and (b) the strands may not be completely
complementary, i.e., the strands may contain simple mismatch
pairings.
[0280] In a preferred embodiment, the Dicer-substrate dsRNA has an
asymmetric structure, with the sense strand having a 25-base pair
length, and the antisense strand having a 27-base pair length with
a 2 base 3'-overhang. In certain instances, this dsRNA having an
asymmetric structure further contains 2 deoxynucleotides at the
3'-end of the sense strand in place of two of the ribonucleotides.
In certain other instances, this dsRNA having an asymmetric
structure further contains 2'OMe modifications at positions 9, 11,
13, 15, 17, 19, 21, 23, and 25 of the antisense strand (wherein the
first base at the 5'-end of the antisense strand is position 1). In
certain additional instances, this dsRNA having an asymmetric
structure further contains a 3'-overhang on the antisense strand
comprising 1, 2, 3, or 4 2'OMe nucleotides (e.g., a 3'-overhang of
2'OMe nucleotides at positions 26 and 27 on the antisense
strand).
[0281] In another embodiment, Dicer-substrate dsRNAs may be
designed by first selecting an antisense strand siRNA sequence
having a length of at least 19 nucleotides. In some instances, the
antisense siRNA is modified to include about 5 to about 11
ribonucleotides on the 5'-end to provide a length of about 24 to
about 30 nucleotides. When the antisense strand has a length of 21
nucleotides, 3-9, preferably 4-7, or more preferably 6 nucleotides
may be added on the 5'-end. Although the added ribonucleotides may
be complementary to the target gene sequence, full complementarity
between the target sequence and the antisense siRNA is not
required. That is, the resultant antisense siRNA is sufficiently
complementary with the target sequence. A sense strand is then
produced that has about 22 to about 28 nucleotides. The sense
strand is substantially complementary with the antisense strand to
anneal to the antisense strand under biological conditions. In one
embodiment, the sense strand is synthesized to contain a modified
3'-end to direct Dicer processing of the antisense strand. In
another embodiment, the antisense strand of the dsRNA has a
3'-overhang. In a further embodiment, the sense strand is
synthesized to contain a modified 3'-end for Dicer binding and
processing and the antisense strand of the dsRNA has a
3'-overhang.
[0282] In a related embodiment, the antisense siRNA may be modified
to include about 1 to about 9 ribonucleotides on the 5'-end to
provide a length of about 22 to about 28 nucleotides. When the
antisense strand has a length of 21 nucleotides, 1-7, preferably
2-5, or more preferably 4 ribonucleotides may be added on the
3'-end. The added ribonucleotides may have any sequence. Although
the added ribonucleotides may be complementary to the target gene
sequence, full complementarity between the target sequence and the
antisense siRNA is not required. That is, the resultant antisense
siRNA is sufficiently complementary with the target sequence. A
sense strand is then produced that has about 24 to about 30
nucleotides. The sense strand is substantially complementary with
the antisense strand to anneal to the antisense strand under
biological conditions. In one embodiment, the antisense strand is
synthesized to contain a modified 3'-end to direct Dicer
processing. In another embodiment, the sense strand of the dsRNA
has a 3'-overhang. In a further embodiment, the antisense strand is
synthesized to contain a modified 3'-end for Dicer binding and
processing and the sense strand of the dsRNA has a 3'-overhang.
[0283] Suitable Dicer-substrate dsRNA sequences can be identified,
synthesized, and modified using any means known in the art for
designing, synthesizing, and modifying siRNA sequences. In
particular embodiments, Dicer-substrate dsRNAs are administered
using a carrier system such as a nucleic acid-lipid particle. In a
preferred embodiment, the nucleic acid-lipid particle comprises:
(a) one or more Dicer-substrate dsRNA molecules; (b) a cationic
lipid of Formula I or a salt thereof; and (c) a non-cationic lipid
(e.g., DPPC, DSPC, DSPE, and/or cholesterol). In certain instances,
the nucleic acid-lipid particle may further comprise a conjugated
lipid that prevents aggregation of particles (e.g., PEG-DAA and/or
POZ-DAA).
[0284] Additional embodiments related to the Dicer-substrate dsRNAs
of the invention, as well as methods of designing and synthesizing
such dsRNAs, are described in U.S. Patent Publication Nos.
20050244858, 20050277610, and 20070265220, and U.S. application
Ser. No. 12/794,701, filed Jun. 4, 2010, the disclosures of which
are herein incorporated by reference in their entirety for all
purposes.
[0285] 3. shRNA
[0286] A "small hairpin RNA" or "short hairpin RNA" or "shRNA"
includes a short RNA sequence that makes a tight hairpin turn that
can be used to silence gene expression via RNA interference. The
shRNAs of the invention may be chemically synthesized or
transcribed from a transcriptional cassette in a DNA plasmid. The
shRNA hairpin structure is cleaved by the cellular machinery into
siRNA, which is then bound to the RNA-induced silencing complex
(RISC).
[0287] The shRNAs of the invention are typically about 15-60,
15-50, or 15-40 (duplex) nucleotides in length, more typically
about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and
are preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in
length (e.g., each complementary sequence of the double-stranded
shRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in
length, preferably about 20-24, 21-22, or 21-23 nucleotides in
length, and the double-stranded shRNA is about 15-60, 15-50, 15-40,
15-30, 15-25, or 19-25 base pairs in length, preferably about
18-22, 19-20, or 19-21 base pairs in length). shRNA duplexes may
comprise 3' overhangs of about 1 to about 4 nucleotides or about 2
to about 3 nucleotides on the antisense strand and/or 5'-phosphate
termini on the sense strand. In some embodiments, the shRNA
comprises a sense strand and/or antisense strand sequence of from
about 15 to about 60 nucleotides in length (e.g., about 15-60,
15-55, 15-50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in
length), preferably from about 19 to about 40 nucleotides in length
(e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length),
more preferably from about 19 to about 23 nucleotides in length
(e.g., 19, 20, 21, 22, or 23 nucleotides in length).
[0288] Non-limiting examples of shRNA include a double-stranded
polynucleotide molecule assembled from a single-stranded molecule,
where the sense and antisense regions are linked by a nucleic
acid-based or non-nucleic acid-based linker; and a double-stranded
polynucleotide molecule with a hairpin secondary structure having
self-complementary sense and antisense regions. In preferred
embodiments, the sense and antisense strands of the shRNA are
linked by a loop structure comprising from about 1 to about 25
nucleotides, from about 2 to about 20 nucleotides, from about 4 to
about 15 nucleotides, from about 5 to about 12 nucleotides, or 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, or more nucleotides.
[0289] Suitable shRNA sequences can be identified, synthesized, and
modified using any means known in the art for designing,
synthesizing, and modifying siRNA sequences. In particular
embodiments, shRNAs are administered using a carrier system such as
a nucleic acid-lipid particle. In a preferred embodiment, the
nucleic acid-lipid particle comprises: (a) one or more shRNA
molecules; (b) a cationic lipid of Formula I or a salt thereof; and
(c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or
cholesterol). In certain instances, the nucleic acid-lipid particle
may further comprise a conjugated lipid that prevents aggregation
of particles (e.g., PEG-DAA and/or POZ-DAA).
[0290] Additional embodiments related to the shRNAs of the
invention, as well as methods of designing and synthesizing such
shRNAs, are described in U.S. application Ser. No. 12/794,701,
filed Jun. 4, 2010, the disclosure of which is herein incorporated
by reference in its entirety for all purposes.
[0291] 4. aiRNA
[0292] Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit
the RNA-induced silencing complex (RISC) and lead to effective
silencing of a variety of genes in mammalian cells by mediating
sequence-specific cleavage of the target sequence between
nucleotide 10 and 11 relative to the 5' end of the antisense strand
(Sun et al., Nat. Biotech., 26:1379-1382 (2008)). Typically, an
aiRNA molecule comprises a short RNA duplex having a sense strand
and an antisense strand, wherein the duplex contains overhangs at
the 3' and 5' ends of the antisense strand. The aiRNA is generally
asymmetric because the sense strand is shorter on both ends when
compared to the complementary antisense strand. In some aspects,
aiRNA molecules may be designed, synthesized, and annealed under
conditions similar to those used for siRNA molecules. As a
non-limiting example, aiRNA sequences may be selected and generated
using the methods described above for selecting siRNA
sequences.
[0293] In another embodiment, aiRNA duplexes of various lengths
(e.g., about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base
pairs, more typically 12, 13, 14, 15, 16, 17, 18, 19, or base
pairs) may be designed with overhangs at the 3' and 5' ends of the
antisense strand to target an mRNA of interest. In certain
instances, the sense strand of the aiRNA molecule is about 10-25,
12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more
typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in
length. In certain other instances, the antisense strand of the
aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in
length, more typically about 15-30, 15-25, or 19-25 nucleotides in
length, and is preferably about 20-24, 21-22, or 21-23 nucleotides
in length.
[0294] In some embodiments, the 5' antisense overhang contains one,
two, three, four, or more nontargeting nucleotides (e.g., "AA",
"UU", "dTdT", etc.). In other embodiments, the 3' antisense
overhang contains one, two, three, four, or more nontargeting
nucleotides (e.g., "AA", "UU", "dTdT", etc.). In certain aspects,
the aiRNA molecules described herein may comprise one or more
modified nucleotides, e.g., in the double-stranded (duplex) region
and/or in the antisense overhangs. As a non-limiting example, aiRNA
sequences may comprise one or more of the modified nucleotides
described above for siRNA sequences. In a preferred embodiment, the
aiRNA molecule comprises 2'OMe nucleotides such as, for example,
2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures
thereof.
[0295] In certain embodiments, aiRNA molecules may comprise an
antisense strand which corresponds to the antisense strand of an
siRNA molecule, e.g., one of the siRNA molecules described herein.
In particular embodiments, aiRNAs are administered using a carrier
system such as a nucleic acid-lipid particle. In a preferred
embodiment, the nucleic acid-lipid particle comprises: (a) one or
more aiRNA molecules; (b) a cationic lipid of Formula I or a salt
thereof; and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE,
and/or cholesterol). In certain instances, the nucleic acid-lipid
particle may further comprise a conjugated lipid that prevents
aggregation of particles (e.g., PEG-DAA and/or POZ-DAA).
[0296] Suitable aiRNA sequences can be identified, synthesized, and
modified using any means known in the art for designing,
synthesizing, and modifying siRNA sequences. Additional embodiments
related to the aiRNA molecules of the invention are described in
U.S. Patent Publication No. 20090291131 and PCT Publication No. WO
09/127,060, the disclosures of which are herein incorporated by
reference in their entirety for all purposes.
[0297] 5. miRNA
[0298] Generally, microRNAs (miRNA) are single-stranded RNA
molecules of about 21-23 nucleotides in length which regulate gene
expression. miRNAs are encoded by genes from whose DNA they are
transcribed, but miRNAs are not translated into protein (non-coding
RNA); instead, each primary transcript (a pri-miRNA) is processed
into a short stem-loop structure called a pre-miRNA and finally
into a functional mature miRNA. Mature miRNA molecules are either
partially or completely complementary to one or more messenger RNA
(mRNA) molecules, and their main function is to downregulate gene
expression. The identification of miRNA molecules is described,
e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau et al.,
Science, 294:858-862; and Lee et al., Science, 294:862-864.
[0299] The genes encoding miRNA are much longer than the processed
mature miRNA molecule. miRNA are first transcribed as primary
transcripts or pri-miRNA with a cap and poly-A tail and processed
to short, .about.70-nucleotide stem-loop structures known as
pre-miRNA in the cell nucleus. This processing is performed in
animals by a protein complex known as the Microprocessor complex,
consisting of the nuclease Drosha and the double-stranded RNA
binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)).
These pre-miRNA are then processed to mature miRNA in the cytoplasm
by interaction with the endonuclease Dicer, which also initiates
the formation of the RNA-induced silencing complex (RISC)
(Bernstein et al., Nature, 409:363-366 (2001). Either the sense
strand or antisense strand of DNA can function as templates to give
rise to miRNA.
[0300] When Dicer cleaves the pre-miRNA stem-loop, two
complementary short RNA molecules are formed, but only one is
integrated into the RISC complex. This strand is known as the guide
strand and is selected by the argonaute protein, the catalytically
active RNase in the RISC complex, on the basis of the stability of
the 5' end (Preall et al., Curr. Biol., 16:530-535 (2006)). The
remaining strand, known as the anti-guide or passenger strand, is
degraded as a RISC complex substrate (Gregory et al., Cell,
123:631-640 (2005)). After integration into the active RISC
complex, miRNAs base pair with their complementary mRNA molecules
and induce target mRNA degradation and/or translational
silencing.
[0301] Mammalian miRNA molecules are usually complementary to a
site in the 3' UTR of the target mRNA sequence. In certain
instances, the annealing of the miRNA to the target mRNA inhibits
protein translation by blocking the protein translation machinery.
In certain other instances, the annealing of the miRNA to the
target mRNA facilitates the cleavage and degradation of the target
mRNA through a process similar to RNA interference (RNAi). miRNA
may also target methylation of genomic sites which correspond to
targeted mRNA. Generally, miRNA function in association with a
complement of proteins collectively termed the miRNP.
[0302] In certain aspects, the miRNA molecules described herein are
about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40
nucleotides in length, more typically about 15-30, 15-25, or 19-25
nucleotides in length, and are preferably about 20-24, 21-22, or
21-23 nucleotides in length. In certain other aspects, miRNA
molecules may comprise one or more modified nucleotides. As a
non-limiting example, miRNA sequences may comprise one or more of
the modified nucleotides described above for siRNA sequences. In a
preferred embodiment, the miRNA molecule comprises 2'OMe
nucleotides such as, for example, 2'OMe-guanosine nucleotides,
2'OMe-uridine nucleotides, or mixtures thereof.
[0303] In particular embodiments, miRNAs are administered using a
carrier system such as a nucleic acid-lipid particle. In a
preferred embodiment, the nucleic acid-lipid particle comprises:
(a) one or more miRNA molecules; (b) a cationic lipid of Formula I
or a salt thereof; and (c) a non-cationic lipid (e.g., DPPC, DSPC,
DSPE, and/or cholesterol). In certain instances, the nucleic
acid-lipid particle may further comprise a conjugated lipid that
prevents aggregation of particles (e.g., PEG-DAA and/or
POZ-DAA).
[0304] In other embodiments, one or more agents that block the
activity of an miRNA targeting an mRNA of interest are administered
using a lipid particle of the invention (e.g., a nucleic acid-lipid
particle such as SNALP). Examples of blocking agents include, but
are not limited to, steric blocking oligonucleotides, locked
nucleic acid oligonucleotides, and Morpholino oligonucleotides.
Such blocking agents may bind directly to the miRNA or to the miRNA
binding site on the target mRNA.
[0305] Additional embodiments related to the miRNA molecules of the
invention are described in U.S. Patent Publication No. 20090291131
and PCT Publication No. WO 09/127,060, the disclosures of which are
herein incorporated by reference in their entirety for all
purposes.
[0306] 6. Antisense Oligonucleotides
[0307] In one embodiment, the nucleic acid is an antisense
oligonucleotide directed to a target gene or sequence of interest.
The terms "antisense oligonucleotide" or "antisense" include
oligonucleotides that are complementary to a targeted
polynucleotide sequence. Antisense oligonucleotides are single
strands of DNA or RNA that are complementary to a chosen sequence.
Antisense RNA oligonucleotides prevent the translation of
complementary RNA strands by binding to the RNA. Antisense DNA
oligonucleotides can be used to target a specific, complementary
(coding or non-coding) RNA. If binding occurs, this DNA/RNA hybrid
can be degraded by the enzyme RNase H. In a particular embodiment,
antisense oligonucleotides comprise from about 10 to about 60
nucleotides, more preferably from about 15 to about 30 nucleotides.
The term also encompasses antisense oligonucleotides that may not
be exactly complementary to the desired target gene. Thus, the
invention can be utilized in instances where non-target
specific-activities are found with antisense, or where an antisense
sequence containing one or more mismatches with the target sequence
is the most preferred for a particular use.
[0308] Antisense oligonucleotides have been demonstrated to be
effective and targeted inhibitors of protein synthesis, and,
consequently, can be used to specifically inhibit protein synthesis
by a targeted gene. The efficacy of antisense oligonucleotides for
inhibiting protein synthesis is well established. For example, the
synthesis of polygalactauronase and the muscarine type 2
acetylcholine receptor are inhibited by antisense oligonucleotides
directed to their respective mRNA sequences (see, U.S. Pat. Nos.
5,739,119 and 5,759,829). Furthermore, examples of antisense
inhibition have been demonstrated with the nuclear protein cyclin,
the multiple drug resistance gene (MDR1), ICAM-1, E-selectin,
STK-1, striatal GABAA receptor, and human EGF (see, Jaskulski et
al., Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer
Commun., 1:225-32 (1989); Penis et al., Brain Res Mol Brain Res.,
15; 57:310-20 (1998); and U.S. Pat. Nos. 5,801,154; 5,789,573;
5,718,709 and 5,610,288). Moreover, antisense constructs have also
been described that inhibit and can be used to treat a variety of
abnormal cellular proliferations, e.g., cancer (see, U.S. Pat. Nos.
5,747,470; 5,591,317; and 5,783,683). The disclosures of these
references are herein incorporated by reference in their entirety
for all purposes.
[0309] Methods of producing antisense oligonucleotides are known in
the art and can be readily adapted to produce an antisense
oligonucleotide that targets any polynucleotide sequence. Selection
of antisense oligonucleotide sequences specific for a given target
sequence is based upon analysis of the chosen target sequence and
determination of secondary structure, T.sub.m, binding energy, and
relative stability. Antisense oligonucleotides may be selected
based upon their relative inability to form dimers, hairpins, or
other secondary structures that would reduce or prohibit specific
binding to the target mRNA in a host cell. Highly preferred target
regions of the mRNA include those regions at or near the AUG
translation initiation codon and those sequences that are
substantially complementary to 5' regions of the mRNA. These
secondary structure analyses and target site selection
considerations can be performed, for example, using v.4 of the
OLIGO primer analysis software (Molecular Biology Insights) and/or
the BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids
Res., 25:3389-402 (1997)).
[0310] 7. Ribozymes
[0311] According to another embodiment of the invention, nucleic
acid-lipid particles are associated with ribozymes. Ribozymes are
RNA-protein complexes having specific catalytic domains that
possess endonuclease activity (see, Kim et al., Proc. Natl. Acad.
Sci. USA., 84:8788-92 (1987); and Forster et al., Cell, 49:211-20
(1987)). For example, a large number of ribozymes accelerate
phosphoester transfer reactions with a high degree of specificity,
often cleaving only one of several phosphoesters in an
oligonucleotide substrate (see, Cech et al., Cell, 27:487-96
(1981); Michel et al., J. Mol. Biol., 216:585-610 (1990);
Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity
has been attributed to the requirement that the substrate bind via
specific base-pairing interactions to the internal guide sequence
("IGS") of the ribozyme prior to chemical reaction.
[0312] At least six basic varieties of naturally-occurring
enzymatic RNA molecules are known presently. Each can catalyze the
hydrolysis of RNA phosphodiester bonds in trans (and thus can
cleave other RNA molecules) under physiological conditions. In
general, enzymatic nucleic acids act by first binding to a target
RNA. Such binding occurs through the target binding portion of an
enzymatic nucleic acid which is held in close proximity to an
enzymatic portion of the molecule that acts to cleave the target
RNA. Thus, the enzymatic nucleic acid first recognizes and then
binds a target RNA through complementary base-pairing, and once
bound to the correct site, acts enzymatically to cut the target
RNA. Strategic cleavage of such a target RNA will destroy its
ability to direct synthesis of an encoded protein. After an
enzymatic nucleic acid has bound and cleaved its RNA target, it is
released from that RNA to search for another target and can
repeatedly bind and cleave new targets.
[0313] The enzymatic nucleic acid molecule may be formed in a
hammerhead, hairpin, hepatitis .delta. virus, group I intron or
RNaseP RNA (in association with an RNA guide sequence), or
Neurospora VS RNA motif, for example. Specific examples of
hammerhead motifs are described in, e.g., Rossi et al., Nucleic
Acids Res., 20:4559-65 (1992). Examples of hairpin motifs are
described in, e.g., EP 0360257, Hampel et al., Biochemistry,
28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304
(1990); and U.S. Pat. No. 5,631,359. An example of the hepatitis
.delta. virus motif is described in, e.g., Perrotta et al.,
Biochemistry, 31:11843-52 (1992). An example of the RNaseP motif is
described in, e.g., Guerrier-Takada et al., Cell, 35:849-57 (1983).
Examples of the Neurospora VS RNA ribozyme motif is described in,
e.g., Saville et al., Cell, 61:685-96 (1990); Saville et al., Proc.
Natl. Acad. Sci. USA, 88:8826-30 (1991); Collins et al.,
Biochemistry, 32:2795-9 (1993). An example of the Group I intron is
described in, e.g., U.S. Pat. No. 4,987,071. Important
characteristics of enzymatic nucleic acid molecules used according
to the invention are that they have a specific substrate binding
site which is complementary to one or more of the target gene DNA
or RNA regions, and that they have nucleotide sequences within or
surrounding that substrate binding site which impart an RNA
cleaving activity to the molecule. Thus, the ribozyme constructs
need not be limited to specific motifs mentioned herein. The
disclosures of these references are herein incorporated by
reference in their entirety for all purposes.
[0314] Methods of producing a ribozyme targeted to any
polynucleotide sequence are known in the art. Ribozymes may be
designed as described in, e.g., PCT Publication Nos. WO 93/23569
and WO 94/02595, and synthesized to be tested in vitro and/or in
vivo as described therein. The disclosures of these PCT
publications are herein incorporated by reference in their entirety
for all purposes.
[0315] Ribozyme activity can be optimized by altering the length of
the ribozyme binding arms or chemically synthesizing ribozymes with
modifications that prevent their degradation by serum ribonucleases
(see, e.g., PCT Publication Nos. WO 92/07065, WO 93/15187, WO
91/03162, and WO 94/13688; EP 92110298.4; and U.S. Pat. No.
5,334,711, which describe various chemical modifications that can
be made to the sugar moieties of enzymatic RNA molecules, the
disclosures of which are each herein incorporated by reference in
their entirety for all purposes), modifications which enhance their
efficacy in cells, and removal of stem II bases to shorten RNA
synthesis times and reduce chemical requirements.
[0316] 8. Immunostimulatory Oligonucleotides
[0317] Nucleic acids associated with the lipid particles of the
present invention may be immunostimulatory, including
immunostimulatory oligonucleotides (ISS; single- or
double-stranded) capable of inducing an immune response when
administered to a subject, which may be a mammal such as a human.
ISS include, e.g., certain palindromes leading to hairpin secondary
structures (see, Yamamoto et al., J. Immunol., 148:4072-6 (1992)),
or CpG motifs, as well as other known ISS features (such as multi-G
domains; see; PCT Publication No. WO 96/11266, the disclosure of
which is herein incorporated by reference in its entirety for all
purposes).
[0318] Immunostimulatory nucleic acids are considered to be
non-sequence specific when it is not required that they
specifically bind to and reduce the expression of a target sequence
in order to provoke an immune response. Thus, certain
immunostimulatory nucleic acids may comprise a sequence
corresponding to a region of a naturally-occurring gene or mRNA,
but they may still be considered non-sequence specific
immunostimulatory nucleic acids.
[0319] In one embodiment, the immunostimulatory nucleic acid or
oligonucleotide comprises at least one CpG dinucleotide. The
oligonucleotide or CpG dinucleotide may be unmethylated or
methylated. In another embodiment, the immunostimulatory nucleic
acid comprises at least one CpG dinucleotide having a methylated
cytosine. In one embodiment, the nucleic acid comprises a single
CpG dinucleotide, wherein the cytosine in the CpG dinucleotide is
methylated. In an alternative embodiment, the nucleic acid
comprises at least two CpG dinucleotides, wherein at least one
cytosine in the CpG dinucleotides is methylated. In a further
embodiment, each cytosine in the CpG dinucleotides present in the
sequence is methylated. In another embodiment, the nucleic acid
comprises a plurality of CpG dinucleotides, wherein at least one of
the CpG dinucleotides comprises a methylated cytosine. Examples of
immunostimulatory oligonucleotides suitable for use in the
compositions and methods of the present invention are described in
PCT Publication Nos. WO 02/069369, WO 01/15726, and WO 09/086,558;
U.S. Pat. No. 6,406,705; and Raney et al., J. Pharm. Exper. Ther.,
298:1185-92 (2001), the disclosures of which are herein
incorporated by reference in their entirety for all purposes. In
certain embodiments, the oligonucleotides used in the compositions
and methods of the invention have a phosphodiester ("PO") backbone
or a phosphorothioate ("PS") backbone, and/or at least one
methylated cytosine residue in a CpG motif.
[0320] B. Other Active Agents
[0321] In certain embodiments, the active agent associated with the
lipid particles of the invention may comprise one or more
therapeutic proteins, polypeptides, or small organic molecules or
compounds. Non-limiting examples of such therapeutically effective
agents or drugs include oncology drugs (e.g., chemotherapy drugs,
hormonal therapeutic agents, immunotherapeutic agents,
radiotherapeutic agents, etc.), lipid-lowering agents, anti-viral
drugs, anti-inflammatory compounds, antidepressants, stimulants,
analgesics, antibiotics, birth control medication, antipyretics,
vasodilators, anti-angiogenics, cytovascular agents, signal
transduction inhibitors, cardiovascular drugs such as
anti-arrhythmic agents, hormones, vasoconstrictors, and steroids.
These active agents may be administered alone in the lipid
particles of the invention, or in combination (e.g.,
co-administered) with lipid particles of the invention comprising
nucleic acid such as interfering RNA.
[0322] Non-limiting examples of chemotherapy drugs include
platinum-based drugs (e.g., oxaliplatin, cisplatin, carboplatin,
spiroplatin, iproplatin, satraplatin, etc.), alkylating agents
(e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan,
melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas,
etc.), anti-metabolites (e.g., 5-fluorouracil (5-FU), azathioprine,
methotrexate, leucovorin, capecitabine, cytarabine, floxuridine,
fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.), plant
alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine,
podophyllotoxin, paclitaxel (taxol), docetaxel, etc.),
topoisomerase inhibitors (e.g., irinotecan (CPT-11; Camptosar),
topotecan, amsacrine, etoposide (VP16), etoposide phosphate,
teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,
adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin,
mitomycin, mitoxantrone, plicamycin, etc.), tyrosine kinase
inhibitors (e.g., gefitinib (Iressa.RTM.), sunitinib (Sutent.RTM.;
SU11248), erlotinib (Tarceva.RTM.; OSI-1774), lapatinib (GW572016;
GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib
(PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec.RTM.;
STI571), dasatinib (BMS-354825), leflunomide (SU101), vandetanib
(Zactima.TM.; ZD6474), etc.), pharmaceutically acceptable salts
thereof, stereoisomers thereof, derivatives thereof, analogs
thereof, and combinations thereof.
[0323] Examples of conventional hormonal therapeutic agents
include, without limitation, steroids (e.g., dexamethasone),
finasteride, aromatase inhibitors, tamoxifen, and goserelin as well
as other gonadotropin-releasing hormone agonists (GnRH).
[0324] Examples of conventional immunotherapeutic agents include,
but are not limited to, immunostimulants (e.g., Bacillus
Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon,
etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2,
anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies),
immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin
conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin
conjugate, etc.), and radioimmunotherapy (e.g., anti-CD20
monoclonal antibody conjugated to .sup.111In, .sup.90Y, or
.sup.131I, etc.).
[0325] Examples of conventional radiotherapeutic agents include,
but are not limited to, radionucleotides such as .sup.47Sc,
.sup.64Cu, .sup.67Cu, .sup.89Sr, .sup.86Y, .sup.87Y, .sup.90Y,
.sup.105Rh, .sup.111Ag, .sup.111In, .sup.117mSn, .sup.149Pm,
.sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.186Re, .sup.188Re,
.sup.211At, and .sup.212Bi, optionally conjugated to antibodies
directed against tumor antigens.
[0326] Additional oncology drugs that may be used according to the
invention include, but are not limited to, alkeran, allopurinol,
altretamine, amifostine, anastrozole, araC, arsenic trioxide,
bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine,
cyclosporin A, cytosine arabinoside, cytoxan, dexrazoxane, DTIC,
estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydrea,
hydroxyurea, idarubicin, interferon, letrozole, leustatin,
leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen,
mithramycin, nitrogen mustard, pamidronate, Pegademase,
pentostatin, porfimer sodium, prednisone, rituxan, streptozocin,
STI-571, taxotere, temozolamide, VM-26, toremifene, tretinoin,
ATRA, valrubicin, and velban. Other examples of oncology drugs that
may be used according to the invention are ellipticin and
ellipticin analogs or derivatives, epothilones, intracellular
kinase inhibitors, and camptothecins.
[0327] Non-limiting examples of lipid-lowering agents for treating
a lipid disease or disorder associated with elevated triglycerides,
cholesterol, and/or glucose include statins, fibrates, ezetimibe,
thiazolidinediones, niacin, beta-blockers, nitroglycerin, calcium
antagonists, fish oil, and mixtures thereof.
[0328] Examples of anti-viral drugs include, but are not limited
to, abacavir, aciclovir, acyclovir, adefovir, amantadine,
amprenavir, arbidol, atazanavir, atripla, cidofovir, combivir,
darunavir, delavirdine, didanosine, docosanol, edoxudine,
efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors,
famciclovir, fixed dose combinations, fomivirsen, fosamprenavir,
foscarnet, fosfonet, fusion inhibitors, ganciclovir, ibacitabine,
immunovir, idoxuridine, imiquimod, indinavir, inosine, integrase
inhibitors, interferon type III (e.g., IFN-.lamda., molecules such
as IFN-.lamda.1, IFN-.lamda.2, and IFN-.lamda.3), interferon type
II (e.g., IFN-.gamma.), interferon type I (e.g., IFN-.alpha. such
as PEGylated IFN-.alpha., IFN-.beta., IFN-.kappa., IFN-.delta.,
IFN-.epsilon., IFN-.tau., IFN-.omega., and IFN-.THETA., interferon,
lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine,
nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir,
penciclovir, peramivir, pleconaril, podophyllotoxin, protease
inhibitors, reverse transcriptase inhibitors, ribavirin,
rimantadine, ritonavir, saquinavir, stavudine, synergistic
enhancers, tenofovir, tenofovir disoproxil, tipranavir,
trifluridine, trizivir, tromantadine, truvada, valaciclovir,
valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,
zanamivir, zidovudine, pharmaceutically acceptable salts thereof,
stereoisomers thereof, derivatives thereof, analogs thereof, and
mixtures thereof.
V. Lipid Particles
[0329] In certain aspects, the present invention provides lipid
particles comprising one or more of the cationic (amino) lipids or
salts thereof described herein. In some embodiments, the lipid
particles of the invention further comprise one or more
non-cationic lipids. In other embodiments, the lipid particles
further comprise one or more conjugated lipids capable of reducing
or inhibiting particle aggregation. In additional embodiments, the
lipid particles further comprise one or more active agents or
therapeutic agents such as therapeutic nucleic acids (e.g.,
interfering RNA such as siRNA).
[0330] Lipid particles include, but are not limited to, lipid
vesicles such as liposomes. As used herein, a lipid vesicle
includes a structure having lipid-containing membranes enclosing an
aqueous interior. In particular embodiments, lipid vesicles
comprising one or more of the cationic lipids described herein are
used to encapsulate nucleic acids within the lipid vesicles. In
other embodiments, lipid vesicles comprising one or more of the
cationic lipids described herein are complexed with nucleic acids
to form lipoplexes.
[0331] The lipid particles of the invention typically comprise an
active agent or therapeutic agent, a cationic lipid, a non-cationic
lipid, and a conjugated lipid that inhibits aggregation of
particles. In some embodiments, the active agent or therapeutic
agent is fully encapsulated within the lipid portion of the lipid
particle such that the active agent or therapeutic agent in the
lipid particle is resistant in aqueous solution to enzymatic
degradation, e.g., by a nuclease or protease. In other embodiments,
the lipid particles described herein are substantially non-toxic to
mammals such as humans. The lipid particles of the invention
typically have a mean diameter of from about 30 nm to about 150 nm,
from about 40 nm to about 150 nm, from about 50 nm to about 150 nm,
from about 60 nm to about 130 nm, from about 70 nm to about 110 nm,
or from about 70 to about 90 nm. The lipid particles of the
invention also typically have a lipid:therapeutic agent (e.g.,
lipid:nucleic acid) ratio (mass/mass ratio) of from about 1:1 to
about 100:1, from about 1:1 to about 50:1, from about 2:1 to about
25:1, from about 3:1 to about 20:1, from about 5:1 to about 15:1,
or from about 5:1 to about 10:1.
[0332] In preferred embodiments, the lipid particles of the
invention are serum-stable nucleic acid-lipid particles (SNALP)
which comprise an interfering RNA (e.g., siRNA, Dicer-substrate
dsRNA, shRNA, aiRNA, and/or miRNA), a cationic lipid (e.g., one or
more cationic lipids of Formula I or salts thereof as set forth
herein), a non-cationic lipid (e.g., mixtures of one or more
phospholipids and cholesterol), and a conjugated lipid that
inhibits aggregation of the particles (e.g., one or more PEG-lipid
and/or POZ-lipid conjugates). The SNALP may comprise at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, or more unmodified and/or modified
interfering RNA molecules. Nucleic acid-lipid particles and their
method of preparation are described in, e.g., U.S. Pat. Nos.
5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745;
and 6,320,017; and PCT Publication No. WO 96/40964, the disclosures
of which are each herein incorporated by reference in their
entirety for all purposes.
[0333] In the nucleic acid-lipid particles of the invention, the
nucleic acid may be fully encapsulated within the lipid portion of
the particle, thereby protecting the nucleic acid from nuclease
degradation. In preferred embodiments, a SNALP comprising a nucleic
acid such as an interfering RNA is fully encapsulated within the
lipid portion of the particle, thereby protecting the nucleic acid
from nuclease degradation. In certain instances, the nucleic acid
in the SNALP is not substantially degraded after exposure of the
particle to a nuclease at 37.degree. C. for at least about 20, 30,
45, or 60 minutes. In certain other instances, the nucleic acid in
the SNALP is not substantially degraded after incubation of the
particle in serum at 37.degree. C. for at least about 30, 45, or 60
minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other
embodiments, the nucleic acid is complexed with the lipid portion
of the particle. One of the benefits of the formulations of the
present invention is that the nucleic acid-lipid particle
compositions are substantially non-toxic to mammals such as
humans.
[0334] The term "fully encapsulated" indicates that the nucleic
acid in the nucleic acid-lipid particle is not significantly
degraded after exposure to serum or a nuclease assay that would
significantly degrade free DNA or RNA. In a fully encapsulated
system, preferably less than about 25% of the nucleic acid in the
particle is degraded in a treatment that would normally degrade
100% of free nucleic acid, more preferably less than about 10%, and
most preferably less than about 5% of the nucleic acid in the
particle is degraded. "Fully encapsulated" also indicates that the
nucleic acid-lipid particles are serum-stable, that is, that they
do not rapidly decompose into their component parts upon in vivo
administration.
[0335] In the context of nucleic acids, full encapsulation may be
determined by performing a membrane-impermeable fluorescent dye
exclusion assay, which uses a dye that has enhanced fluorescence
when associated with nucleic acid. Specific dyes such as
OliGreen.RTM. and RiboGreen.RTM. (Invitrogen Corp.; Carlsbad,
Calif.) are available for the quantitative determination of plasmid
DNA, single-stranded deoxyribonucleotides, and/or single- or
double-stranded ribonucleotides. Encapsulation is determined by
adding the dye to a liposomal formulation, measuring the resulting
fluorescence, and comparing it to the fluorescence observed upon
addition of a small amount of nonionic detergent.
Detergent-mediated disruption of the liposomal bilayer releases the
encapsulated nucleic acid, allowing it to interact with the
membrane-impermeable dye. Nucleic acid encapsulation may be
calculated as E=(I.sub.o-I)/I.sub.o, where I and I.sub.o refer to
the fluorescence intensities before and after the addition of
detergent (see, Wheeler et al., Gene Ther., 6:271-281 (1999)).
[0336] In other embodiments, the present invention provides a
nucleic acid-lipid particle (e.g., SNALP) composition comprising a
plurality of nucleic acid-lipid particles.
[0337] In some instances, the SNALP composition comprises nucleic
acid that is fully encapsulated within the lipid portion of the
particles, such that from about 30% to about 100%, from about 40%
to about 100%, from about 50% to about 100%, from about 60% to
about 100%, from about 70% to about 100%, from about 80% to about
100%, from about 90% to about 100%, from about 30% to about 95%,
from about 40% to about 95%, from about 50% to about 95%, from
about 60% to about 95%, from about 70% to about 95%, from about 80%
to about 95%, from about 85% to about 95%, from about 90% to about
95%, from about 30% to about 90%, from about 40% to about 90%, from
about 50% to about 90%, from about 60% to about 90%, from about 70%
to about 90%, from about 80% to about 90%, or at least about 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof
or range therein) of the particles have the nucleic acid
encapsulated therein.
[0338] In other instances, the SNALP composition comprises nucleic
acid that is fully encapsulated within the lipid portion of the
particles, such that from about 30% to about 100%, from about 40%
to about 100%, from about 50% to about 100%, from about 60% to
about 100%, from about 70% to about 100%, from about 80% to about
100%, from about 90% to about 100%, from about 30% to about 95%,
from about 40% to about 95%, from about 50% to about 95%, from
about 60% to about 95%, from about 70% to about 95%, from about 80%
to about 95%, from about 85% to about 95%, from about 90% to about
95%, from about 30% to about 90%, from about 40% to about 90%, from
about 50% to about 90%, from about 60% to about 90%, from about 70%
to about 90%, from about 80% to about 90%, or at least about 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof
or range therein) of the input nucleic acid is encapsulated in the
particles.
[0339] Depending on the intended use of the lipid particles of the
invention, the proportions of the components can be varied and the
delivery efficiency of a particular formulation can be measured
using, e.g., an endosomal release parameter (ERP) assay.
[0340] In particular embodiments, the present invention provides a
lipid particle (e.g., SNALP) composition comprising a plurality of
lipid particles described herein and an antioxidant. In certain
instances, the antioxidant in the lipid particle composition
reduces, prevents, and/or inhibits the degradation of a cationic
lipid present in the lipid particle. In instances wherein the
active agent is a therapeutic nucleic acid such as an interfering
RNA (e.g., siRNA), the antioxidant in the lipid particle
composition reduces, prevents, and/or inhibits the degradation of
the nucleic acid payload, e.g., by reducing, preventing, and/or
inhibiting the formation of adducts between the nucleic acid and
the cationic lipid. Non-limiting examples of antioxidants include
hydrophilic antioxidants such as chelating agents (e.g., metal
chelators such as ethylenediaminetetraacetic acid (EDTA), citrate,
and the like), lipophilic antioxidants (e.g., vitamin E isomers,
polyphenols, and the like), salts thereof; and mixtures thereof. If
needed, the antioxidant is typically present in an amount
sufficient to prevent, inhibit, and/or reduce the degradation of
the cationic lipid and/or active agent present in the particle,
e.g., at least about 20 mM EDTA or a salt thereof, or at least
about 100 mM citrate or a salt thereof. An antioxidant such as EDTA
and/or citrate may be included at any step or at multiple steps in
the lipid particle formation process described in Section VI (e.g.,
prior to, during, and/or after lipid particle formation).
[0341] Additional embodiments related to methods of preventing the
degradation of cationic lipids and/or active agents (e.g.,
therapeutic nucleic acids) present in lipid particles, compositions
comprising lipid particles stabilized by these methods, methods of
making these lipid particles, and methods of delivering and/or
administering these lipid particles are described in U.S.
Provisional Application No. 61/265,671, entitled "SNALP
Formulations Containing Antioxidants," filed Dec. 1, 2009, the
disclosure of which is herein incorporated by reference in its
entirety for all purposes.
[0342] A. Cationic Lipids
[0343] Any of the novel cationic lipids of Formula I or salts
thereof as set forth herein may be used in the lipid particles of
the present invention (e.g., SNALP), either alone or in combination
with one or more other cationic lipid species or non-cationic lipid
species.
[0344] Other cationic lipids or salts thereof which may also be
included in the lipid particles of the present invention include,
but are not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane
(DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-di-.gamma.-linolenyloxy-N,N-dimethylaminopropane
(.gamma.-DLenDMA), 1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine
(C2-DLinDMA), 1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine
(C2-DLinDAP),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-K-C2-DMA; also known as "XTC2" or "C2K"),
2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane
(DLin-K-C3-DMA; "C3K"),
2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane
(DLin-K-C4-DMA; "C4K"),
2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA),
2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ),
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),
2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DO-K-DMA),
2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DS-K-DMA),
2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),
2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride
(DLin-K-TMA.Cl),
2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)[1,3]-dioxolane
(DLin-K.sup.2-DMA),
2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane
(D-Lin-K-N-methylpiperzine),
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)
butanoate (DLin-M-C3-DMA; "MC3"),
dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA; also
known as DLin-M-K-DMA or DLin-M-DMA),
1,2-dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP),
1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),
1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl),
1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),
1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-dioleylamino)-1,2-propanedio (DOAP),
1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA),
3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (CLinDMA),
2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-
-2'-octadecadienoxy)propane (CpLinDMA),
N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),
1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),
1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),
1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), 3-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
(DC-Chol),
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE),
2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-
iumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine
(DOGS), and mixtures thereof.
[0345] Additional cationic lipids or salts thereof which may be
present in the lipid particles described herein include novel
cyclic cationic lipids such as CP-LenMC3, CP-.gamma.-LenMC3,
CP-MC3, CP-DLen-C2K-DMA, CP-.gamma.DLen-C2K-DMA, CP-C2K-DMA,
CP-DODMA, CP-DPetroDMA, CP-DLinDMA, CP-DLenDMA, CP-.gamma.DLenDMA,
analogs thereof, and combinations thereof, as described in U.S.
Provisional Application No. 61/334,096, entitled "Novel Cyclic
Cationic Lipids and Methods of Use Thereof," bearing Attorney
Docket No. 020801-010100US, filed May 12, 2010. Additional cationic
lipids or salts thereof which may be present in the lipid particles
described herein include novel cationic lipids such as 4-B 10, 4-B
12, 4-B 13, 4-B 14, 4-B 15, 4-B 16, .gamma.-4-B 10,
N,N-dimethyl-2-((11Z,14Z)-3-((9Z,12Z)-octadeca-9,12-dienyloxy)icosa-11,14-
-dienyloxy)ethanamine (4-B10 Ether),
(11Z,14Z)-3-(dimethylamino)propyl
3-((9Z,12Z)-octadeca-9,12-dienoyloxy)icosa-11,14-dienoate,
CP-4-B10, analogs thereof, and combinations thereof, as described
in U.S. Provisional Application No. 61/334,087, entitled "Novel
Cationic Lipids and Methods of Use Thereof," bearing Attorney
Docket No. 020801-010800US, filed May 12, 2010. Additional cationic
lipids or salts thereof which may be present in the lipid particles
described herein include the novel cationic lipids described in
U.S. Provisional Application No. 61/295,134, entitled "Improved
Cationic Lipids and Methods for the Delivery of Nucleic Acids,"
filed Jan. 14, 2010. Additional cationic lipids or salts thereof
which may be present in the lipid particles described herein
include the cationic lipids described in U.S. Patent Publication
No. 20090023673. The disclosures of each of these patent documents
are herein incorporated by reference in their entirety for all
purposes.
[0346] In some embodiments, the additional cationic lipid forms a
salt (preferably a crystalline salt) with one or more anions. In
one particular embodiment, the additional cationic lipid is the
oxalate (e.g., hemioxalate) salt thereof, which is preferably a
crystalline salt.
[0347] The synthesis of cationic lipids such as DLinDMA and
DLenDMA, as well as additional cationic lipids, is described in
U.S. Patent Publication No. 20060083780, the disclosure of which is
herein incorporated by reference in its entirety for all
purposes.
[0348] The synthesis of cationic lipids such as .gamma.-DLenDMA,
C2-DLinDMA and C2-DLinDAP, as well as additional cationic lipids,
is described in U.S. Provisional Application No. 61/295,134,
entitled "Improved Cationic Lipids and Methods for the Delivery of
Nucleic Acids," filed Jan. 14, 2010, the disclosure of which is
herein incorporated by reference in its entirety for all
purposes.
[0349] The synthesis of cationic lipids such as DLin-K-DMA, as well
as additional cationic lipids, is described in PCT Publication No.
WO 09/086,558, the disclosure of which is herein incorporated by
reference in its entirety for all purposes.
[0350] The synthesis of cationic lipids such as DLin-K-C2-DMA,
DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DO-K-DMA,
DS-K-DMA, DLin-K-MA, DLin-K-TMA.Cl, DLin-K.sup.2-DMA,
D-Lin-K-N-methylpiperzine, DLin-M-C2-DMA, DO-C-DAP, DMDAP, and
DOTAP.Cl, as well as additional cationic lipids, is described in
PCT Publication No. WO 2010/042877, entitled "Improved Amino Lipids
and Methods for the Delivery of Nucleic Acids," filed Oct. 9, 2009,
the disclosure of which is incorporated herein by reference in its
entirety for all purposes.
[0351] The synthesis of DLin-M-C3-DMA ("MC3"), as well as
additional cationic lipids (e.g., certain analogs of MC3), is
described herein and in U.S. Provisional Application No.
61/185,800, entitled "Novel Lipids and Compositions for the
Delivery of Therapeutics," filed Jun. 10, 2009, and U.S.
Provisional Application No. 61/287,995, entitled "Methods and
Compositions for Delivery of Nucleic Acids," filed Dec. 18, 2009,
the disclosures of which are herein incorporated by reference in
their entirety for all purposes.
[0352] The synthesis of cationic lipids such as DLin-C-DAP,
DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl,
DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as
additional cationic lipids, is described in PCT Publication No. WO
09/086,558, the disclosure of which is herein incorporated by
reference in its entirety for all purposes.
[0353] The synthesis of cationic lipids such as CLinDMA, as well as
additional cationic lipids, is described in U.S. Patent Publication
No. 20060240554, the disclosure of which is herein incorporated by
reference in its entirety for all purposes.
[0354] The synthesis of a number of other cationic lipids and
related analogs has been described in U.S. Pat. Nos. 5,208,036;
5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT
Publication No. WO 96/10390, the disclosures of which are each
herein incorporated by reference in their entirety for all
purposes. Additionally, a number of commercial preparations of
cationic lipids can be used, such as, e.g., LIPOFECTIN.RTM.
(including DOTMA and DOPE, available from GIBCO/BRL);
LIPOFECTAMINE.RTM. (including DOSPA and DOPE, available from
GIBCO/BRL); and TRANSFECTAM.RTM. (including DOGS, available from
Promega Corp.).
[0355] In some embodiments, the cationic lipid comprises from about
50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %,
from about 50 mol % to about 80 mol %, from about 50 mol % to about
75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol
% to about 65 mol %, from about 50 mol % to about 60 mol %, from
about 55 mol % to about 65 mol %, or from about 55 mol % to about
70 mol % (or any fraction thereof or range therein) of the total
lipid present in the particle. In particular embodiments, the
cationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol
%, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60
mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any
fraction thereof) of the total lipid present in the particle.
[0356] In other embodiments, the cationic lipid comprises from
about 2 mol % to about 60 mol %, from about 5 mol % to about 50 mol
%, from about 10 mol % to about 50 mol %, from about 20 mol % to
about 50 mol %, from about 20 mol % to about 40 mol %, from about
30 mol % to about 40 mol %, or about 40 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle.
[0357] Additional percentages and ranges of cationic lipids
suitable for use in the lipid particles of the present invention
are described in PCT Publication No. WO 09/127,060, U.S.
application Ser. No. 12/794,701, filed Jun. 4, 2010, U.S.
Provisional Application No. 61/295,134, filed Jan. 14, 2010, and
U.S. application Ser. No. 12/828,189, filed Jun. 30, 2010, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes.
[0358] It should be understood that the percentage of cationic
lipid present in the lipid particles of the invention is a target
amount, and that the actual amount of cationic lipid present in the
formulation may vary, for example, by .+-.5 mol %. For example, in
the 1:57 lipid particle (e.g., SNALP) formulation, the target
amount of cationic lipid is 57.1 mol %, but the actual amount of
cationic lipid may be .+-.5 mol %, .+-.4 mol %, .+-.3 mol %, .+-.2
mol %, .+-.1 mol %, .+-.0.75 mol %, .+-.0.5 mol %, .+-.0.25 mol %,
or .+-.0.1 mol % of that target amount, with the balance of the
formulation being made up of other lipid components (adding up to
100 mol % of total lipids present in the particle).
[0359] B. Non-Cationic Lipids
[0360] The non-cationic lipids used in the lipid particles of the
invention (e.g., SNALP) can be any of a variety of neutral
uncharged, zwitterionic, or anionic lipids capable of producing a
stable complex.
[0361] Non-limiting examples of non-cationic lipids include
phospholipids such as lecithin, phosphatidylethanolamine,
lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM),
cephalin, cardiolipin, phosphatidic acid, cerebrosides,
dicetylphosphate, distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoyl-phosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
palmitoyloleyol-phosphatidylglycerol (POPG),
dioleoylphosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl-phosphatidylethanolamine (DPPE),
dimyristoyl-phosphatidylethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE),
monomethyl-phosphatidylethanolamine,
dimethyl-phosphatidylethanolamine,
dielaidoyl-phosphatidylethanolamine (DEPE),
stearoyloleoyl-phosphatidylethanolamine (SOPE),
lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and
mixtures thereof. Other diacylphosphatidylcholine and
diacylphosphatidylethanolamine phospholipids can also be used. The
acyl groups in these lipids are preferably acyl groups derived from
fatty acids having C.sub.10-C.sub.24 carbon chains, e.g., lauroyl,
myristoyl, palmitoyl, stearoyl, or oleoyl.
[0362] Additional examples of non-cationic lipids include sterols
such as cholesterol and derivatives thereof. Non-limiting examples
of cholesterol derivatives include polar analogues such as
5.alpha.-cholestanol, 5.beta.-coprostanol,
cholesteryl-(2'-hydroxy)-ethyl ether,
cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol;
non-polar analogues such as 5.alpha.-cholestane, cholestenone,
5.alpha.-cholestanone, 5.beta.-cholestanone, and cholesteryl
decanoate; and mixtures thereof. In preferred embodiments, the
cholesterol derivative is a polar analogue such as
cholesteryl-(4'-hydroxy)-butyl ether. The synthesis of
cholesteryl-(2'-hydroxy)-ethyl ether is described in PCT
Publication No. WO 09/127,060, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
[0363] In some embodiments, the non-cationic lipid present in the
lipid particles (e.g., SNALP) comprises or consists of a mixture of
one or more phospholipids and cholesterol or a derivative thereof.
In other embodiments, the non-cationic lipid present in the lipid
particles (e.g., SNALP) comprises or consists of one or more
phospholipids, e.g., a cholesterol-free lipid particle formulation.
In yet other embodiments, the non-cationic lipid present in the
lipid particles (e.g., SNALP) comprises or consists of cholesterol
or a derivative thereof, e.g., a phospholipid-free lipid particle
formulation.
[0364] Other examples of non-cationic lipids suitable for use in
the present invention include nonphosphorous containing lipids such
as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl
palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl
myristate, amphoteric acrylic polymers, triethanolamine-lauryl
sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides,
dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and
the like.
[0365] In some embodiments, the non-cationic lipid comprises from
about 10 mol % to about 60 mol %, from about 20 mol % to about 55
mol %, from about 20 mol % to about 45 mol %, from about 20 mol %
to about 40 mol %, from about 25 mol % to about 50 mol %, from
about 25 mol % to about 45 mol %, from about 30 mol % to about 50
mol %, from about 30 mol % to about 45 mol %, from about 30 mol %
to about 40 mol %, from about 35 mol % to about 45 mol %, from
about 37 mol % to about 42 mol %, or about 35 mol %, 36 mol %, 37
mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %,
44 mol %, or 45 mol % (or any fraction thereof or range therein) of
the total lipid present in the particle.
[0366] In embodiments where the lipid particles contain a mixture
of phospholipid and cholesterol or a cholesterol derivative, the
mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55
mol %, or 60 mol % of the total lipid present in the particle.
[0367] In some embodiments, the phospholipid component in the
mixture may comprise from about 2 mol % to about 20 mol %, from
about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol
%, from about 4 mol % to about 15 mol %, or from about 4 mol % to
about 10 mol % (or any fraction thereof or range therein) of the
total lipid present in the particle. In certain preferred
embodiments, the phospholipid component in the mixture comprises
from about 5 mol % to about 10 mol %, from about 5 mol % to about 9
mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to
about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol
%, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle. As a non-limiting example, a 1:57 lipid particle
formulation comprising a mixture of phospholipid and cholesterol
may comprise a phospholipid such as DPPC or DSPC at about 7 mol %
(or any fraction thereof), e.g., in a mixture with cholesterol or a
cholesterol derivative at about 34 mol % (or any fraction thereof)
of the total lipid present in the particle. As another non-limiting
example, a 7:54 lipid particle formulation comprising a mixture of
phospholipid and cholesterol may comprise a phospholipid such as
DPPC or DSPC at about 7 mol % (or any fraction thereof), e.g., in a
mixture with cholesterol or a cholesterol derivative at about 32
mol % (or any fraction thereof) of the total lipid present in the
particle.
[0368] In other embodiments, the cholesterol component in the
mixture may comprise from about 25 mol % to about 45 mol %, from
about 25 mol % to about 40 mol %, from about 30 mol % to about 45
mol %, from about 30 mol % to about 40 mol %, from about 27 mol %
to about 37 mol %, from about 25 mol % to about 30 mol %, or from
about 35 mol % to about 40 mol % (or any fraction thereof or range
therein) of the total lipid present in the particle. In certain
preferred embodiments, the cholesterol component in the mixture
comprises from about 25 mol % to about 35 mol %, from about 27 mol
% to about 35 mol %, from about 29 mol % to about 35 mol %, from
about 30 mol % to about 35 mol %, from about 30 mol % to about 34
mol %, from about 31 mol % to about 33 mol %, or about 30 mol %, 31
mol %, 32 mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle. Typically, a 1:57 lipid particle formulation comprising a
mixture of phospholipid and cholesterol may comprise cholesterol or
a cholesterol derivative at about 34 mol % (or any fraction
thereof), e.g., in a mixture with a phospholipid such as DPPC or
DSPC at about 7 mol % (or any fraction thereof) of the total lipid
present in the particle. Typically, a 7:54 lipid particle
formulation comprising a mixture of phospholipid and cholesterol
may comprise cholesterol or a cholesterol derivative at about 32
mol % (or any fraction thereof), e.g., in a mixture with a
phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction
thereof) of the total lipid present in the particle.
[0369] In embodiments where the lipid particles are
phospholipid-free, the cholesterol or derivative thereof may
comprise up to about 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol
%, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in
the particle.
[0370] In some embodiments, the cholesterol or derivative thereof
in the phospholipid-free lipid particle formulation may comprise
from about 25 mol % to about 45 mol %, from about mol % to about 40
mol %, from about 30 mol % to about 45 mol %, from about 30 mol %
to about 40 mol %, from about 31 mol % to about 39 mol %, from
about 32 mol % to about 38 mol %, from about 33 mol % to about 37
mol %, from about 35 mol % to about 45 mol %, from about 30 mol %
to about 35 mol %, from about 35 mol % to about 40 mol %, or about
mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36 mol %,
37 mol %, 38 mol %, 39 mol %, or 40 mol % (or any fraction thereof
or range therein) of the total lipid present in the particle. As a
non-limiting example, a 1:62 lipid particle formulation may
comprise cholesterol at about 37 mol % (or any fraction thereof) of
the total lipid present in the particle. As another non-limiting
example, a 7:58 lipid particle formulation may comprise cholesterol
at about 35 mol % (or any fraction thereof) of the total lipid
present in the particle.
[0371] In other embodiments, the non-cationic lipid comprises from
about 5 mol % to about 90 mol %, from about 10 mol % to about 85
mol %, from about 20 mol % to about 80 mol %, about 10 mol % (e.g.,
phospholipid only), or about 60 mol % (e.g., phospholipid and
cholesterol or derivative thereof) (or any fraction thereof or
range therein) of the total lipid present in the particle.
[0372] Additional percentages and ranges of non-cationic lipids
suitable for use in the lipid particles of the present invention
are described in PCT Publication No. WO 09/127,060, U.S.
application Ser. No. 12/794,701, filed Jun. 4, 2010, U.S.
Provisional Application No. 61/295,134, filed Jan. 14, 2010, and
U.S. application Ser. No. 12/828,189, filed Jun. 30, 2010, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes.
[0373] It should be understood that the percentage of non-cationic
lipid present in the lipid particles of the invention is a target
amount, and that the actual amount of non-cationic lipid present in
the formulation may vary, for example, by .+-.5 mol %. For example,
in the 1:57 lipid particle (e.g., SNALP) formulation, the target
amount of phospholipid is 7.1 mol % and the target amount of
cholesterol is 34.3 mol %, but the actual amount of phospholipid
may be .+-.2 mol %, .+-.1.5 mol %, .+-.1 mol %, .+-.0.75 mol %,
.+-.0.5 mol %, .+-.0.25 mol %, or .+-.0.1 mol % of that target
amount, and the actual amount of cholesterol may be .+-.3 mol %,
.+-.2 mol %, .+-.1 mol %, .+-.0.75 mol %, .+-.0.5 mol %, .+-.0.25
mol %, or .+-.0.1 mol % of that target amount, with the balance of
the formulation being made up of other lipid components (adding up
to 100 mol % of total lipids present in the particle). Similarly,
in the 7:54 lipid particle (e.g., SNALP) formulation, the target
amount of phospholipid is 6.75 mol % and the target amount of
cholesterol is 32.43 mol %, but the actual amount of phospholipid
may be .+-.2 mol %, .+-.1.5 mol %, .+-.1 mol %, .+-.0.75 mol %,
.+-.0.5 mol %, .+-.0.25 mol %, or .+-.0.1 mol % of that target
amount, and the actual amount of cholesterol may be .+-.3 mol %,
.+-.2 mol %, .+-.1 mol %, .+-.0.75 mol %, .+-.0.5 mol %, .+-.0.25
mol %, or .+-.0.1 mol % of that target amount, with the balance of
the formulation being made up of other lipid components (adding up
to 100 mol % of total lipids present in the particle).
[0374] C. Lipid Conjugates
[0375] In addition to cationic and non-cationic lipids, the lipid
particles of the invention (e.g., SNALP) may further comprise a
lipid conjugate. The conjugated lipid is useful in that it prevents
the aggregation of particles. Suitable conjugated lipids include,
but are not limited to, PEG-lipid conjugates, POZ-lipid conjugates,
ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs),
and mixtures thereof. In certain embodiments, the particles
comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate
together with a CPL.
[0376] In a preferred embodiment, the lipid conjugate is a
PEG-lipid. Examples of PEG-lipids include, but are not limited to,
PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g.,
PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol
(PEG-DAG) as described in, e.g., U.S. Patent Publication Nos.
20030077829 and 2005008689, PEG coupled to phospholipids such as
phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides as
described in, e.g., U.S. Pat. No. 5,885,613, PEG conjugated to
cholesterol or a derivative thereof, and mixtures thereof. The
disclosures of these patent documents are herein incorporated by
reference in their entirety for all purposes.
[0377] Additional PEG-lipids suitable for use in the invention
include, without limitation,
mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The
synthesis of PEG-C-DOMG is described in PCT Publication No. WO
09/086,558, the disclosure of which is herein incorporated by
reference in its entirety for all purposes. Yet additional suitable
PEG-lipid conjugates include, without limitation,
1-[8'-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaoctanyl]carbam-
oyl-w-methyl-poly(ethylene glycol) (2 KPEG-DMG). The synthesis of 2
KPEG-DMG is described in U.S. Pat. No. 7,404,969, the disclosure of
which is herein incorporated by reference in its entirety for all
purposes.
[0378] PEG is a linear, water-soluble polymer of ethylene PEG
repeating units with two terminal hydroxyl groups. PEGs are
classified by their molecular weights; for example, PEG 2000 has an
average molecular weight of about 2,000 daltons, and PEG 5000 has
an average molecular weight of about 5,000 daltons. PEGs are
commercially available from Sigma Chemical Co. and other companies
and include, but are not limited to, the following:
monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene
glycol-succinate (MePEG-S), monomethoxypolyethylene
glycol-succinimidyl succinate (MePEG-S-NHS),
monomethoxypolyethylene glycol-amine (MePEG-NH.sub.2),
monomethoxypolyethylene glycol-tresylate (MePEG-TRES),
monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as
well as such compounds containing a terminal hydroxyl group instead
of a terminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS,
HO-PEG-NH.sub.2, etc.). Other PEGs such as those described in U.S.
Pat. Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are
also useful for preparing the PEG-lipid conjugates of the present
invention. The disclosures of these patents are herein incorporated
by reference in their entirety for all purposes. In addition,
monomethoxypolyethyleneglycol-acetic acid (MePEG-CH.sub.2COOH) is
particularly useful for preparing PEG-lipid conjugates including,
e.g., PEG-DAA conjugates.
[0379] The PEG moiety of the PEG-lipid conjugates described herein
may comprise an average molecular weight ranging from about 550
daltons to about 10,000 daltons. In certain instances, the PEG
moiety has an average molecular weight of from about 750 daltons to
about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000
daltons, from about 1,500 daltons to about 3,000 daltons, from
about 750 daltons to about 3,000 daltons, from about 750 daltons to
about 2,000 daltons, etc.). In preferred embodiments, the PEG
moiety has an average molecular weight of about 2,000 daltons or
about 750 daltons.
[0380] In certain instances, the PEG can be optionally substituted
by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated
directly to the lipid or may be linked to the lipid via a linker
moiety. Any linker moiety suitable for coupling the PEG to a lipid
can be used including, e.g., non-ester containing linker moieties
and ester-containing linker moieties. In a preferred embodiment,
the linker moiety is a non-ester containing linker moiety. As used
herein, the term "non-ester containing linker moiety" refers to a
linker moiety that does not contain a carboxylic ester bond
(--OC(O)--). Suitable non-ester containing linker moieties include,
but are not limited to, amido (--C(O)NH--), amino (--NR--),
carbonyl (--C(O)--), carbamate (--NHC(O)O--), urea (--NHC(O)NH--),
disulphide (--S--S--), ether (--O--), succinyl
(--(O)CCH.sub.2CH.sub.2C(O)--), succinamidyl
(--NHC(O)CH.sub.2CH.sub.2C(O)NH--), ether, disulphide, as well as
combinations thereof (such as a linker containing both a carbamate
linker moiety and an amido linker moiety). In a preferred
embodiment, a carbamate linker is used to couple the PEG to the
lipid.
[0381] In other embodiments, an ester containing linker moiety is
used to couple the PEG to the lipid. Suitable ester containing
linker moieties include, e.g., carbonate (--OC(O)O--), succinoyl,
phosphate esters (--O--(O)POH--O--), sulfonate esters, and
combinations thereof.
[0382] Phosphatidylethanolamines having a variety of acyl chain
groups of varying chain lengths and degrees of saturation can be
conjugated to PEG to form the lipid conjugate. Such
phosphatidylethanolamines are commercially available, or can be
isolated or synthesized using conventional techniques known to
those of skilled in the art. Phosphatidyl-ethanolamines containing
saturated or unsaturated fatty acids with carbon chain lengths in
the range of C.sub.10 to C.sub.20 are preferred.
Phosphatidylethanolamines with mono- or diunsaturated fatty acids
and mixtures of saturated and unsaturated fatty acids can also be
used. Suitable phosphatidylethanolamines include, but are not
limited to, dimyristoyl-phosphatidylethanolamine (DMPE),
dipalmitoyl-phosphatidylethanolamine (DPPE),
dioleoylphosphatidylethanolamine (DOPE), and
distearoyl-phosphatidylethanolamine (DSPE).
[0383] The term "ATTA" or "polyamide" includes, without limitation,
compounds described in U.S. Pat. Nos. 6,320,017 and 6,586,559, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes. These compounds include a compound
having the formula:
##STR00040##
[0384] wherein R is a member selected from the group consisting of
hydrogen, alkyl and acyl; R.sup.1 is a member selected from the
group consisting of hydrogen and alkyl; or optionally, R and
R.sup.1 and the nitrogen to which they are bound form an azido
moiety; R.sup.2 is a member of the group selected from hydrogen,
optionally substituted alkyl, optionally substituted aryl and a
side chain of an amino acid; R.sup.3 is a member selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto,
hydrazino, amino and NR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5
are independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p
is 1 to 4; and q is 0 or 1. It will be apparent to those of skill
in the art that other polyamides can be used in the compounds of
the present invention.
[0385] The term "diacylglycerol" or "DAG" includes a compound
having 2 fatty acyl chains, R.sup.1 and R.sup.2, both of which have
independently between 2 and 30 carbons bonded to the 1- and
2-position of glycerol by ester linkages. The acyl groups can be
saturated or have varying degrees of unsaturation. Suitable acyl
groups include, but are not limited to, lauroyl (C.sub.12),
myristoyl (C.sub.14), palmitoyl (C.sub.16), stearoyl (C.sub.18),
and icosoyl (C.sub.20). In preferred embodiments, R.sup.1 and
R.sup.2 are the same, i.e., R.sup.1 and R.sup.2 are both myristoyl
(i.e., dimyristoyl), R.sup.1 and R.sup.2 are both stearoyl (i.e.,
distearoyl), etc. Diacylglycerols have the following general
formula:
##STR00041##
[0386] The term "dialkyloxypropyl" or "DAA" includes a compound
having 2 alkyl chains, R.sup.1 and R.sup.2, both of which have
independently between 2 and 30 carbons. The alkyl groups can be
saturated or have varying degrees of unsaturation.
Dialkyloxypropyls have the following general formula:
##STR00042##
[0387] In a preferred embodiment, the PEG-lipid is a PEG-DAA
conjugate having the following formula:
##STR00043##
[0388] wherein R.sup.1 and R.sup.2 are independently selected and
are long-chain alkyl groups having from about 10 to about 22 carbon
atoms; PEG is a polyethyleneglycol; and L is a non-ester containing
linker moiety or an ester containing linker moiety as described
above. The long-chain alkyl groups can be saturated or unsaturated.
Suitable alkyl groups include, but are not limited to, decyl
(C.sub.10), lauryl (C.sub.12), myristyl (C.sub.14), palmityl
(C.sub.16), stearyl (C.sub.18), and icosyl (C.sub.20). In preferred
embodiments, R.sup.1 and R.sup.2 are the same, i.e., R.sup.1 and
R.sup.2 are both myristyl (i.e., dimyristyl), R.sup.1 and R.sup.2
are both stearyl (i.e., distearyl), etc.
[0389] In Formula X above, the PEG has an average molecular weight
ranging from about 550 daltons to about 10,000 daltons. In certain
instances, the PEG has an average molecular weight of from about
750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons
to about 5,000 daltons, from about 1,500 daltons to about 3,000
daltons, from about 750 daltons to about 3,000 daltons, from about
750 daltons to about 2,000 daltons, etc.). In preferred
embodiments, the PEG has an average molecular weight of about 2,000
daltons or about 750 daltons. The PEG can be optionally substituted
with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments,
the terminal hydroxyl group is substituted with a methoxy or methyl
group.
[0390] In a preferred embodiment, "L" is a non-ester containing
linker moiety. Suitable non-ester containing linkers include, but
are not limited to, an amido linker moiety, an amino linker moiety,
a carbonyl linker moiety, a carbamate linker moiety, a urea linker
moiety, an ether linker moiety, a disulphide linker moiety, a
succinamidyl linker moiety, and combinations thereof. In a
preferred embodiment, the non-ester containing linker moiety is a
carbamate linker moiety (i.e., a PEG-C-DAA conjugate). In another
preferred embodiment, the non-ester containing linker moiety is an
amido linker moiety (i.e., a PEG-A-DAA conjugate). In yet another
preferred embodiment, the non-ester containing linker moiety is a
succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).
[0391] In particular embodiments, the PEG-lipid conjugate is
selected from:
##STR00044##
[0392] The PEG-DAA conjugates are synthesized using standard
techniques and reagents known to those of skill in the art. It will
be recognized that the PEG-DAA conjugates will contain various
amide, amine, ether, thio, carbamate, and urea linkages. Those of
skill in the art will recognize that methods and reagents for
forming these bonds are well known and readily available. See,
e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock,
COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Furniss,
VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed. (Longman
1989). It will also be appreciated that any functional groups
present may require protection and deprotection at different points
in the synthesis of the PEG-DAA conjugates. Those of skill in the
art will recognize that such techniques are well known. See, e.g.,
Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley
1991).
[0393] Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl
(C.sub.10) conjugate, a PEG-dilauryloxypropyl (C.sub.12) conjugate,
a PEG-dimyristyloxypropyl (C.sub.14) conjugate, a
PEG-dipalmityloxypropyl (C.sub.16) conjugate, or a
PEG-distearyloxypropyl (C.sub.18) conjugate. In these embodiments,
the PEG preferably has an average molecular weight of about 750 or
about 2,000 daltons. In one particularly preferred embodiment, the
PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the "2000"
denotes the average molecular weight of the PEG, the "C" denotes a
carbamate linker moiety, and the "DMA" denotes dimyristyloxypropyl.
In another particularly preferred embodiment, the PEG-lipid
conjugate comprises PEG750-C-DMA, wherein the "750" denotes the
average molecular weight of the PEG, the "C" denotes a carbamate
linker moiety, and the "DMA" denotes dimyristyloxypropyl. In
particular embodiments, the terminal hydroxyl group of the PEG is
substituted with a methyl group. Those of skill in the art will
readily appreciate that other dialkyloxypropyls can be used in the
PEG-DAA conjugates of the present invention.
[0394] In addition to the foregoing, it will be readily apparent to
those of skill in the art that other hydrophilic polymers can be
used in place of PEG. Examples of suitable polymers that can be
used in place of PEG include, but are not limited to,
polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyl methacrylamide, polymethacrylamide and
polydimethylacrylamide, polylactic acid, polyglycolic acid, and
derivatized celluloses such as hydroxymethylcellulose or
hydroxyethylcellulose.
[0395] In addition to the foregoing components, the lipid particles
(e.g., SNALP) of the present invention can further comprise
cationic poly(ethylene glycol) (PEG) lipids or CPLs (see, e.g.,
Chen et al., Bioconj. Chem., 11:433-437 (2000); U.S. Pat. No.
6,852,334; PCT Publication No. WO 00/62813, the disclosures of
which are herein incorporated by reference in their entirety for
all purposes).
[0396] Suitable CPLs include compounds of Formula XI:
A-W-Y (XI),
[0397] wherein A, W, and Y are as described below.
[0398] With reference to Formula XI, "A" is a lipid moiety such as
an amphipathic lipid, a neutral lipid, or a hydrophobic lipid that
acts as a lipid anchor. Suitable lipid examples include, but are
not limited to, diacylglycerolyls, dialkylglycerolyls,
N--N-dialkylaminos, 1,2-diacyloxy-3-aminopropanes, and
1,2-dialkyl-3-aminopropanes.
[0399] "W" is a polymer or an oligomer such as a hydrophilic
polymer or oligomer. Preferably, the hydrophilic polymer is a
biocompatible polymer that is nonimmunogenic or possesses low
inherent immunogenicity. Alternatively, the hydrophilic polymer can
be weakly antigenic if used with appropriate adjuvants. Suitable
nonimmunogenic polymers include, but are not limited to, PEG,
polyamides, polylactic acid, polyglycolic acid, polylactic
acid/polyglycolic acid copolymers, and combinations thereof. In a
preferred embodiment, the polymer has a molecular weight of from
about 250 to about 7,000 daltons.
[0400] "Y" is a polycationic moiety. The term polycationic moiety
refers to a compound, derivative, or functional group having a
positive charge, preferably at least 2 positive charges at a
selected pH, preferably physiological pH. Suitable polycationic
moieties include basic amino acids and their derivatives such as
arginine, asparagine, glutamine, lysine, and histidine; spermine;
spermidine; cationic dendrimers; polyamines; polyamine sugars; and
amino polysaccharides. The polycationic moieties can be linear,
such as linear tetralysine, branched or dendrimeric in structure.
Polycationic moieties have between about 2 to about 15 positive
charges, preferably between about 2 to about 12 positive charges,
and more preferably between about 2 to about 8 positive charges at
selected pH values. The selection of which polycationic moiety to
employ may be determined by the type of particle application which
is desired.
[0401] The charges on the polycationic moieties can be either
distributed around the entire particle moiety, or alternatively,
they can be a discrete concentration of charge density in one
particular area of the particle moiety e.g., a charge spike. If the
charge density is distributed on the particle, the charge density
can be equally distributed or unequally distributed. All variations
of charge distribution of the polycationic moiety are encompassed
by the present invention.
[0402] The lipid "A" and the nonimmunogenic polymer "W" can be
attached by various methods and preferably by covalent attachment.
Methods known to those of skill in the art can be used for the
covalent attachment of "A" and "W." Suitable linkages include, but
are not limited to, amide, amine, carboxyl, carbonate, carbamate,
ester, and hydrazone linkages. It will be apparent to those skilled
in the art that "A" and "W" must have complementary functional
groups to effectuate the linkage. The reaction of these two groups,
one on the lipid and the other on the polymer, will provide the
desired linkage. For example, when the lipid is a diacylglycerol
and the terminal hydroxyl is activated, for instance with NHS and
DCC, to form an active ester, and is then reacted with a polymer
which contains an amino group, such as with a polyamide (see, e.g.,
U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which
are herein incorporated by reference in their entirety for all
purposes), an amide bond will form between the two groups.
[0403] In certain instances, the polycationic moiety can have a
ligand attached, such as a targeting ligand or a chelating moiety
for complexing calcium. Preferably, after the ligand is attached,
the cationic moiety maintains a positive charge. In certain
instances, the ligand that is attached has a positive charge.
Suitable ligands include, but are not limited to, a compound or
device with a reactive functional group and include lipids,
amphipathic lipids, carrier compounds, bioaffinity compounds,
biomaterials, biopolymers, biomedical devices, analytically
detectable compounds, therapeutically active compounds, enzymes,
peptides, proteins, antibodies, immune stimulators, radiolabels,
fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides,
liposomes, virosomes, micelles, immunoglobulins, functional groups,
other targeting moieties, or toxins.
[0404] In some embodiments, the lipid conjugate (e.g., PEG-lipid)
comprises from about 0.1 mol % to about 2 mol %, from about 0.5 mol
% to about 2 mol %, from about 1 mol % to about 2 mol %, from about
0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol
%, from about 0.8 mol % to about 1.7 mol %, from about 0.9 mol % to
about 1.6 mol %, from about 0.9 mol % to about 1.8 mol %, from
about 1 mol % to about 1.8 mol %, from about 1 mol % to about 1.7
mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol
% to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, or
from about 1.4 mol % to about 1.5 mol % (or any fraction thereof or
range therein) of the total lipid present in the particle.
[0405] In other embodiments, the lipid conjugate (e.g., PEG-lipid)
comprises from about 0 mol % to about 20 mol %, from about 0.5 mol
% to about 20 mol %, from about 2 mol % to about 20 mol %, from
about 1.5 mol % to about 18 mol %, from about 2 mol % to about 15
mol %, from about 4 mol % to about 15 mol %, from about 2 mol % to
about 12 mol %, from about 5 mol % to about 12 mol %, or about 2
mol % (or any fraction thereof or range therein) of the total lipid
present in the particle.
[0406] In further embodiments, the lipid conjugate (e.g.,
PEG-lipid) comprises from about 4 mol % to about 10 mol %, from
about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol
%, from about 5 mol % to about 8 mol %, from about 6 mol % to about
9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6
mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle.
[0407] Additional examples, percentages, and/or ranges of lipid
conjugates suitable for use in the lipid particles of the present
invention are described in, e.g., PCT Publication No. WO
09/127,060, U.S. application Ser. No. 12/794,701, filed Jun. 4,
2010, U.S. Provisional Application No. 61/295,134, filed Jan. 14,
2010, U.S. application Ser. No. 12/828,189, filed Jun. 30, 2010,
U.S. Provisional Application No. 61/294,828, filed Jan. 13, 2010,
U.S. Provisional Application No. 61/295,140, filed Jan. 14, 2010,
and PCT Publication No. WO 2010/006282, the disclosures of which
are herein incorporated by reference in their entirety for all
purposes.
[0408] It should be understood that the percentage of lipid
conjugate (e.g., PEG-lipid) present in the lipid particles of the
invention is a target amount, and that the actual amount of lipid
conjugate present in the formulation may vary, for example, by
.+-.2 mol %. For example, in the 1:57 lipid particle (e.g., SNALP)
formulation, the target amount of lipid conjugate is 1.4 mol %, but
the actual amount of lipid conjugate may be .+-.0.5 mol %, .+-.0.4
mol %, .+-.0.3 mol %, .+-.0.2 mol %, .+-.0.1 mol %, or .+-.0.05 mol
% of that target amount, with the balance of the formulation being
made up of other lipid components (adding up to 100 mol % of total
lipids present in the particle). Similarly, in the 7:54 lipid
particle (e.g., SNALP) formulation, the target amount of lipid
conjugate is 6.76 mol %, but the actual amount of lipid conjugate
may be .+-.2 mol %, .+-.1.5 mol %, .+-.1 mol %, .+-.0.75 mol %,
.+-.0.5 mol %, .+-.0.25 mol %, or .+-.0.1 mol % of that target
amount, with the balance of the formulation being made up of other
lipid components (adding up to 100 mol % of total lipids present in
the particle).
[0409] One of ordinary skill in the art will appreciate that the
concentration of the lipid conjugate can be varied depending on the
lipid conjugate employed and the rate at which the lipid particle
is to become fusogenic.
[0410] By controlling the composition and concentration of the
lipid conjugate, one can control the rate at which the lipid
conjugate exchanges out of the lipid particle and, in turn, the
rate at which the lipid particle becomes fusogenic. For instance,
when a PEG-DAA conjugate is used as the lipid conjugate, the rate
at which the lipid particle becomes fusogenic can be varied, for
example, by varying the concentration of the lipid conjugate, by
varying the molecular weight of the PEG, or by varying the chain
length and degree of saturation of the alkyl groups on the PEG-DAA
conjugate. In addition, other variables including, for example, pH,
temperature, ionic strength, etc. can be used to vary and/or
control the rate at which the lipid particle becomes fusogenic.
Other methods which can be used to control the rate at which the
lipid particle becomes fusogenic will become apparent to those of
skill in the art upon reading this disclosure. Also, by controlling
the composition and concentration of the lipid conjugate, one can
control the lipid particle (e.g., SNALP) size.
VI. Preparation of Lipid Particles
[0411] The lipid particles of the present invention, e.g., SNALP,
in which an active agent or therapeutic agent such as an
interfering RNA (e.g., siRNA) is entrapped within the lipid portion
of the particle and is protected from degradation, can be formed by
any method known in the art including, but not limited to, a
continuous mixing method, a direct dilution process, and an in-line
dilution process.
[0412] In particular embodiments, the cationic lipids may comprise
lipids of Formula I or salts thereof, alone or in combination with
other cationic lipids. In other embodiments, the non-cationic
lipids are egg sphingomyelin (ESM), distearoylphosphatidylcholine
(DSPC), dioleoylphosphatidylcholine (DOPC),
1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),
dipalmitoyl-phosphatidylcholine (DPPC),
monomethyl-phosphatidylethanolamine,
dimethyl-phosphatidylethanolamine, 14:0 PE
(1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE
(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE
(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE
(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE
(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE
(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE
(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)),
polyethylene glycol-based polymers (e.g., PEG 2000, PEG 5000,
PEG-modified diacylglycerols, or PEG-modified dialkyloxypropyls),
cholesterol, derivatives thereof, or combinations thereof.
[0413] In certain embodiments, the present invention provides
nucleic acid-lipid particles (e.g., SNALP) produced via a
continuous mixing method, e.g., a process that includes providing
an aqueous solution comprising a nucleic acid (e.g., interfering
RNA) in a first reservoir, providing an organic lipid solution in a
second reservoir (wherein the lipids present in the organic lipid
solution are solubilized in an organic solvent, e.g., a lower
alkanol such as ethanol), and mixing the aqueous solution with the
organic lipid solution such that the organic lipid solution mixes
with the aqueous solution so as to substantially instantaneously
produce a lipid vesicle (e.g., liposome) encapsulating the nucleic
acid within the lipid vesicle. This process and the apparatus for
carrying out this process are described in detail in U.S. Patent
Publication No. 20040142025, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
[0414] The action of continuously introducing lipid and buffer
solutions into a mixing environment, such as in a mixing chamber,
causes a continuous dilution of the lipid solution with the buffer
solution, thereby producing a lipid vesicle substantially
instantaneously upon mixing. As used herein, the phrase
"continuously diluting a lipid solution with a buffer solution"
(and variations) generally means that the lipid solution is diluted
sufficiently rapidly in a hydration process with sufficient force
to effectuate vesicle generation. By mixing the aqueous solution
comprising a nucleic acid with the organic lipid solution, the
organic lipid solution undergoes a continuous stepwise dilution in
the presence of the buffer solution (i.e., aqueous solution) to
produce a nucleic acid-lipid particle.
[0415] The nucleic acid-lipid particles formed using the continuous
mixing method typically have a size of from about 30 nm to about
150 nm, from about 40 nm to about 150 nm, from about 50 nm to about
150 nm, from about 60 nm to about 130 nm, from about 70 nm to about
110 nm, from about 70 nm to about 100 nm, from about 80 nm to about
100 nm, from about 90 nm to about 100 nm, from about 70 to about 90
nm, from about 80 nm to about 90 nm, from about 70 nm to about 80
nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or
about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70
nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115
nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm (or
any fraction thereof or range therein). The particles thus formed
do not aggregate and are optionally sized to achieve a uniform
particle size.
[0416] In another embodiment, the present invention provides
nucleic acid-lipid particles (e.g., SNALP) produced via a direct
dilution process that includes forming a lipid vesicle (e.g.,
liposome) solution and immediately and directly introducing the
lipid vesicle solution into a collection vessel containing a
controlled amount of dilution buffer. In preferred aspects, the
collection vessel includes one or more elements configured to stir
the contents of the collection vessel to facilitate dilution. In
one aspect, the amount of dilution buffer present in the collection
vessel is substantially equal to the volume of lipid vesicle
solution introduced thereto. As a non-limiting example, a lipid
vesicle solution in 45% ethanol when introduced into the collection
vessel containing an equal volume of dilution buffer will
advantageously yield smaller particles.
[0417] In yet another embodiment, the present invention provides
nucleic acid-lipid particles (e.g., SNALP) produced via an in-line
dilution process in which a third reservoir containing dilution
buffer is fluidly coupled to a second mixing region. In this
embodiment, the lipid vesicle (e.g., liposome) solution formed in a
first mixing region is immediately and directly mixed with dilution
buffer in the second mixing region. In preferred aspects, the
second mixing region includes a T-connector arranged so that the
lipid vesicle solution and the dilution buffer flows meet as
opposing 180.degree. flows; however, connectors providing shallower
angles can be used, e.g., from about 27.degree. to about
180.degree. (e.g., about 90.degree.). A pump mechanism delivers a
controllable flow of buffer to the second mixing region. In one
aspect, the flow rate of dilution buffer provided to the second
mixing region is controlled to be substantially equal to the flow
rate of lipid vesicle solution introduced thereto from the first
mixing region. This embodiment advantageously allows for more
control of the flow of dilution buffer mixing with the lipid
vesicle solution in the second mixing region, and therefore also
the concentration of lipid vesicle solution in buffer throughout
the second mixing process. Such control of the dilution buffer flow
rate advantageously allows for small particle size formation at
reduced concentrations.
[0418] These processes and the apparatuses for carrying out these
direct dilution and in-line dilution processes are described in
detail in U.S. Patent Publication No. 20070042031, the disclosure
of which is herein incorporated by reference in its entirety for
all purposes.
[0419] The nucleic acid-lipid particles formed using the direct
dilution and in-line dilution processes typically have a size of
from about 30 nm to about 150 nm, from about 40 nm to about 150 nm,
from about 50 nm to about 150 nm, from about 60 nm to about 130 nm,
from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,
from about 80 nm to about 100 nm, from about 90 nm to about 100 nm,
from about 70 to about 90 nm, from about 80 nm to about 90 nm, from
about 70 nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm,
90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm,
60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105
nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm,
or 150 nm (or any fraction thereof or range therein). The particles
thus formed do not aggregate and are optionally sized to achieve a
uniform particle size.
[0420] If needed, the lipid particles of the invention (e.g.,
SNALP) can be sized by any of the methods available for sizing
liposomes. The sizing may be conducted in order to achieve a
desired size range and relatively narrow distribution of particle
sizes.
[0421] Several techniques are available for sizing the particles to
a desired size. One sizing method, used for liposomes and equally
applicable to the present particles, is described in U.S. Pat. No.
4,737,323, the disclosure of which is herein incorporated by
reference in its entirety for all purposes. Sonicating a particle
suspension either by bath or probe sonication produces a
progressive size reduction down to particles of less than about 50
nm in size. Homogenization is another method which relies on
shearing energy to fragment larger particles into smaller ones. In
a typical homogenization procedure, particles are recirculated
through a standard emulsion homogenizer until selected particle
sizes, typically between about 60 and about 80 nm, are observed. In
both methods, the particle size distribution can be monitored by
conventional laser-beam particle size discrimination, or QELS.
[0422] Extrusion of the particles through a small-pore
polycarbonate membrane or an asymmetric ceramic membrane is also an
effective method for reducing particle sizes to a relatively
well-defined size distribution. Typically, the suspension is cycled
through the membrane one or more times until the desired particle
size distribution is achieved. The particles may be extruded
through successively smaller-pore membranes, to achieve a gradual
reduction in size.
[0423] In some embodiments, the nucleic acids present in the
particles are precondensed as described in, e.g., U.S. patent
application Ser. No. 09/744,103, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
[0424] In other embodiments, the methods may further comprise
adding non-lipid polycations which are useful to effect the
lipofection of cells using the present compositions.
[0425] Examples of suitable non-lipid polycations include,
hexadimethrine bromide (sold under the brand name POLYBRENE.RTM.,
from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of
hexadimethrine. Other suitable polycations include, for example,
salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine,
poly-D-lysine, polyallylamine, and polyethyleneimine Addition of
these salts is preferably after the particles have been formed.
[0426] In some embodiments, the nucleic acid to lipid ratios
(mass/mass ratios) in a formed nucleic acid-lipid particle (e.g.,
SNALP) will range from about 0.01 to about 0.2, from about 0.05 to
about 0.2, from about 0.02 to about 0.1, from about 0.03 to about
0.1, or from about 0.01 to about 0.08. The ratio of the starting
materials (input) also falls within this range. In other
embodiments, the particle preparation uses about 400 .mu.g nucleic
acid per 10 mg total lipid or a nucleic acid to lipid mass ratio of
about 0.01 to about 0.08 and, more preferably, about 0.04, which
corresponds to 1.25 mg of total lipid per 50 .mu.g of nucleic acid.
In other preferred embodiments, the particle has a nucleic
acid:lipid mass ratio of about 0.08.
[0427] In other embodiments, the lipid to nucleic acid ratios
(mass/mass ratios) in a formed nucleic acid-lipid particle (e.g.,
SNALP) will range from about 1 (1:1) to about 100 (100:1), from
about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50
(50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1)
to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from
about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25
(25:1), from about 2 (2:1) to about 25 (25:1), from about 3 (3:1)
to about 25 (25:1), from about 4 (4:1) to about 25 (25:1), from
about 5 (5:1) to about 25 (25:1), from about 5 (5:1) to about 20
(20:1), from about 5 (5:1) to about 15 (15:1), from about 5 (5:1)
to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9
(9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15
(15:1), 16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21
(21:1), 22 (22:1), 23 (23:1), 24 (24:1), or 25 (25:1), or any
fraction thereof or range therein. The ratio of the starting
materials (input) also falls within this range.
[0428] As previously discussed, the conjugated lipid may further
include a CPL. A variety of general methods for making SNALP-CPLs
(CPL-containing SNALP) are discussed herein. Two general techniques
include the "post-insertion" technique, that is, insertion of a CPL
into, for example, a pre-formed SNALP, and the "standard"
technique, wherein the CPL is included in the lipid mixture during,
for example, the SNALP formation steps. The post-insertion
technique results in SNALP having CPLs mainly in the external face
of the SNALP bilayer membrane, whereas standard techniques provide
SNALP having CPLs on both internal and external faces. The method
is especially useful for vesicles made from phospholipids (which
can contain cholesterol) and also for vesicles containing
PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of making
SNALP-CPLs are taught, for example, in U.S. Pat. Nos. 5,705,385;
6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent
Publication No. 20020072121; and PCT Publication No. WO 00/62813,
the disclosures of which are herein incorporated by reference in
their entirety for all purposes.
VII. Kits
[0429] The present invention also provides lipid particles (e.g.,
SNALP) in kit form. In some embodiments, the kit comprises a
container which is compartmentalized for holding the various
elements of the lipid particles (e.g., the active agents or
therapeutic agents such as nucleic acids and the individual lipid
components of the particles). Preferably, the kit comprises a
container (e.g., a vial or ampoule) which holds the lipid particles
of the invention (e.g., SNALP), wherein the particles are produced
by one of the processes set forth herein. In certain embodiments,
the kit may further comprise an endosomal membrane destabilizer
(e.g., calcium ions). The kit typically contains the particle
compositions of the invention, either as a suspension in a
pharmaceutically acceptable carrier or in dehydrated form, with
instructions for their rehydration (if lyophilized) and
administration.
[0430] The lipid particles of the present invention can be tailored
to preferentially target particular tissues, organs, or tumors of
interest. In some instances, the 1:57 lipid particle (e.g., SNALP)
formulation can be used to preferentially target the liver (e.g.,
normal liver tissue). In other instances, the 7:54 lipid particle
(e.g., SNALP) formulation can be used to preferentially target
solid tumors such as liver tumors and tumors outside of the liver.
In preferred embodiments, the kits of the invention comprise these
liver-directed and/or tumor-directed lipid particles, wherein the
particles are present in a container as a suspension or in
dehydrated form.
[0431] In certain other instances, it may be desirable to have a
targeting moiety attached to the surface of the lipid particle to
further enhance the targeting of the particle. Methods of attaching
targeting moieties (e.g., antibodies, proteins, etc.) to lipids
(such as those used in the present particles) are known to those of
skill in the art.
VIII. Administration of Lipid Particles
[0432] Once formed, the lipid particles of the invention (e.g.,
SNALP) are useful for the introduction of active agents or
therapeutic agents (e.g., nucleic acids such as interfering RNA)
into cells. Accordingly, the present invention also provides
methods for introducing an active agent or therapeutic agent such
as a nucleic acid (e.g., interfering RNA) into a cell. In some
instances, the cell is a liver cell such as, e.g., a hepatocyte
present in liver tissue. In other instances, the cell is a tumor
cell such as, e.g., a tumor cell present in a solid tumor. The
methods are carried out in vitro or in vivo by first forming the
particles as described above and then contacting the particles with
the cells for a period of time sufficient for delivery of the
active agent or therapeutic agent to the cells to occur.
[0433] The lipid particles of the invention (e.g., SNALP) can be
adsorbed to almost any cell type with which they are mixed or
contacted. Once adsorbed, the particles can either be endocytosed
by a portion of the cells, exchange lipids with cell membranes, or
fuse with the cells. Transfer or incorporation of the active agent
or therapeutic agent (e.g., nucleic acid) portion of the particle
can take place via any one of these pathways. In particular, when
fusion takes place, the particle membrane is integrated into the
cell membrane and the contents of the particle combine with the
intracellular fluid.
[0434] The lipid particles of the invention (e.g., SNALP) can be
administered either alone or in a mixture with a pharmaceutically
acceptable carrier (e.g., physiological saline or phosphate buffer)
selected in accordance with the route of administration and
standard pharmaceutical practice. Generally, normal buffered saline
(e.g., 135-150 mM NaCl) will be employed as the pharmaceutically
acceptable carrier. Other suitable carriers include, e.g., water,
buffered water, 0.4% saline, 0.3% glycine, and the like, including
glycoproteins for enhanced stability, such as albumin, lipoprotein,
globulin, etc. Additional suitable carriers are described in, e.g.,
REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company,
Philadelphia, Pa., 17th ed. (1985). As used herein, "carrier"
includes any and all solvents, dispersion media, vehicles,
coatings, diluents, antibacterial and antifungal agents, isotonic
and absorption delaying agents, buffers, carrier solutions,
suspensions, colloids, and the like. The phrase "pharmaceutically
acceptable" refers to molecular entities and compositions that do
not produce an allergic or similar untoward reaction when
administered to a human.
[0435] The pharmaceutically acceptable carrier is generally added
following lipid particle formation. Thus, after the lipid particle
(e.g., SNALP) is formed, the particle can be diluted into
pharmaceutically acceptable carriers such as normal buffered
saline.
[0436] The concentration of particles in the pharmaceutical
formulations can vary widely, i.e., from less than about 0.05%,
usually at or at least about 2 to 5%, to as much as about 10 to 90%
by weight, and will be selected primarily by fluid volumes,
viscosities, etc., in accordance with the particular mode of
administration selected. For example, the concentration may be
increased to lower the fluid load associated with treatment. This
may be particularly desirable in patients having
atherosclerosis-associated congestive heart failure or severe
hypertension. Alternatively, particles composed of irritating
lipids may be diluted to low concentrations to lessen inflammation
at the site of administration.
[0437] The pharmaceutical compositions of the present invention may
be sterilized by conventional, well-known sterilization techniques.
Aqueous solutions can be packaged for use or filtered under aseptic
conditions and lyophilized, the lyophilized preparation being
combined with a sterile aqueous solution prior to administration.
The compositions can contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, and calcium chloride.
Additionally, the particle suspension may include lipid-protective
agents which protect lipids against free-radical and
lipid-peroxidative damages on storage. Lipophilic free-radical
quenchers, such as alphatocopherol, and water-soluble iron-specific
chelators, such as ferrioxamine, are suitable.
[0438] In some embodiments, the lipid particles of the invention
(e.g., SNALP) are particularly useful in methods for the
therapeutic delivery of one or more nucleic acids comprising an
interfering RNA sequence (e.g., siRNA). In particular, it is an
object of this invention to provide in vitro and in vivo methods
for treatment of a disease or disorder in a mammal (e.g., a rodent
such as a mouse or a primate such as a human, chimpanzee, or
monkey) by downregulating or silencing the transcription and/or
translation of one or more target nucleic acid sequences or genes
of interest. As a non-limiting example, the methods of the
invention are useful for in vivo delivery of interfering RNA (e.g.,
siRNA) to the liver and/or tumor of a mammalian subject. In certain
embodiments, the disease or disorder is associated with expression
and/or overexpression of a gene and expression or overexpression of
the gene is reduced by the interfering RNA (e.g., siRNA). In
certain other embodiments, a therapeutically effective amount of
the lipid particle may be administered to the mammal. In some
instances, an interfering RNA (e.g., siRNA) is formulated into a
SNALP, and the particles are administered to patients requiring
such treatment. In other instances, cells are removed from a
patient, the interfering RNA is delivered in vitro (e.g., using a
SNALP described herein), and the cells are reinjected into the
patient.
[0439] A. In Vivo Administration
[0440] Systemic delivery for in vivo therapy, e.g., delivery of a
therapeutic nucleic acid to a distal target cell via body systems
such as the circulation, has been achieved using nucleic acid-lipid
particles such as those described in PCT Publication Nos. WO
05/007196, WO 05/121348, WO 05/120152, and WO 04/002453, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes. The present invention also provides
fully encapsulated lipid particles that protect the nucleic acid
from nuclease degradation in serum, are non-immunogenic, are small
in size, and are suitable for repeat dosing.
[0441] For in vivo administration, administration can be in any
manner known in the art, e.g., by injection, oral administration,
inhalation (e.g., intransal or intratracheal), transdermal
application, or rectal administration. Administration can be
accomplished via single or divided doses. The pharmaceutical
compositions can be administered parenterally, i.e.,
intraarticularly, intravenously, intraperitoneally, subcutaneously,
or intramuscularly. In some embodiments, the pharmaceutical
compositions are administered intravenously or intraperitoneally by
a bolus injection (see, e.g., U.S. Pat. No. 5,286,634).
Intracellular nucleic acid delivery has also been discussed in
Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et
al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther.
Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274
(1993). Still other methods of administering lipid-based
therapeutics are described in, for example, U.S. Pat. Nos.
3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and
4,588,578. The lipid particles can be administered by direct
injection at the site of disease or by injection at a site distal
from the site of disease (see, e.g., Culver, HUMAN GENE THERAPY,
MaryAnn Liebert, Inc., Publishers, New York. pp. 70-71 (1994)). The
disclosures of the above-described references are herein
incorporated by reference in their entirety for all purposes.
[0442] In embodiments where the lipid particles of the present
invention (e.g., SNALP) are administered intravenously, at least
about 5%, 10%, 15%, 20%, or 25% of the total injected dose of the
particles is present in plasma about 8, 12, 24, 36, or 48 hours
after injection. In other embodiments, more than about 20%, 30%,
40% and as much as about 60%, 70% or 80% of the total injected dose
of the lipid particles is present in plasma about 8, 12, 24, 36, or
48 hours after injection. In certain instances, more than about 10%
of a plurality of the particles is present in the plasma of a
mammal about 1 hour after administration. In certain other
instances, the presence of the lipid particles is detectable at
least about 1 hour after administration of the particle. In certain
embodiments, the presence of a therapeutic agent such as a nucleic
acid is detectable in cells of the lung, liver, tumor, or at a site
of inflammation at about 8, 12, 24, 36, 48, 60, 72 or 96 hours
after administration. In other embodiments, downregulation of
expression of a target sequence by an interfering RNA (e.g., siRNA)
is detectable at about 8, 12, 24, 36, 48, 60, 72 or 96 hours after
administration. In yet other embodiments, downregulation of
expression of a target sequence by an interfering RNA (e.g., siRNA)
occurs preferentially in liver cells (e.g., hepatocytes), tumor
cells, or in cells at a site of inflammation. In further
embodiments, the presence or effect of an interfering RNA (e.g.,
siRNA) in cells at a site proximal or distal to the site of
administration or in cells of the lung, liver, or a tumor is
detectable at about 12, 24, 48, 72, or 96 hours, or at about 6, 8,
10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after
administration. In additional embodiments, the lipid particles
(e.g., SNALP) of the invention are administered parenterally or
intraperitoneally.
[0443] The compositions of the present invention, either alone or
in combination with other suitable components, can be made into
aerosol formulations (i.e., they can be "nebulized") to be
administered via inhalation (e.g., intranasally or intratracheally)
(see, Brigham et al., Am. J. Sci., 298:278 (1989)). Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0444] In certain embodiments, the pharmaceutical compositions may
be delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering nucleic acid compositions
directly to the lungs via nasal aerosol sprays have been described,
e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212. Likewise, the
delivery of drugs using intranasal microparticle resins and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are
also well-known in the pharmaceutical arts. Similarly, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. 5,780,045. The disclosures of
the above-described patents are herein incorporated by reference in
their entirety for all purposes.
[0445] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions are preferably administered, for
example, by intravenous infusion, orally, topically,
intraperitoneally, intravesically, or intrathecally.
[0446] Generally, when administered intravenously, the lipid
particle formulations are formulated with a suitable pharmaceutical
carrier. Many pharmaceutically acceptable carriers may be employed
in the compositions and methods of the present invention. Suitable
formulations for use in the present invention are found, for
example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing
Company, Philadelphia, Pa., 17th ed. (1985). A variety of aqueous
carriers may be used, for example, water, buffered water, 0.4%
saline, 0.3% glycine, and the like, and may include glycoproteins
for enhanced stability, such as albumin, lipoprotein, globulin,
etc. Generally, normal buffered saline (135-150 mM NaCl) will be
employed as the pharmaceutically acceptable carrier, but other
suitable carriers will suffice. These compositions can be
sterilized by conventional liposomal sterilization techniques, such
as filtration. The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents, wetting agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine
oleate, etc. These compositions can be sterilized using the
techniques referred to above or, alternatively, they can be
produced under sterile conditions. The resulting aqueous solutions
may be packaged for use or filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration.
[0447] In certain applications, the lipid particles disclosed
herein may be delivered via oral administration to the individual.
The particles may be incorporated with excipients and used in the
form of ingestible tablets, buccal tablets, troches, capsules,
pills, lozenges, elixirs, mouthwash, suspensions, oral sprays,
syrups, wafers, and the like (see, e.g., U.S. Pat. Nos. 5,641,515,
5,580,579, and 5,792,451, the disclosures of which are herein
incorporated by reference in their entirety for all purposes).
These oral dosage forms may also contain the following: binders,
gelatin; excipients, lubricants, and/or flavoring agents. When the
unit dosage form is a capsule, it may contain, in addition to the
materials described above, a liquid carrier. Various other
materials may be present as coatings or to otherwise modify the
physical form of the dosage unit. Of course, any material used in
preparing any unit dosage form should be pharmaceutically pure and
substantially non-toxic in the amounts employed.
[0448] Typically, these oral formulations may contain at least
about 0.1% of the lipid particles or more, although the percentage
of the particles may, of course, be varied and may conveniently be
between about 1% or 2% and about 60% or 70% or more of the weight
or volume of the total formulation. Naturally, the amount of
particles in each therapeutically useful composition may be
prepared is such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
[0449] Formulations suitable for oral administration can consist
of: (a) liquid solutions, such as an effective amount of a packaged
therapeutic agent such as nucleic acid (e.g., interfering RNA)
suspended in diluents such as water, saline, or PEG 400; (b)
capsules, sachets, or tablets, each containing a predetermined
amount of a therapeutic agent such as nucleic acid (e.g.,
interfering RNA), as liquids, solids, granules, or gelatin; (c)
suspensions in an appropriate liquid; and (d) suitable emulsions.
Tablet forms can include one or more of lactose, sucrose, mannitol,
sorbitol, calcium phosphates, corn starch, potato starch,
microcrystalline cellulose, gelatin, colloidal silicon dioxide,
talc, magnesium stearate, stearic acid, and other excipients,
colorants, fillers, binders, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, dyes, disintegrating
agents, and pharmaceutically compatible carriers. Lozenge forms can
comprise a therapeutic agent such as nucleic acid (e.g.,
interfering RNA) in a flavor, e.g., sucrose, as well as pastilles
comprising the therapeutic agent in an inert base, such as gelatin
and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in addition to the therapeutic agent, carriers known in
the art.
[0450] In another example of their use, lipid particles can be
incorporated into a broad range of topical dosage forms. For
instance, a suspension containing nucleic acid-lipid particles such
as SNALP can be formulated and administered as gels, oils,
emulsions, topical creams, pastes, ointments, lotions, foams,
mousses, and the like.
[0451] When preparing pharmaceutical preparations of the lipid
particles of the invention, it is preferable to use quantities of
the particles which have been purified to reduce or eliminate empty
particles or particles with therapeutic agents such as nucleic acid
associated with the external surface.
[0452] The methods of the present invention may be practiced in a
variety of hosts. Preferred hosts include mammalian species, such
as primates (e.g., humans and chimpanzees as well as other nonhuman
primates), canines, felines, equines, bovines, ovines, caprines,
rodents (e.g., rats and mice), lagomorphs, and swine.
[0453] The amount of particles administered will depend upon the
ratio of therapeutic agent (e.g., nucleic acid) to lipid, the
particular therapeutic agent (e.g., nucleic acid) used, the disease
or disorder being treated, the age, weight, and condition of the
patient, and the judgment of the clinician, but will generally be
between about 0.01 and about 50 mg per kilogram of body weight,
preferably between about 0.1 and about 5 mg/kg of body weight, or
about 10.sup.8-10.sup.10 particles per administration (e.g.,
injection).
[0454] B. In Vitro Administration
[0455] For in vitro applications, the delivery of therapeutic
agents such as nucleic acids (e.g., interfering RNA) can be to any
cell grown in culture, whether of plant or animal origin,
vertebrate or invertebrate, and of any tissue or type. In preferred
embodiments, the cells are animal cells, more preferably mammalian
cells, and most preferably human cells (e.g., tumor cells or
hepatocytes).
[0456] Contact between the cells and the lipid particles, when
carried out in vitro, takes place in a biologically compatible
medium. The concentration of particles varies widely depending on
the particular application, but is generally between about 1 mmol
and about 10 mmol Treatment of the cells with the lipid particles
is generally carried out at physiological temperatures (about
37.degree. C.) for periods of time of from about 1 to 48 hours,
preferably of from about 2 to 4 hours.
[0457] In one group of preferred embodiments, a lipid particle
suspension is added to 60-80% confluent plated cells having a cell
density of from about 10.sup.3 to about 10.sup.5 cells/ml, more
preferably about 2.times.10.sup.4 cells/ml. The concentration of
the suspension added to the cells is preferably of from about 0.01
to 0.2 .mu.g/ml, more preferably about 0.1 .mu.g/ml.
[0458] To the extent that tissue culture of cells may be required,
it is well-known in the art. For example, Freshney, Culture of
Animal Cells, a Manual of Basic Technique, 3rd Ed., Wiley-Liss, New
York (1994), Kuchler et al., Biochemical Methods in Cell Culture
and Virology, Dowden, Hutchinson and Ross, Inc. (1977), and the
references cited therein provide a general guide to the culture of
cells. Cultured cell systems often will be in the form of
monolayers of cells, although cell suspensions are also used.
[0459] Using an Endosomal Release Parameter (ERP) assay, the
delivery efficiency of the SNALP or other lipid particle of the
invention can be optimized. An ERP assay is described in detail in
U.S. Patent Publication No. 20030077829, the disclosure of which is
herein incorporated by reference in its entirety for all purposes.
More particularly, the purpose of an ERP assay is to distinguish
the effect of various cationic lipids and helper lipid components
of SNALP or other lipid particle based on their relative effect on
binding/uptake or fusion with/destabilization of the endosomal
membrane. This assay allows one to determine quantitatively how
each component of the SNALP or other lipid particle affects
delivery efficiency, thereby optimizing the SNALP or other lipid
particle. Usually, an ERP assay measures expression of a reporter
protein (e.g., luciferase, .beta.-galactosidase, green fluorescent
protein (GFP), etc.), and in some instances, a SNALP formulation
optimized for an expression plasmid will also be appropriate for
encapsulating an interfering RNA. In other instances, an ERP assay
can be adapted to measure downregulation of transcription or
translation of a target sequence in the presence or absence of an
interfering RNA (e.g., siRNA). By comparing the ERPs for each of
the various SNALP or other lipid particles, one can readily
determine the optimized system, e.g., the SNALP or other lipid
particle that has the greatest uptake in the cell.
[0460] C. Cells for Delivery of Lipid Particles
[0461] The compositions and methods of the present invention are
used to treat a wide variety of cell types, in vivo and in vitro.
Suitable cells include, but are not limited to, hepatocytes,
reticuloendothelial cells (e.g., monocytes, macrophages, etc.),
fibroblast cells, endothelial cells, platelet cells, other cell
types infected and/or susceptible of being infected with viruses,
hematopoietic precursor (stem) cells, keratinocytes, skeletal and
smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes,
terminally differentiated cells, slow or noncycling primary cells,
parenchymal cells, lymphoid cells, epithelial cells, bone cells,
and the like.
[0462] In particular embodiments, an active agent or therapeutic
agent such as a nucleic acid (e.g., an interfering RNA) is
delivered to cancer cells (e.g., cells of a solid tumor) including,
but not limited to, liver cancer cells, lung cancer cells, colon
cancer cells, rectal cancer cells, anal cancer cells, bile duct
cancer cells, small intestine cancer cells, stomach (gastric)
cancer cells, esophageal cancer cells, gallbladder cancer cells,
pancreatic cancer cells, appendix cancer cells, breast cancer
cells, ovarian cancer cells, cervical cancer cells, prostate cancer
cells, renal cancer cells, cancer cells of the central nervous
system, glioblastoma tumor cells, skin cancer cells, lymphoma
cells, choriocarcinoma tumor cells, head and neck cancer cells,
osteogenic sarcoma tumor cells, and blood cancer cells.
[0463] In vivo delivery of lipid particles such as SNALP
encapsulating a nucleic acid (e.g., an interfering RNA) is suited
for targeting cells of any cell type. The methods and compositions
can be employed with cells of a wide variety of vertebrates,
including mammals, such as, e.g, canines, felines, equines,
bovines, ovines, caprines, rodents (e.g., mice, rats, and guinea
pigs), lagomorphs, swine, and primates (e.g. monkeys, chimpanzees,
and humans).
[0464] D. Detection of Lipid Particles
[0465] In some embodiments, the lipid particles of the present
invention (e.g., SNALP) are detectable in the subject at about 1,
2, 3, 4, 5, 6, 7, 8 or more hours. In other embodiments, the lipid
particles of the present invention (e.g., SNALP) are detectable in
the subject at about 8, 12, 24, 48, 60, 72, or 96 hours, or about
6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25, or 28 days after
administration of the particles. The presence of the particles can
be detected in the cells, tissues, or other biological samples from
the subject. The particles may be detected, e.g., by direct
detection of the particles, detection of a therapeutic nucleic acid
such as an interfering RNA (e.g., siRNA) sequence, detection of the
target sequence of interest (i.e., by detecting expression or
reduced expression of the sequence of interest), or a combination
thereof
[0466] 1. Detection of Particles
[0467] Lipid particles of the invention such as SNALP can be
detected using any method known in the art. For example, a label
can be coupled directly or indirectly to a component of the lipid
particle using methods well-known in the art. A wide variety of
labels can be used, with the choice of label depending on
sensitivity required, ease of conjugation with the lipid particle
component, stability requirements, and available instrumentation
and disposal provisions. Suitable labels include, but are not
limited to, spectral labels such as fluorescent dyes (e.g.,
fluorescein and derivatives, such as fluorescein isothiocyanate
(FITC) and Oregon Green.TM.; rhodamine and derivatives such Texas
red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin,
biotin, phycoerythrin, AMCA, CyDyes.TM., and the like; radiolabels
such as .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, .sup.33P,
etc.; enzymes such as horse radish peroxidase, alkaline
phosphatase, etc.; spectral colorimetric labels such as colloidal
gold or colored glass or plastic beads such as polystyrene,
polypropylene, latex, etc. The label can be detected using any
means known in the art.
[0468] 2. Detection of Nucleic Acids
[0469] Nucleic acids (e.g., interfering RNA) are detected and
quantified herein by any of a number of means well-known to those
of skill in the art. The detection of nucleic acids may proceed by
well-known methods such as Southern analysis, Northern analysis,
gel electrophoresis, PCR, radiolabeling, scintillation counting,
and affinity chromatography. Additional analytic biochemical
methods such as spectrophotometry, radiography, electrophoresis,
capillary electrophoresis, high performance liquid chromatography
(HPLC), thin layer chromatography (TLC), and hyperdiffusion
chromatography may also be employed.
[0470] The selection of a nucleic acid hybridization format is not
critical. A variety of nucleic acid hybridization formats are known
to those skilled in the art. For example, common formats include
sandwich assays and competition or displacement assays.
Hybridization techniques are generally described in, e.g., "Nucleic
Acid Hybridization, A Practical Approach," Eds. Hames and Higgins,
IRL Press (1985). The sensitivity of the hybridization assays may
be enhanced through the use of a nucleic acid amplification system
which multiplies the target nucleic acid being detected. In vitro
amplification techniques suitable for amplifying sequences for use
as molecular probes or for generating nucleic acid fragments for
subsequent subcloning are known. Examples of techniques sufficient
to direct persons of skill through such in vitro amplification
methods, including the polymerase chain reaction (PCR), the ligase
chain reaction (LCR), Q.beta.-replicase amplification, and other
RNA polymerase mediated techniques (e.g., NASBA.TM.) are found in
Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (2000); and Ausubel et al., SHORT
PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002);
as well as U.S. Pat. No. 4,683,202; PCR Protocols, A Guide to
Methods and Applications (Innis et al. eds.) Academic Press Inc.
San Diego, Calif. (1990); Arnheim & Levinson (Oct. 1, 1990),
C&EN 36; The Journal Of NIH Research, 3:81 (1991); Kwoh et al.,
Proc. Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli et al., Proc.
Natl. Acad. Sci. USA, 87:1874 (1990); Lomell et al., J. Clin.
Chem., 35:1826 (1989); Landegren et al., Science, 241:1077 (1988);
Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene, 4:560
(1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan and
Malek, Biotechnology, 13:563 (1995). Improved methods of cloning in
vitro amplified nucleic acids are described in U.S. Pat. No.
5,426,039. Other methods described in the art are the nucleic acid
sequence based amplification (NASBA.TM., Cangene, Mississauga,
Ontario) and Q.beta.-replicase systems. These systems can be used
to directly identify mutants where the PCR or LCR primers are
designed to be extended or ligated only when a select sequence is
present. Alternatively, the select sequences can be generally
amplified using, for example, nonspecific PCR primers and the
amplified target region later probed for a specific sequence
indicative of a mutation. The disclosures of the above-described
references are herein incorporated by reference in their entirety
for all purposes.
[0471] Nucleic acids for use as probes, e.g., in in vitro
amplification methods, for use as gene probes, or as inhibitor
components are typically synthesized chemically according to the
solid phase phosphoramidite triester method described by Beaucage
et al., Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an
automated synthesizer, as described in Needham VanDevanter et al.,
Nucleic Acids Res., 12:6159 (1984). Purification of
polynucleotides, where necessary, is typically performed by either
native acrylamide gel electrophoresis or by anion exchange HPLC as
described in Pearson et al., J. Chrom., 255:137 149 (1983). The
sequence of the synthetic polynucleotides can be verified using the
chemical degradation method of Maxam and Gilbert (1980) in Grossman
and Moldave (eds.) Academic Press, New York, Methods in Enzymology,
65:499.
[0472] An alternative means for determining the level of
transcription is in situ hybridization. In situ hybridization
assays are well-known and are generally described in Angerer et
al., Methods Enzymol., 152:649 (1987). In an in situ hybridization
assay, cells are fixed to a solid support, typically a glass slide.
If DNA is to be probed, the cells are denatured with heat or
alkali. The cells are then contacted with a hybridization solution
at a moderate temperature to permit annealing of specific probes
that are labeled. The probes are preferably labeled with
radioisotopes or fluorescent reporters.
IX. Examples
[0473] The present invention will be described in greater detail by
way of specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of noncritical parameters which can be changed or modified
to yield essentially the same results.
Example 1
Synthesis of 13-B1
[0474] Synthesis of compound 13-B1,
(13Z,16Z)-4-hydroxy-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12Z)-octadec-
a-9,12-dienyloxy)docosa-13,16-dienyl 4-(dimethylamino)butanoate,
having structure (B) below, was synthesized as described in Scheme
7 below:
##STR00045##
[0475] Step 1: Synthesis of
3-((9Z,12Z)-octadeca-9,12-dienyloxy)dihydrofuran-2(3H)-one, having
the structure below:
##STR00046##
[0476] To a solution of .alpha.-hydroxybutyrolactone (2.0 g, 19 6
mmol) and linoleyl methane sulfonate (5.4 g, 15.7 mmol) in
anhydrous DMF (60 mL), was added cesium carbonate (8.0 g, 24.5
mmol). The solution was stirred overnight under nitrogen at
80.degree. C. Upon completion, the reaction was poured into water
(100 mL) and extracted with ethyl acetate (3.times.100 mL). The
combined ethyl acetate extracts were washed with brine (3.times.50
mL), dried on magnesium sulfate, filtered and concentrated in vacuo
to dryness. The residue was purified by column chromatography on
silica gel 60 (eluted with 10% ethyl acetate in hexanes) to afford
3-((9Z,12Z)-octadeca-9,12-dienyloxy)dihydrofuran-2(3H)-one as a
pale yellow oil (3.5 g, 51%).
[0477] Step 2: Synthesis of
(14Z,17Z)-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12Z)-octadeca-9,12-die-
nyloxy)tricosa-14,17-diene-1,4-diol, having the following
structure:
##STR00047##
[0478] A 100 mL round bottom flask charged with magnesium turnings
(250 mg) and a stir bar was dried with a heat gun for 5 minutes
then cooled under nitrogen. The flask was charged with dry THF (10
mL) and a small grain of iodine. A solution of linoleyl bromide (3
g, 9.1 mmol) in diethyl ether was added slowly and the mixture was
stirred at room temperature. The Grignard did not initiate so the
solution was concentrated to .about.5 mL then stirred for 2 minutes
until the solution turned opaque and the iodine color disappeared.
After 20 minutes, a colorless solid precipitated so the reaction
was diluted with THF (15 mL) and stirred overnight. The next day
3-((9Z,12Z)-octadeca-9,12-dienyloxy)dihydrofuran-2(3H)-one (1.3 g,
3.6 mmol) was added and the solution was stirred for 3 hours. The
reaction was diluted with ether (100 mL) and washed with 5% HCl (50
mL). The ether layer was dried on magnesium sulfate, filtered and
concentrated in vacuo to dryness. The residue was purified by
column chromatography on silica gel 60 (eluted with 10% ether in
hexanes) to afford
(14Z,17Z)-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12Z)-octadeca-9,12-die-
nyloxy)tricosa-14,17-diene-1,4-diol as a colorless oil (1.75 g,
56%).
[0479] Step 3: Synthesis of compound 13-B1,
(13Z,16Z)-4-hydroxy-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12Z)-octadec-
a-9,12-dienyloxy)docosa-13,16-dienyl 4-(dimethylamino)butanoate,
having the following structure:
##STR00048##
[0480] To a solution of
(14Z,17Z)-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12Z)-octadeca-9,12-die-
nyloxy)tricosa-14,17-diene-1,4-diol (450 mg, 0.52 mmol),
4-(dimethylamino)butanoic acid hydrochloride (105 mg, 0.62 mmol),
EDCI hydrochloride (120 mg, 0.62 mmol), DIPEA (225 .mu.L, 1.3 mmol)
in anhydrous dichloromethane (10 mL) was added DMAP (5 mg). The
solution was stirred at room temperature under nitrogen for 64
hours. Upon completion, the solution was concentrated in vacuo to
dryness and purified by column chromatography on silica gel 60
(eluted with 1:1 hexanes/Ethyl acetate) to afford
(13Z,16Z)-4-hydroxy-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12-
Z)-octadeca-9,12-dienyloxy)docosa-13,16-dienyl
4-(dimethylamino)butanoate as a colorless oil (375 mg, 75%).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.43-5.29 (m, 12H),
4.31-4.11 (m, 2H), 3.60-5.29 (m, 2H), 3.27-3.22 (m, 1H), 2.78 (t,
6H, J=6.4 Hz), 2.39-2.28 (m, 3H), 2.24 (s, 6H), 2.09-2.02 (m, 12H),
1.91-1.72 (m, 4H), 1.61-1.23 (m, 60H), 0.90 (t, 9H, J=7.0 Hz).
Example 2
Synthesis of 13-B3
[0481] See steps 1 and 2 of Example 1.
[0482] Step 3: Synthesis of compound 13-B3,
(13Z,16Z)-4-hydroxy-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12Z)-octadec-
a-9,12-dienyloxy)docosa-13,16-dienyl 3-(dimethylamino)propanoate,
having the following structure, was synthesized as described above
in Scheme 7 for structure (A):
##STR00049##
[0483] To a solution of
(14Z,17Z)-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12Z)-octadeca-9,12-die-
nyloxy)tricosa-14,17-diene-1,4-diol (1.5 g, 1.73 mmol),
4-(dimethylamino)propanoic acid hydrochloride (500 mg, 3.3 mmol),
EDCI hydrochloride (750 mg, 3.9 mmol), DIPEA (225 4, 1.3 mmol) in
anhydrous dichloromethane (15 mL) was added DMAP (5 mg). The
solution was stirred at room temperature under nitrogen for 20
hours. Upon completion, the solution was concentrated in vacuo to
dryness and purified by column chromatography on silica gel 60
(eluted with 1:1 hexanes/Ethyl acetate) to afford
(13Z,16Z)-4-hydroxy-4-((9Z,12Z)-octadeca-9,12-dienyl)-3-((9Z,12-
Z)-octadeca-9,12-dienyloxy)docosa-13,16-dienyl
3-(dimethylamino)propanoate as a colorless oil (275 mg). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 5.43-5.29 (m, 12H), 4.34-4.14 (m,
2H), 3.60-3.43 (m, 2H), 3.25 (dd, 1H, J=8.8, 2.8 Hz), 2.81-2.68 (m,
8H), 2.62-2.53 (m, 2H), 2.33 (s, 6H), 2.10-2.00 (m, 12H), 1.04-1.72
(m, 2H), 1.62-1.15 (m, 60H), 0.90 (t, 9H, J=7.0 Hz).
Example 3
Synthesis of 13-B2
[0484] Compound 13-B2, having structure (C) below, was synthesized
as described in Scheme 8 below.
##STR00050##
[0485] Step 1: Synthesis of
(6Z,9Z,29Z,32Z)-19-((9Z,12Z)-octadeca-9,12-dienyl)octatriaconta-6,9,29,32-
-tetraene-19,20-diol, having the following structure:
##STR00051##
[0486] A 100 mL round bottom flask was charged with magnesium
turnings (360 mg, 14.6 mmol) and a stir bar. The flask was dried
with a heat gun for 5 minutes then cooled under nitrogen. The flask
was charged with THF (5 mL) and a small grain of iodine. A solution
of linoleyl bromide (4 g, 12.1 mmol) in THF (5 mL) was added slowly
and after 5 minutes the reaction initiated vigorously. The solution
was stirred at room temperature for 2 hours, and then ethyl
glyoxalate (0.5 mL, 2.42 mmol, 50% solution in toluene) was added.
The exothermic reaction was finished immediately and quenched with
saturated ammonium chloride solution (5 mL). The solution diluted
with water and extracted with diethyl ether (3.times.50 mL). The
combined diethyl ether extracts were dried on magnesium sulfate,
filtered and concentrated in vacuo to dryness. The oil was purified
by column chromatography on silica gel 60 (eluted with 100% Hexanes
to 15% Et.sub.2O in hexanes) to afford
(6Z,9Z,29Z,32Z)-19-((9Z,12Z)-octadeca-9,12-dienyl)octatriaconta-6,9,29,32-
-tetraene-19,20-diol as a colorless oil (945 mg, 48%).
[0487] Step 2: Compound 13-B2, synthesis of
(6Z,9Z,29Z,32Z)-20-hydroxy-20-((9Z,12Z)-octadeca-9,12-dienyl)octatriacont-
a-6,9,29,32-tetraen-19-yl 4-(dimethylamino)butanoate
##STR00052##
[0488] To a solution of
(6Z,9Z,29Z,32Z)-19-((9Z,12Z)-octadeca-9,12-dienyl)octatriaconta-6,9,29,32-
-tetraene-19,20-diol (500 mg, 0.65 mmol), 4-(dimethylamino)butanoic
acid hydrochloride (131 mg, 0.78 mmol), EDCI hydrochloride (150 mg,
0.78 mmol), DIPEA (285 .mu.L, 1.63 mmol) in anhydrous
dichloromethane (10 mL) was added DMAP (5 mg). The solution was
stirred at room temperature under nitrogen for 16 hours. The
reaction was not complete so additional 4-(dimethylamino)butanoic
acid hydrochloride (131 mg, 0.78 mmol), EDCI hydrochloride (150 mg,
0.78 mmol), DIPEA (285 .mu.L, 1.63 mmol) and DMAP (5 mg) were
added. The solution was refluxed for 6 hours and cooled to room
temperature. The reaction mixture (10 mL loaded directly onto
column) was purified by column chromatography on silica gel 60
(eluted with 100% hexanes.fwdarw.2:1.fwdarw.1:1 hexanes/ethyl
acetate) to afford
(6Z,9Z,29Z,32Z)-20-hydroxy-20-((9Z,12Z)-octadeca-9,12-dienyl)octatriacont-
a-6,9,29,32-tetraen-19-yl 4-(dimethylamino)butanoate as a colorless
oil (440 mg, 77%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
5.42-5.31 (m, 12H), 4.98 (dd, 1H, J=10.5, 2.3 Hz), 2.78 (t, 6H,
J=6.9 Hz), 2.45-2.33 (m, 4H), 2.26 (s, 6H), 2.09-2.02 (m, 12H),
1.93-1.77 (m, 2H), 1.62-1.42 (m, 4H), 1.41-1.20 (m, 52H), 0.90 (t,
9H, J=6.7 Hz).
Example 4
Synthesis of 13-B4
[0489] See step 1 of Example 3.
[0490] Step 2: Synthesis of 5-Hydroxy benzylpentanoate, having the
following structure:
##STR00053##
[0491] A solution of .delta.-valerolactone (10.0 g, 100 mmol) in a
1M aqueous sodium hydroxide solution (100 mL) was heated for 16
hours at 65.degree. C. The residue was concentrated in vacuo and
the remaining white powder dried under high vacuum at 60.degree. C.
The white powder was pulverized and suspended in acetone (40 mL).
With stirring, benzyl bromide (17.0 g, 101 mmol) and
tetrabutylammonium bromide (0.82 g) were added. The mixture was
heated with stirring for 72 hours at 45.degree. C. It was then
cooled to room temperature, concentrated in vacuo to dryness and
dissolved in ethyl acetate (250 mL). The ethyl acetate solution was
washed with saturated sodium bicarbonate (2.times.50 mL) and brine
(2.times.50 mL). The organic portion was dried on magnesium
sulfate, filtered, and concentrated in vacuo to dryness. The
resulting yellow oil was purified by column chromatography on
silica gel 60 (eluted with a gradient of 100%
hexanes.fwdarw.30%.fwdarw.50% ethyl acetate in hexanes) to afford
5-hydroxy benzylpentanoate as a yellow oil (3.11 g, 15%).
[0492] Step 3: Synthesis of 5-Methanesulfonyl benzylpentanoate,
having the following structure:
##STR00054##
[0493] To a solution of 5-hydroxy benzylpentanoate (1.46 g, 7.0
mmol) and triethylamine (1.95 mL, 14.0 mmol) in anhydrous
dichloromethane (30 mL) at -10.degree. C. was added a solution of
methanesulfonyl chloride (0.68 mL, 8.8 mmol) in dry dichloromethane
(10 mL) drop wise over 1 hour. The solution was stirred overnight
at room temperature. TLC (3:1 ethyl acetate/hexanes) indicated that
the reaction was complete so the solution was diluted with
dichloromethane and washed saturated sodium bicarbonate (3.times.50
mL). The combined aqueous layers were back extracted twice with
dichloromethane. The combined organic layers were dried on
magnesium sulfate, filtered, and concentrated in vacuo to dryness.
The resulting yellow oil was purified by column chromatography on
silica gel 60 (eluted with a gradient of 10%.fwdarw.20%.fwdarw.30%
ethyl acetate in hexanes) to afford 5-Methanesulfonyl
benzylpentanoate as a pale yellow oil (1.76 g, 88%).
[0494] Step 4: Synthesis of 5-(dimethylamino)benzylpentanoate,
having the following structure:
##STR00055##
[0495] A solution of 5-(Methanesulfonyl)benzylpentanoate (1.76 g,
6.2 mmol) in 5.6M dimethylamine in ethanol (100 mL) was stirred at
room temperature for 72 hours. Upon completion, the solution was
concentrated in vacuo to dryness and the resulting brown oil
purified by column chromatography on silica gel 60 (eluted with 2%
MeOH and 0.3% NH.sub.4OH(aq) in dichloromethane) to afford
5-(dimethylamino)benzylpentanoate as a brown oil (577 mg, 40%).
[0496] Step 5: Synthesis of 5-(Dimethylamino)pentanoic Acid, having
the following structure:
##STR00056##
[0497] To a solution of 5-(dimethylamino)benzylpentanoate (577 mg,
2.45 mmol) in ethyl acetate (20 mL) was added 10% palladium on
carbon (80 mg). The solution was stirred vigorously under hydrogen
and monitored by TLC (10% MeOH, 1% NH4OH in dichloromethane). After
4 hours the reaction was approximately 50% complete, so additional
palladium on carbon (160 mg) was added and hydrogen gas bubbled
through the solution. The solution was then left stirring under
hydrogen overnight. Upon completion, the solution was filtered
through celite and concentrated in vacuo to afford
5-(Dimethylamino)pentanoic Acid as a yellow solid (357 mg,
quantitative).
[0498] Step 6: Synthesis of compound 13-B4,
(6Z,9Z,29Z,32Z)-20-hydroxy-20-((9Z,12Z)-octadeca-9,12-dienyl)octatriacont-
a-6,9,29,32-tetraen-19-yl 5-(dimethylamino)pentanoate, having the
following structure:
##STR00057##
[0499] To a solution of
(6Z,9Z,29Z,32Z)-19-((9Z,12Z)-octadeca-9,12-dienyl)octatriaconta-6,9,29,32-
-tetraene-19,20-diol (0.75 g, 0.98 mmol),
5-(dimethylamino)pentanoic acid (285 mg, 2.0 mmol), EDC
hydrochloride (378 mg, 2.0 mmol), and 4-(N,N-dimethylamino)pyridine
(5 mg) in anhydrous dichloromethane (10 mL) was added DIPEA (0.68
mL, 3.9 mmol). The reaction was stirred for 16 hours at room
temperature under nitrogen. TLC (1:1 hexanes/ethyl acetate)
indicated that the reaction had stalled, so additional
5-(dimethylamino)pentanoic acid (70 mg, 0.5 mmol) and fresh EDC
hydrochloride (300 mg, 1.6 mmol) were added. The reaction was
stirred for an additional 24 hours under nitrogen at room
temperature. The solution was diluted with dichloromethane (100 mL)
and washed with saturated sodium bicarbonate (50 mL), water (50
mL), and brine (50 mL). The combined aqueous layers were back
extracted once with dichloromethane (50 mL). The combined
dichloromethane extracts were dried on magnesium sulfate, filtered,
and concentrated in vacuo to dryness. The resulting orange oil was
purified by column chromatography on silica gel 60 (eluted with a
gradient of 25%.fwdarw.50% ethyl acetate in hexanes) to afford
(6Z,9Z,29Z,32Z)-20-hydroxy-20-((9Z,12Z)-octadeca-9,12-dienyl)octatriacont-
a-6,9,29,32-tetraen-19-yl 5-(dimethylamino)pentanoate as a pale
yellow oil (560 mg, 64%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
5.43-5.29 (m, 12H), 4.95 (dd, 1H, J=10.1, 2.7 Hz), 2.78 (t, 6H,
J=6.5 Hz), 2.38 (t, 2H, J=7.5 Hz), 2.28 (t, 2H, J=7.5 Hz), 2.22 (s,
6H), 2.09-2.02 (m, 12H), 1.72-1.42 (m, 13H), 1.41-1.19 (m, 56H),
0.90 (t, 9H, J=6.7 Hz).
Example 5
Synthesis of 13-B5
##STR00058##
[0501] Step 1: Synthesis of
(6Z,9Z)-18-(3-(allyloxy)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propoxy)octa-
deca-6,9-diene, having the following structure:
##STR00059##
[0502] A 500 mL round bottom flask was charged with
3-(allyloxy)propane-1,2-diol (2.6 g, 19.8 mmol), linoleyl mesylate
(15.0 g, 43.5 mmol), tetrabutylammonium hydrogen sulfate (3.4 g,
9.9 mmol), toluene (600 mL) and 40% NaOH (30 mL). The reagents were
vigorously stirred (emulsified) for 80 hours at room temperature.
Upon completion, the solution was diluted with water (100 mL) and
toluene (100 mL). The toluene was separated and the remaining
aqueous solution was extracted with toluene (150 mL). The combine
toluene extracts were dried on magnesium sulfate, filtered and
concentrated in vacuo to dryness. The residue was purified by
column chromatography on silica gel 60 (eluted with 5% diethyl
ether in hexanes) to afford
(6Z,9Z)-18-(3-(allyloxy)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propoxy)octa-
deca-6,9-diene as a colorless oil (9.1 g, 73%).
[0503] Step 2: Synthesis of
2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-ol, having the
following structure:
##STR00060##
[0504] To a solution of
(6Z,9Z)-18-(3-(allyloxy)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propoxy)octa-
deca-6,9-diene (9.1 g, 14.3 mmol) in ethanol (100 ml) was added
trifluoroacetic acid (15.0 mL) and
tetrakis(triphenylphosphine)palladium(0) (1.65 g, 1.43 mmol). The
solution was refluxed for 16 hours, cooled to room temperature and
concentrated in vacuo to dryness. The residue was purified by
column chromatography on silica gel 60 (eluted with a gradient of
100% hexanes to 1:1 hexanes/DCM to 100% DCM) to afford
2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-ol as a colorless
oil (6.1 g, 54%).
[0505] Step 3: Synthesis of
2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propanal, having the
following structure:
##STR00061##
[0506] To a solution of
2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-ol (4.0 g, 7.0
mmol) and PCC (4.5 g, 21.0 mmol) in dichloromethane (75 mL) was
added sodium carbonate (375 mg, 3.5 mmol). The solution was stirred
for 6 hours at room temperature then filtered through silica with
diethyl ether rinses (250 mL). The filtrate was concentrated in
vacuo to dryness and purified by column chromatography on silica
gel 60 (eluted with a gradient of 100% hexanes to 5% ether in
hexanes) to afford
2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propanal as a colorless
oil (1.6 g, 39%)
[0507] Step 4: Synthesis of
(12Z,15Z)-1,2-bis((9Z,12Z)-octadeca-9,12-dienyloxy)henicosa-12,15-dien-3--
ol, having the following structure:
##STR00062##
[0508] To a solution of
2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propanal (1.5 g, 2.62
mmol) in anhydrous diethyl ether (10 mL) was added LinMgBr Grignard
(2.56 mL, 5.23 mmol). Upon completion, the solution was quenched
with 5% HCl (50 mL) and washed with diethyl ether (3.times.50 mL).
The combined ethyl ether extracts were dried on magnesium sulfate,
filtered and concentrated in vacuo to dryness. The residue was
purified by column chromatography on silica gel 60 (eluted with a
gradient of 100% hexanes to 2% ethyl acetate in hexanes) to afford
(12Z,15Z)-1,2-bis((9Z,12Z)-octadeca-9,12-dienyloxy)henicosa-12,15-dien-3--
ol as a colorless oil (460 mg, 21%).
[0509] Step 5: Synthesis of compound 13-B5,
(12Z,15Z)-1,2-bis((9Z,12Z)-octadeca-9,12-dienyloxy)henicosa-12,15-dien-3--
yl 4-(dimethylamino)butanoate, having the following structure:
##STR00063##
[0510] To a solution of
(12Z,15Z)-1,2-bis((9Z,12Z)-octadeca-9,12-dienyloxy)henicosa-12,15-dien-3--
ol (450 mg, 0.54 mmol), 4-(dimethylamino)butanoic acid
hydrochloride (135 mg, 0.8 mmol), EDCI hydrochloride (153 mg, 0.8
mmol), DIPEA (260 .mu.L, 1.6 mmol) in anhydrous dichloromethane (10
mL) was added DMAP (5 mg). The solution was refluxed for 3 hours,
cooled to room temperature and concentrated in vacuo to dryness.
The reaction mixture was purified by column chromatography on
silica gel 60 (eluted with a gradient of 100% hexanes to 1:1
hexanes/ethyl acetate) to afford
(12Z,15Z)-1,2-bis((9Z,12Z)-octadeca-9,12-dienyloxy)henicosa-12,15-dien-3--
yl 4-(dimethylamino)butanoate as a colorless oil (236 mg).
5.43-5.29 (m, 2H), 5.05-4.98 (m, 1H), 3.68-3.64 (m, 1H), 3.52-3.46
(m, 1H), 3.40 (t, 2H, J=6.7 Hz), 2.78 (t, 6H, J=6.4 Hz), 2.46-2.35
(m, 4H), 2.32 (s, 6H), 2.09-2.02 (m, 12H), 1.91-1.81 (m, 2H),
1.70-1.48 (m, 6H), 1.41-1.23 (m, 50H), 0.90 (t, 9H, J=6.7 Hz).
Example 6
Synthesis of 13-B6
[0511] Step 1: Synthesis of Di-Linoleyl acetonitrile, having the
following structure:
##STR00064##
[0512] A solution of di-lin-OMs (2.0 g, 3.3 mmol) in anhydrous DMF
(15 mL) was treated with NaCN (809 mg, 16.5 mmol) and heated
(55.degree. C., 16 h). The DMF was then removed under reduced
pressure. The residue was dissolved in EtOAc and washed with
H.sub.2O and brine, then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The crude material was subjected to chromatography
(heptane.fwdarw.2% EtOAc-heptane) to yield Di-Linoleyl acetonitrile
(1.2 g, 69%) as a pale yellow oil. Rf 0.84 (10% EtOAc-hexanes), FW
537.95, C.sub.38H.sub.67N.
[0513] Step 2: Synthesis of the Di-Linoleyl acetylaldehyde, having
the following structure:
##STR00065##
[0514] A solution of the nitrile, Di-Linoleyl acetonitrile, (1.01
g, 1.88 mmol) in anhydrous CH.sub.2Cl.sub.2 (25 mL) was cooled
(-78.degree. C.) and treated, dropwise, with DIBAL (3.75 mL, 3.75
mmol; 1.0M in hexanes). The solution was warmed (-25.degree.
C.--30.degree. C.) and stirred (3 h), then carefully poured into
cold HCl (5%, aq.) and extracted with EtOAc. The organic extract
was washed with brine, dried (Na.sub.2SO.sub.4), filtered and
concentrated. The crude material was subjected to chromatography
(2% EtOAc-hexanes) to yield Di-Linoleyl acetylaldehyde (552 mg,
54%) as a colorless oil. Rf 0.62 (10% EtOAc-hexanes), FW 540.95,
C.sub.38H.sub.68O.
[0515] Step 3: Synthesis of the Tri-linoleyl alcohol, referred to
as Compound AM-246 below, having the following structure:
##STR00066##
[0516] A solution of the above di-linoleyl acetylaldehyde from step
2 (520 mg, 0.96 mmol) in anhydrous THF (5 mL) was cooled (0.degree.
C.) and treated, dropwise, with LinMgBr (0.93 mL, 1.90 mmol; 2.04M
in THF), and stirred (16 h). The solution was poured into H.sub.2O
and extracted with hexanes. The organic extract was washed with HCL
(5M, aq.) and H.sub.2O, dried (Na.sub.2SO.sub.4), filtered and
concentrated. The crude material was subjected to chromatography
(1% EtOAc-hexanes) to yield AM-246 (600 mg, 79%) as a colorless
oil. Rf 0.66 (10% EtOAc-hexanes), FW 791.41,
C.sub.56H.sub.102O.
[0517] Step 4: Synthesis of compound 13-B6, having the following
structure:
##STR00067##
[0518] A solution of the Tri-linoleyl alcohol of step 3 above,
AM-246, (600 mg, 0.76 mmol) and N,N-dimethylamino-butyric acid
hydrochloride (165 mg, 0.99 mmol) in anhydrous CH.sub.2Cl.sub.2 (5
mL) was treated with EDC (189 mg, 0.99 mmol), Hunig's base (400
.mu.L, 2.3 mmol) and DMAP (7 mg). After stirring (16 h) the
solution was diluted with CH.sub.2Cl.sub.2, washed with NaHCO.sub.3
(sat. aq.) and brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The crude material was subjected to chromatography
(CH.sub.2Cl.sub.2.fwdarw.2% CH.sub.3OH--CH.sub.2Cl.sub.2) to yield
AM-250 (499 mg, 73%) as a pale yellow oil. Rf 0.51 (8%
CH.sub.3OH--CHCl.sub.3), .sup.1H NMR (400 MHz, CDCl.sub.3,
.delta.H) 5.43-5.30 (m, 12H, C.dbd.CH.times.12), 4.96-4.91 (m 1H,
CHO.sub.2CR), 2.82-2.75 (app. t, 6H,
C.dbd.CHCH.sub.2HC.dbd.C.times.3) 2.35-2.28 (m, 4H,
CH.sub.2CO.sub.2R, CH.sub.2N(CH.sub.3).sub.2), 2.23 (s, 6H,
N(CH.sub.3).sub.2), 2.10-2.04 (m, 13H, CH.sub.2HC.dbd.C.times.6,
O.sub.2CHCH(CH.sub.2).sub.2), 1.84-1.75 (m, 2H, CH.sub.2),
1.55-1.42 (m, 2H, CH.sub.2), 1.41-1.14 (m, 58H, CH.sub.2.times.29),
0.90 (t, 9H, CH.sub.3.times.3); FW 904.57,
C.sub.62H.sub.113NO.sub.2.
Example 7
Synthesis of 13-B7
[0519] Synthesis of Compound 13-B7,
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
3-(dimethylamino)propyl((9Z,12Z)-octadeca-9,12-dienyl)carbamate,
having the following structure:
##STR00068##
[0520] Step 1: Synthesis of (9Z,12Z)-octadeca-9,12-dienal, having
the following structure:
##STR00069##
[0521] A solution of linoleyl alcohol (15 g, 56 mmol) in anhydrous
CH.sub.2Cl.sub.2 (400 mL) was treated with pyridinium
chlorochromate (36.4 g, 169 mmol) and Na.sub.2CO.sub.3 (2.98 g,
28.2 mmol) and then stirred (4 h). The reaction mixture was diluted
with EtOAc and filtered through a pad of silica and then
concentrated. The crude material was subjected to chromatography
(1%.fwdarw.3%.fwdarw.6% EtOAc-heptane) to yield
(9Z,12Z)-octadeca-9,12-dienal (10.5 g, 71%) as a colorless oil. Rf
0.58 (10% EtOAc-hexanes), FW 264.45, C.sub.18H.sub.32O.
##STR00070##
[0522] Step 2: Synthesis of
N,N-dimethyl-N'-((9Z,12Z)-octadeca-9,12-dienyl)propane-1,3-diamine,
having the following structure:
##STR00071##
[0523] A solution of (9Z,12Z)-octadeca-9,12-dienal (1.06 g, 4.0
mmol) and N,N-dimethylpropane-1,3-diamine (408 mg, 4.0 mmol) in
anhydrous tetrahydrofuran (15 mL) was stirred under nitrogen for 15
minutes then sodium triacetoxyborohydride (1.7 g, 8.0 mmol). The
solution was stirred for 2 hours at room temperature. Upon
completion, the solution was diluted with ethyl acetate (20 mL) and
washed with sodium bicarbonate (2.times.15 mL) and brine (15 mL).
The ethyl acetate layer was dried on magnesium sulfate, filtered
and concentrated in vacuo to dryness. The oil was purified by
column chromatography on silica gel (eluted with 100%
CH.sub.2Cl.sub.2 to 1/5/94 NH.sub.4OH/MeOH/CH.sub.2Cl.sub.2) to
afford
N,N-dimethyl-N'-((9Z,12Z)-octadeca-9,12-dienyl)propane-1,3-diamine
as a colorless oil (420 mg, 30%).
[0524] Step 3: Synthesis of
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
3-(dimethylamino)propyl((9Z,12Z)-octadeca-9,12-dienyl)carbamate,
having the following structure:
##STR00072##
[0525] To a solution of
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
carbonochloridate (590 mg, 1.0 mmol),
N,N-dimethyl-N'-((9Z,12Z)-octadeca-9,12-dienyl)propane-1,3-diamine
(350 mg, 1 mmol) and triethylamine (500 .mu.L, 3.6 mmol) in
dichloromethane (8 mL) was stirred overnight at room temperature.
Upon completion, the solution was concentrated in vacuo to dryness
and purified by column chromatography on silica gel to afford
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
3-(dimethylamino)propyl((9Z,12Z)-octadeca-9,12-dienyl)carbamate as
a colorless oil (500 mg, 55%). .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 5.42-5.28 (m, 12H), 4.79-4.70 (m, 1H), 3.32-3.12 (m, 4H),
2.78 (t, 6H, J=6.3 Hz), 2.38-2.20 (m, 8H), 2.09-2.02 (m, 12H),
1.82-1.63 (m, 2H), 1.57-1.43 (m, 6H), 1.41-1.23 (m, 52H), 0.89 (t,
9H, J=7.0 Hz).
Example 8
Synthesis of 13-B8
[0526] Compound 13B-8, having structure (G) below, was synthesized
as described in Scheme 10 below:
##STR00073##
[0527] Step 1: Preparation of
4,5-di((8Z,11Z)-heptadeca-8,11-dienyl)-4-((9Z,12Z)-octadeca-9,12-dienyl)--
1,3-dioxolan-2-one
##STR00074##
[0528] To a solution of
(6Z,9Z,28Z,31Z)-19-((8Z,11Z)-heptadeca-8,11-dienyl)heptatriaconta-6,9,28,-
31-tetraene-18,19-diol (540 mg, 0.67 mmol) in anhydrous ether (5
mL) was added pyridine (81 .mu.L, 1.0 mmol). To this solution was
added diphosgene (132 .mu.L, 1.1 mmol) slowly over 5 minutes. The
solution was stirred for 2 hours at room temperature then
concentrated in vacuo to dryness and purified by column
chromatography on silica gel 60 (eluted with 5% ether in hexanes)
to afford
4,5-di((8Z,11Z)-heptadeca-8,11-dienyl)-4-((9Z,12Z)-octadeca-9,12-dienyl)--
1,3-dioxolan-2-one as a colorless oil (310 mg, 57%).
[0529] Step 2: Synthesis of Compound 13-B8,
(6Z,9Z,28Z,31Z)-19-((9Z,12Z)-heptadeca-9,12-dienyl)-19-hydroxyhexatriacon-
ta-6,9,28,31-tetraen-18-yl
3-(dimethylamino)propyl(methyl)carbamate, having the following
structure:
##STR00075##
[0530] To a solution of N,N,N-Trimethyl propane-1,3-diamine (87 mg,
0.75 mmol) in anhydrous THF (5 mL) cooled to -78.degree. C. was
added slowly 2.5M n-BuLi in hexanes (0.3 mL, 0.75 mmol). The
solution was stirred for 30 minutes at -78.degree. C. then a
solution of the cyclic carbamate (200 mg, 0.25 mmol) in THF (5 mL).
The solution was slowly warmed to room temperature and stirred for
1 hour. The solution was diluted with ethyl acetate (75 mL) and
washed with water (1.times.50 mL) and brine (1.times.50 mL). The
ethyl acetate solution was dried on magnesium sulfate, filtered and
concentrated in vacuo to dryness. The residue was purified by
column chromatography on silica gel 60 (eluted with 100% ethyl
acetate) to afford
(6Z,9Z,28Z,31Z)-19-((9Z,12Z)-heptadeca-9,12-dienyl)-19-hydroxyhexatriacon-
ta-6,9,28,31-tetraen-18-yl 3-(dimethylamino)propyl(methyl)carbamate
(196 mg, 86%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.43-5.29
(m, 12H), 4.81-4.78 (m, 1H), 3.43-3.25 (m, 2H), 2.93 (s, 3H), 2.77
(t, 6H, J=6.4 Hz), 2.39-2.20 (m, 12H), 1.76-1.45 (m, 9H), 1.42-1.21
(m, 54H), 0.90 (t, 9H, J=6.8 Hz).
Example 9
Lipid Encapsulation of siRNA
[0531] All siRNA molecules used in these studies were chemically
synthesized and annealed using standard procedures.
[0532] In some embodiments, siRNA molecules were encapsulated into
serum-stable nucleic acid-lipid particles (SNALP) composed of the
following lipids: (1) the lipid conjugate PEG2000-C-DMA
(3-N-[(-methoxypoly(ethylene
glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); (2) one or
more cationic lipids or salts thereof (e.g., cationic lipids of
Formula I of the invention and/or other cationic lipids described
herein); (3) the phospholipid DPPC
(1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (Avanti Polar Lipids;
Alabaster, Ala.); and (4) synthetic cholesterol (Sigma-Aldrich
Corp.; St. Louis, Mo.) in the molar ratio 1.4:57.1:7.1:34.3,
respectively. In other words, siRNA molecules were encapsulated
into SNALP of the following "1:57" formulation: 1.4% PEG2000-C-DMA;
57.1% cationic lipid; 7.1% DPPC; and 34.3% cholesterol. It should
be understood that the 1:57 formulation is a target formulation,
and that the amount of lipid (both cationic and non-cationic)
present and the amount of lipid conjugate present in the
formulation may vary. Typically, in the 1:57 formulation, the
amount of cationic lipid will be 57.1 mol %.+-.5 mol %, and the
amount of lipid conjugate will be 1.4 mol %.+-.0.5 mol %, with the
balance of the 1:57 formulation being made up of non-cationic lipid
(e.g., phospholipid, cholesterol, or a mixture of the two).
[0533] In other embodiments, siRNA may be encapsulated into SNALP
composed of the following lipids: (1) the lipid conjugate
PEG750-C-DMA (3-N-[(-Methoxypoly(ethylene
glycol)750)carbamoyl]-1,2-dimyristyloxypropylamine); (2) one or
more cationic lipids or salts thereof (e.g., cationic lipids of
Formula I of the invention and/or other cationic lipids described
herein); (3) the phospholipid DPPC; and (4) synthetic cholesterol
in the molar ratio 6.76:54.06:6.75:32.43, respectively. In other
words, siRNA may be encapsulated into SNALP of the following "7:54"
formulation: 6.76 mol % PEG750-C-DMA; 54.06 mol % cationic lipid;
6.75 mol % DPPC; and 32.43 mol % cholesterol. Typically, in the
7:54 formulation, the amount of cationic lipid will be 54.06 mol
%.+-.5 mol %, and the amount of lipid conjugate will be 6.76 mol
%.+-.1 mol %, with the balance of the 7:54 formulation being made
up of non-cationic lipid (e.g., phospholipid, cholesterol, or a
mixture of the two).
[0534] For vehicle controls, empty particles with identical lipid
composition may be formed in the absence of siRNA.
Example 10
pKa Measurements of SNALP Formulations Containing Novel Trialkyl
Cationic Lipids
[0535] This example demonstrates the determination of pK.sub.a
values of various SNALP formulations containing the novel trialkyl
cationic lipids described herein. In particular, SNALP formulations
containing encapsulated siRNA may be prepared as described in
Example 9 and section IV above with one or more cationic lipids of
Formula I of the invention.
[0536] In certain embodiments, the apparent pK.sub.a of the
cationic lipids present in these SNALP formulations may be
determined using a 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate
(TNS) assay. TNS is a negatively-charged indicator of membrane
potential that is electrostatically attracted to positively charged
membranes (see, Bailey and Cullis, Biochem., 33 12573-80 (1994)).
Subsequent adsorption to the lipid membrane results in the
immediate environment of the TNS becoming more lipophilic, removing
the water molecules that otherwise quench TNS fluorescence. As a
result, TNS measures the surface potential of the particle, wherein
the more positive the surface potential, the greater the level of
fluorescence. The surface pK.sub.a values of a SNALP formulation
may be determined by varying the local pH in the presence of TNS.
By plotting fluorescence versus pH, the pK.sub.a of a cationic
lipid can be estimated in the particle as the pH where fluorescence
equals 50% of total fluorescence. In other words, the pK.sub.a of a
cationic lipid is the pH at which 50% of the cationic lipid present
in the particle is charged.
Example 11
Characterization of SNALP Formulations Containing Novel Trialkyl
Cationic Lipids
[0537] This example demonstrates the efficacy of 1:57 SNALP
formulations containing various novel trialkyl cationic lipids of
Formula I described herein with an siRNA targeting ApoB in a mouse
liver model. In particular, the objective of this study was to
assess the ApoB dose response gene silencing activity in the mouse
liver of 1:57 SNALP containing various novel trialkyl cationic
lipids of Formula I. The ApoB siRNA sequence used in this study is
provided in Table 1.
TABLE-US-00002 TABLE 1 % 2'OMe- % Modified siRNA ApoB siRNA
Sequence Modified in DS Region ApoB-10164
5'-AGUGUCAUCACACUGAAUACC-3' 7/42 = 16.7% 7/38 = 18.4% (SEQ ID NO:
1) 3'-GUUCACAGUAGUGUGACUUAU-5' (SEQ ID NO: 2) Column 1: The number
after "ApoB" refers to the nucleotide position of the 5' base of
the sense strand relative to the human ApoB mRNA sequence
NM_000384. Column 2: 2'OMe nucleotides are indicated in bold and
underlined. The 3'-overhangs on one or both strands of the siRNA
molecule may alternatively comprise 1-4 deoxythymidine (dT)
nucleotides, 1-4 modified and/or unmodified uridine (U)
ribonucleotides, or 1-2 additional ribonucleotides having
complementarity to the target sequence or the complementary strand
thereof. Column 3: The number and percentage of 2'OMe-modified
nucleotides in the siRNA molecule are provided. Column 4: The
number and percentage of modified nucleotides in the
double-stranded (DS) region of the siRNA molecule are provided.
I. Experimental Design
[0538] The experimental design for this study is set forth in Table
2.
TABLE-US-00003 TABLE 2 IV Bolus siRNA Dosing End Group Test Article
Dose days Point Collections 1 PBS -- Day 0 Day 2 Plasma for 2 1:57
C2K 0.05 mg/kg 48 hours cholesterol 3 1:57 MC3 Half of Left 4 1:57
Lateral MC3MC Lobe into 5 1:57 13-B1 RNAlater 6 1:57 13-B2
(formulated at low pH) 7 1:57 13-B3 (formulated at low pH) 8 1:57
13-B4 9 1:57 1-B17 10 1:57 MC3 Thioester In Table 2, Group Size: n
= 3, female BALB/c mice
II. Test Articles
[0539] Ready-to-inject vials of test article, diluted to the
appropriate concentrations, were prepared no sooner than 3 days
before use. Test articles were provided as 0.22 .mu.m filter
sterilized liquids in crimp top glass vials. All vials were labeled
with the study #, group #, lot #, and siRNA concentration. For
control formulations lacking siRNA, the total lipid concentration
was provided instead. Test article vials were stored at 2-8.degree.
C. and removed from storage 30-60 minutes before use in order to
equilibrate to room temperature.
[0540] Brief description of formulations & controls used:
[0541] The PBS used for dilution of SNALP (test article
preparation) is Hyclone lot#ATL33632 [0542] "Base" lipid
composition 1.4|57.1|7.1|34.3 is described in molar percentages of
PEG.sub.2000-C-DMA, cationic lipid, DPPC, and cholesterol (in that
order). Input lipid:drug ratio is 6. [0543] siRNA: Test articles
contain GMP-grade siApoB-8 manufactured by Avecia Lot 100,000
(11.632 mg/mL solution). [0544] Drug concentration is expressed as
total siRNA content. [0545] Batch size is 0.34 mg, ethanol is
removed via Slide-A-Lyzer bag dialysis.
[0546] 1:57 SNALP formulations containing encapsulated ApoB siRNA
were prepared as described in Sections V and VI above with the
following cationic lipids: (1) DLin-C2K-DMA ("C2K"); (2) MC3; (3)
MC3MC; (4) 13-B1; (5) 13-B2; (6) 13-B3; (7) 13-B4; (8) MC4 Ether
("1-B17"); and (9) MC3 Thioester. The synthesis of MC3, MC3MC, MC4
Ether, and MC3 Thioester is described in U.S. Provisional
Application No. ______, entitled "Novel Cationic Lipids and Methods
of Use Thereof," bearing Attorney Docket No. 020801-010610US, filed
Sep. 17, 2010. The synthesis of 13-B1,13-B2,13-B3, and 13-B4 is
described herein. Table 3 provides exemplary features of these
SNALP formulations, including particle size (Z-average),
polydispersity, and percent encapsulation.
TABLE-US-00004 TABLE 3 Total siRNA Encap. Z-average Description
(mg/mL) (%) (nm) Polydispersity PBS N/A N/A N/A N/A 1:57 C2K 0.135
85 82.60 0.059 1:57 MC3 0.128 79 80.05 0.048 1:57 MC3MC 0.137 87
90.19 0.019 1:57 13-B1 0.122 86 88.88 0.094 1:57 13-B2 0.141 86
84.73 0.060 (formulated at low pH) 1:57 13-B3 0.131 84 81.54 0.030
(formulated at low pH) 1:57 13-B4 0.131 87 95.07 0.033 1:57 MC4
Ether 0.132 85 84.85 0.034 1:57 MC3 Thioester 0.126 75 80.57
0.069
III. Procedures
[0547] Identification:
[0548] Groups were assigned numbers 1-10 as listed in Section I
Animal IDs were assigned two ways: a) for visual identification
during the in-life phase, animals within a cage were differentiated
by `color` code (R=red, G=green, W=no color); and b) for specimen
identification during the analytical phase, animals were assigned a
number based on group order.
[0549] Cage-Sorting:
[0550] At least 1 day (preferably at least 2-3 days, and as much as
7 days) before the study begins, animals were sorted in order to
normalize body weight distribution such that a) the mean for each
cage was similar by targeting all cage means to be within 2.0 g and
b) the range within a cage was similar between cages. Also, a
routine health check was performed and any abnormal animals
(including unusually heavy or light animals) were noted and
replaced with spares as available.
[0551] Treatment:
[0552] Just prior to the first treatment, animals were weighed and
dose amounts were calculated based on the weight of individual
animals (equivalent to 10 mL/kg, rounded to the nearest 10 .mu.L).
The test article was administered by IV injection through the tail
vein once on Day 0 (1 dose total per animal).
[0553] Monitoring:
[0554] Body weight was measured at cage-sort, as well as on every
day from Day 0 through Day 2. Weights for all groups were taken at
the same time of day (for convenience), at a consistent time of day
to minimize effects of circadian fluctuation in this parameter.
Cageside observations were taken in concert with body weight
measurements, at 1 hour after treatment, at end-of-day on the day
of dosing, and additionally as warranted (recorded for all on-study
animals whenever abnormal behavior in a subset of animals was
detected).
[0555] Nonterminal Collections:
[0556] None.
[0557] Terminal Collection:
[0558] Animals were sacrificed on Day 2, 48 h after test article
administration. Animals were anaesthetized with a lethal dose of
ketamine/xylazine; then exsanguination was performed via cardiac
puncture (no cervical dislocation). Blood was collected by cardiac
puncture. For each animal, blood (target 0.5 mL) was collected into
a lavender EDTA microtainer for plasma: Invert 10.times., then
immediately centrifuge for 5 min at 16,000.times.g and 16.degree.
C., transfer plasma into a clean 1.5 mL microfuge tube (target 250
.mu.L, record volume and appearance), store at 4.degree. C. prior
to further analyses. A brief necropsy was performed and
observations recorded. Approximately 200-300 mg of the bottom
(unattached) half of the left liver lobe was cut off and submerged
in >5 volumes of RNAlater (<0.3 g into a 2.0 mL tube
containing 1.5 mL RNAlater), stored at least 16 hours (and no more
than one month) at +4.degree. C. prior to analysis. Following
analysis, samples were transferred into long term storage at
-80.degree. C. for archival purposes.
[0559] Euthanization:
[0560] Formulations were expected to be well tolerated. Mice which
exhibited signs of distress associated with the treatment were
terminated at the discretion of the core facility staff.
[0561] Data Analysis:
[0562] Body weight was reported for individual animals in addition
to group mean and standard deviation of the group mean. Data were
expressed in terms of the measured unit (grams) and the group mean
difference from PBS vehicle control treatment was calculated for
this expression (include SD). For body weights, individual animal
data were also expressed as % of pre-treatment value and the group
means were expressed as % change from pre-treatment value (include
SD). Cageside observations were reported as raw tabulated data
entries (generally line entries were organized by clock time with
granularity to the level of individual animals) as well as a
tabulated summary containing treatment groups on the left column
and study timeline running left-to-right on the top row as header.
Necropsy gross observations were reported as raw tabulated data
entries (with granularity to the level of individual animals) as
well as a tabulated summary containing treatment groups on the left
column and selected key tissues and/or findings on the top row as
header. Drug activity was measured using a QuantiGene assay for
ApoB mRNA level in the liver (normalized to GAPDH mRNA level). An
ELISA for ApoB-100 protein in plasma and/or colorimetric enzymatic
assay for plasma total cholesterol may also be conducted. For any
given activity assay, individual animal data were reported in
addition to group mean and standard deviation of the group mean.
Data were expressed in terms of the measured unit as well as
difference from PBS vehicle control treatment. KD50 ("50%
knockdown") values were interpolated from QuantiGene measurements
for those test articles for which a dose response was
performed.
IV. Results
[0563] The FIGURE shows a comparison of the liver ApoB mRNA
knockdown activity of each of these SNALP formulations (Error
bars=SD). In particular, the FIGURE shows that a SNALP formulation
containing either 13-B1 or 13-B2 displayed similar ApoB silencing
activity compared to SNALP containing the C2K benchmark cationic
lipid. Notably, the FIGURE also shows that a SNALP formulation
containing 13-B4, in which subscript a of Formula I is 4 (i.e.,
there is a 4-carbon spacer between the amino head group and the
ester linker), displayed unexpectedly improved ApoB silencing
activity compared to SNALP containing the C2K benchmark cationic
lipid.
[0564] It is to be understood that the above description is
intended to be illustrative and not restrictive. Many embodiments
will be apparent to those of skill in the art upon reading the
above description. The scope of the invention should, therefore, be
determined not with reference to the above description, but should
instead be determined with reference to the appended claims, along
with the full scope of equivalents to which such claims are
entitled. The disclosures of all articles and references, including
patent applications, patents, PCT publications, and Genbank
Accession Nos., are incorporated herein by reference for all
purposes.
Sequence CWU 1
1
2121RNAArtificial Sequencesynthetic apolipoprotein B (ApoB, APOB)
siRNA ApoB-10164 1agunucanca cacngaauac c 21221RNAArtificial
Sequencesynthetic apolipoprotein B (ApoB, APOB) siRNA ApoB-10164
2uauncanunu gaugacacnu g 21
* * * * *
References